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https://openalex.org/W2160957612	https://www.solid-earth.net/6/1231/2015/se-6-1231-2015.pdf	English	null	Influence of humic acid applications on modulus of rupture, aggregate stability, electrical conductivity, carbon and nitrogen content of a crusting problem soil	Solid earth	2,015	cc-by	5,342	Inﬂuence of humic acid applications on modulus of rupture, aggregate stability, electrical conductivity, carbon and nitrogen content of a crusting problem soil ˙I. Gümü¸s and C. ¸Seker Department of Soil Science and Plant Nutrition, Faculty of Agriculture, University of Selçuk, 42031 Konya, Turkey Correspondence to: ˙I. Gümü¸s (ersoy@selcuk.edu.tr) Received: 23 July 2015 – Published in Solid Earth Discuss.: 7 September 2015 Revised: 20 October 2015 – Accepted: 21 October 2015 – Published: 11 November 2015 ˙I. Gümü¸s and C. ¸Seker Department of Soil Science and Plant Nutrition, Faculty of Agriculture, University of Selçuk, 420 Received: 23 July 2015 – Published in Solid Earth Discuss.: 7 September 2015 Revised: 20 October 2015 – Accepted: 21 October 2015 – Published: 11 November 2015 agement of cultivable land and by urbanization and soil degradation. There is a general agreement that oil biochem- ical, microbiological and biological properties are more im- portant than physical and chemical properties for the purpose of estimating alterations in soil quality and hence soil degra- dation (Keesstra et al., 2012; Paz-Ferreiro and Fu, 2013). Soil organic matter plays an essential role in nutrient (N, P, S, K) cycles, soil stability and the ecological and environ- mental aspects of sustainability of soil fertility (Garcıa-Gil et al., 2004). Turkish soils generally have low organic matter levels and are commonly treated with mineral fertilizers that may improve yield in the short term but do not enhance the physical properties of the soil and result in soil degradation over the longer term. In many regions in Turkey, especially in Central Anatolia, the organic matter content of soils has fallen below 2 or 1 % (¸Seker and Karakaplan, 1999). Abstract. Soil structure is often said to be the key to soil pro- ductivity since a fertile soil, with desirable soil structure and adequate moisture supply, constitutes a productive soil. Soil structure inﬂuences soil water movement and retention, ero- sion, crusting, nutrient recycling, root penetration and crop yield. The objective of this work is to study humic acid (HA) application on some physical and chemical properties in weakly structured soils. The approach involved establish- ing a plot experiment in laboratory conditions. Different rates of HA (control, 0.5, 1, 2 and 4 %) were applied to soil during three incubation periods (21, 42 and 62 days). At the end of the each incubation period, the changes in physicochemical properties were measured. Generally, HA addition increased electrical conductivity values during all incubation periods. HA applications decreased soil modulus of rupture. Inﬂuence of humic acid applications on modulus of rupture, aggregate stability, electrical conductivity, carbon and nitrogen content of a crusting problem soil Applica- tion of HA at the rate of 4 % signiﬁcantly increased soil or- ganic carbon contents. HA applications at the rate of 4 % sig- niﬁcantly increased both mean soil total nitrogen content and aggregate stability after three incubation periods (p < 0.05). Therefore, HA has the potential to improve the structure of soil in the short term. Organic materials are important soil additives to improve soil physical, chemical and biological properties. This is im- portant to sustain the productivity of soils particularly in semi-arid regions (such as Turkey) where there is low input of organic materials. Usage of organic-based materials has gained importance within the last few years for sustainable agriculture and preventing soil degradation (Alagöz and Er- dem, 2009). 1 Introduction Soil structure is often said to be the key to soil, with its ability to support plant and animal life, and moderate envi- ronmental quality with particular emphasis on soil carbon (C) sequestration and water quality. Understanding soil structural formation involves aspects of biology, chemistry, geology and physics within the context of the soil environment (Bre- vik et al., 2015). Using aggregate size, shape and distinct- The widespread use of unsuitable and unsustainable produc- tion techniques in agricultural systems has resulted in exten- sive deterioration of soil quality and reductions in soil or- ganic matter content and crop production (Verhulst et al., 2010; Martinez-Blanco et al., 2011). Soil quality is threat- ened by the increase in human population, by intensive man- 2.2 Methods Certain components of soil organic matter such as polysac- charides, humic substances, root material and fungal hyphae have an important role in structural stabilization. Some syn- thetic conditioners which have shown promise for use in improving soil structure and physical properties at low ap- plication rates are polyacrylamides and polyvinyl alcohols (Bryan, 1992). The study was carried out in a randomized-plot design with three replications and conducted under laboratory conditions as a pot experiment. Surface soil samples (0–20 cm) were air-dried, ground through a 2 mm sieve and mixed homoge- neously. Firstly, soil samples (2000 g) were placed in each pot (dimensions of pot; 13.5 cm × 17 cm). Five levels of HA (0 % (control), 0.5, 1, 2 and 4 %) were incubated. During the incubation period, the soil moisture level in the pots was maintained at 50–75 % of ﬁeld capacity. After various incubation periods (21, 42 and 62 days), the soil samples in the pots were mixed to ensure homogeneity in physical, chemical and biological properties. The soils were then sub- sampled (250 g) for analyses; 21-, 42- and 62-day incubation periods after the incubation of soil samples were analyzed with three replications. y Humic acids and their salts, derived from coal and other natural sources, which have modes of action similar to syn- thetic conditioners, have been evaluated as potential soil con- ditioners. The advantage of humic substances is the refrac- tory nature of their chemical structures that makes them more resistant to microbial attacks. Piccolo et al. (1997) re- ported that humic substances have a potential as soil con- ditioners in conversation practices aimed at increasing the structural stability of soils. Ersoy and ¸Seker (2004) reported that urban waste compost, cattle manure, chicken manure and leonardite improved soil aggregate stability values. ¸Seker (2003) reported that adding Portland cement and wheat straw to a soil having a crusting problem increased its aggregate stability; in turn seedling emergence of wheat was improved by decreased modulus of rupture and penetration resistance. Imbufe et al. (2005) suggested that potassium humate is po- tentially effective as a soil conditioner in improving aggre- gate stability of acidic and sodic soils against adverse ef- fects of cyclic seasonal wetting and drying conditions. Bal et al. (2011) determined crusting problems of the Konya- Sarıcalar research station soils and offered some recommen- dations for a solution. 2.1 Material Humic acid (trade name DELTA K-Humate) was supplied from a company. The soil sample used in this study has prob- lems such as insufﬁcient seedling emergency, low aggregate stability and crusting problems (Bal et al., 2011) Compos- ited soil samples were taken from a problematic plot in the Agricultural Faculty of Selçuk University experiment station (0–20 cm soil depth) near the Konya Sarıcalar village located in Central Anatolia, Turkey (38◦06′ N, 32◦36′ E; 1010 m). The climate is semi-arid, with an annual precipitation of 379.38 mm, an annual mean temperature of 11.5 ◦C and an annual mean evaporation of 1226.4 mm. ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications Reports have indicated that loss of organic matter is generally associated with a de- cline in soil porosity and wet aggregate stability, as well as an increase in soil strength indices (¸Seker and Karakaplan, 1999). The aim of this study was to determine the effects of hu- mic acid applications on crust resistance, aggregate stability, electrical conductivity (EC), nitrogen and organic carbon of weakly structured soils. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 1232 ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications ness as the basis for classes, types and grades, respectively, soil structure describes the manner in which soil particles are aggregated. Soil structure affects water and air movement through soil, greatly inﬂuencing a soil’s ability to sustain life and to perform other vital soil functions. A well-developed structure and high aggregate stability are important for im- proving soil fertility, increasing agronomic productivity, en- hancing porosity and decreasing erodibility. Aggregate sta- bility is a reﬂection of soil structure and soil health in gen- eral because it depends on an integrated balance of chemical, physical and biological factors (Solera-Mataix, 2011; Brevik et al., 2015). Soil aggregate stability is widely regarded as one of the quality indicators in the soil system (Saygın et al., 2015). The decline in soil structure is increasingly seen as a form of soil degradation (Chan et al., 2003) and is often re- lated to land use and soil–crop management practices. Struc- tural and physical soil degradation is often associated with a decline in the organic matter content. Reports have indicated that loss of organic matter is generally associated with a de- cline in soil porosity and wet aggregate stability, as well as an increase in soil strength indices (¸Seker and Karakaplan, 1999). ness as the basis for classes, types and grades, respectively, soil structure describes the manner in which soil particles are aggregated. Soil structure affects water and air movement through soil, greatly inﬂuencing a soil’s ability to sustain life and to perform other vital soil functions. A well-developed structure and high aggregate stability are important for im- proving soil fertility, increasing agronomic productivity, en- hancing porosity and decreasing erodibility. Aggregate sta- bility is a reﬂection of soil structure and soil health in gen- eral because it depends on an integrated balance of chemical, physical and biological factors (Solera-Mataix, 2011; Brevik et al., 2015). Soil aggregate stability is widely regarded as one of the quality indicators in the soil system (Saygın et al., 2015). The decline in soil structure is increasingly seen as a form of soil degradation (Chan et al., 2003) and is often re- lated to land use and soil–crop management practices. Struc- tural and physical soil degradation is often associated with a decline in the organic matter content. www.solid-earth.net/6/1231/2015/ Table 2. Properties of the humic acid. Table 2. Properties of the humic acid. Table 2. Properties of the humic acid. Properties HA pH (H2O, 1 : 2.5) 11–13 Organic matter (%) 30 Humic and fulvic acid (%) 80 K2O (%) 10 Figure 1. Effects of different rates of HA applications on soil mod- ulus of rupture. Properties HA pH (H2O, 1 : 2.5) 11–13 Organic matter (%) 30 Humic and fulvic acid (%) 80 K2O (%) 10 Figure 1. Effects of different rates of HA applications on soil mod- ulus of rupture. matter addition (Özdemir, 2002; ¸Seker, 2003). The possible mechanisms by which coal-derived humic acids improve soil physical properties are the formation of organomineral com- plexes by functional groups of the humic acids (Glaser et al., 2002). LSD at P < 0.05) according to the procedures outlined by Snedecor and Cochran (1980). All statistical results were cal- culated using the one-way analysis-of-variance procedure on MINITAB statistical software package (Minitab, 1995). 3 Result and discussion Some physical and chemical properties of the soil and humic acid are given in Tables 1 and 2. The soil was characterized by having a clay texture, an alkaline soil pH (7.80) and or- ganic matter and CaCO3 contents of 2.95 and 11.17 %, re- spectively. 2.2 Methods As a result, an increase in the organic matter content and a reduction in agricultural practices to the minimum tillage were required in order to prevent crusting problems in the research soils. Particle-size distribution of the soil was determined by the hydrometer method (Day, 1965). Soil water retention at ﬁeld capacity (−0.33 kPa) suction was determined by using a ce- ramic plate (Peters, 1965). Soil EC values were determined using a glass–calomel electrode in a 1 : 2.5 mixture (v/v) of soil and water (Jackson, 1967). Soil organic carbon was de- termined on sample ground to pass through a 0.5 mm sieve by the using the TruSpec CN Carbon/Nitrogen Determina- tor (LECO Corporation 2006). The methodology used for measuring modulus of rupture (MR) as an index of crust- ing was that proposed by Reeve (1965). Aggregate stabil- ity was determined by immersing the sieves, containing the aggregate samples (between 1 and 2 mm size), in distilled water up and down oscillating on screens through 55 mm at 30 strokes min−1 for 5 min (Kemper, 1965). The data col- lected from the experiment were analyzed using analysis- of-variance tests based on randomized-plot design (using F- Solid Earth, 6, 1231–1236, 2015 www.solid-earth.net/6/1231/2015/ ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications 1233 Table 1. Properties of the soil used in the experiment Soil properties Values Soil properties Values Sand (2–0.05 mm) (%) 7.36 Field capacity (%) 31.14 Silt (0.05–0.002 mm) (%) 37.72 Wilting point (%) 15.39 Clay (< 0.002 mm) (%) 54.92 Aggregate stability (%) 14.37 Textural class Clay Bulk density (g cm−3) 1.34 pH (H2O, 1 : 2.5) 7.80 EC (H2O, 1 : 2.5) d S m−1 0.556 Organic matter (%) 2.95 Carbonates (%) 11.17 CEC (cmol kg−1) 33.6 CEC: cation exchange capacity. a a a a b b c b c c 0 50 100 150 200 250 21 day 42 day 62 day Modulus of rupture ( kPa) ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications Table 1. 3.2 Effects of different rates of humic acid (HA) applications on aggregate stability The effects of HA on soil aggregate stability values are given in Fig. 2. Aggregate stability values of the soil treated with different doses of HA application were measured after 21-, 42- and 62-day incubation periods. The effects of HA ap- plication on soil aggregate stability values were signiﬁcant (P < 0.05). Generally, aggregate stability increased with HA applications. These results may be explained by the fact that biological and physicochemical properties (especially, metal ions, humic or fulvic acids and carbohydrates content) can play a role in initial aggregate formation. Stability of micro- aggregates is strongly correlated with the humic matter con- tent (humic or humic + fulvic acid) (Piccolo and Mbagwu, 1990). Kütük et al. (2000) reported that the highest number of water-resistant aggregates was obtained in the highest dose of humic acid application. Aggregate stability decreased in the 42- and 62-day incubation periods in all humic acid rates compared to 21-day incubation period. It is well known that soil organic matter, especially humic materials, is cementing agents in soil particles; however, certain organic components can paradoxically play a role as a dispersion element in clay– water systems (Tarchitzky et al., 1993). Reduced input rate of organic matter and translocation of surface soils with water erosion are also reasons for low organic matter content of cultivated soils (Ozgöz et al., 2013). Lower organic carbon content in farmland caused a signiﬁcant difference in aggre- gate stability values (Ozgöz et al., 2013). Shanmuganathan and Oades (1983) reported that addition of anions to soils causes dispersion in clay fraction associated with decreas- 3.1 Effects of different rates of humic acid (HA) applications on soil modulus of rupture The effects of HA on soil modulus of rupture are given in Fig. 1. Modulus rupture of the soil treated with different doses of HA application was measured after 21-, 42- and 62-day incubation periods. The effects of HA application on modulus of rupture were signiﬁcant (P < 0.05). Gener- ally, soil modulus of rupture decreased with the increasing amendment rates of HA. These results may be explained by buildup of soil aggregate systems during incubation periods. The modulus of rupture was reduced because of the increase in HA treatments, which allowed less cohesion among the soil particles. Soil degradation caused by which may have led to a reduction in soil organic matter decomposition, soil erosion and nutrient leaching. The decreased soil erosion risk, nutrient leaching an organic matter decomposition rate helped in improving soil quality (Zhang et al., 2015). Similar positive effects were recorded with various forms of organic www.solid-earth.net/6/1231/2015/ 2.2 Methods Properties of the soil used in the experiment Soil properties Values Soil properties Values Sand (2–0.05 mm) (%) 7.36 Field capacity (%) 31.14 Silt (0.05–0.002 mm) (%) 37.72 Wilting point (%) 15.39 Clay (< 0.002 mm) (%) 54.92 Aggregate stability (%) 14.37 Textural class Clay Bulk density (g cm−3) 1.34 pH (H2O, 1 : 2.5) 7.80 EC (H2O, 1 : 2.5) d S m−1 0.556 Organic matter (%) 2.95 Carbonates (%) 11.17 CEC (cmol kg−1) 33.6 CEC: cation exchange capacity. a 0 50 100 150 200 250 2 Modulus of rupture ( kPa) ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications 1233 a a a a b b c b c c 0 50 100 150 200 250 21 day 42 day 62 day Modulus of rupture ( kPa) control 0.5% 1% 2% 4% Figure 1. Effects of different rates of HA applications on soil mod- ulus of rupture. a a a a b b c b c c 0 50 100 150 200 250 21 day 42 day 62 day Modulus of rupture ( kPa) control 0.5% 1% 2% 4% ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications SOC con- tent, nitrogen and phosphorus are some of the most impor- tant chemical soil quality indicators for soil recovery and can drive changes in the biological, chemical and physical soil attributes (Vaconcellos et al., 2013). Similar positive effects were recorded with various forms of organic matter and ar- buscular mycorrhizal fungi addition (Ferreras et al., 2006; Kavdır and Killi, 2008; Yılmaz, 2011; Vaconcellos et al., 2013). ing isoelectric point, and it is known that especially fulvic acids are the most efﬁcient anions. In addition, aggregate sta- bility of the soil samples decreased after incubation periods due, most probably, to mechanical mixing practices (¸Seker, 2003). Aggregate stability should be used judiciously and in concert with other indicators for an overall assessment of the soil physical quality condition (Moncada et al., 2013). 3.3 Effects of different rates of humic acid (HA) applications on soil EC The effects of HA on EC values of the soil are given in Fig. 3. As illustrated in Fig. 3, the EC values signiﬁcantly increased with respect to elevated HA application. According to the investigation at 21 days, the applications signiﬁcantly in- creased the EC, except for 0.5 %. Investigation performed at 42 and 62 days revealed that soil EC linearly increased in re- sponse to increments in HA doses. The increasing EC values in experiment for different doses in HA application may be explained by rich nutrient composition of organic fragments and remains from the materials during incubation periods (Yılmaz, 2010). Imbufe et al. (2004) reported that potassium humate application increased in soil pH and electrical con- ductivity. The present ﬁndings including the previous studies indicate that the increment in HA dose is accompanied by the elevation in soil EC level. For this reason, excessive use of HA should be avoided when HA is considered for use as a solvent substance for calcareous soils in agriculture. ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications 1234 I d d c bc c c c b b ab a a a a a 0 5 10 15 20 21 day 42 day 62 day Aggregate stability (%) control 0.5% 1% 2% 4% Figure 2. Effects of different rates of HA applications on aggregate stability. 1234 Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications d e d e d d c c c b b c a a a 0 500 1000 1500 2000 2500 3000 21 day 42 day 62 day EC (ds m -1) control 0.5% 1% 2% 4% Figure 3. Effects of different rates of HA applications on soil EC. d e d e d d c c c b b c a a a 0 500 1000 1500 2000 2500 3000 21 day 42 day 62 day EC (ds m -1) control 0.5% 1% 2% 4% Figure 3. Effects of different rates of HA applications on soil EC. Figure 3. Effects of different rates of HA applications on soil EC. Figure 3. Effects of different rates of HA applications on soil EC. Figure 2. Effects of different rates of HA applications on aggregate stability. in response to increment in HA dose and the strongest ef- fect obtained with the doses 2 and 4 %, where differences in SOC values depending on incubation periods and rates of HA were noticed. SOC content of soil increased with increasing amendment rates of HA. Generally, SOC content values in experiments increase with the increase of amendment rates of organic materials. Total organic matter has long been rec- ognized as an important determinant of soil performance. It depends on how much organic matter is added to the soil, how quickly it decomposes, and how much can be held by the soil. The amount, type and location of organic matter may be some of the best integrating indicators of many phys- ical, chemical and biological processes (Lewandowski and Zumwinkle, 1999). SOC has been reported as dynamic soil quality indicators (Shukla et al., 2006; Zhao et al., 2015). Therefore, to assess the effect of changes in SOC content on soil structure condition, the aggregate stability can be consid- ered as a good indicator (Moncada et al., 2013). www.solid-earth.net/6/1231/2015/ Solid Earth, 6, 1231–1236, 2015 ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications 4 Conclusions In conclusion, the results of this laboratory study indicate that humic acid applications can improve the stability of structurally soils. Chemical and physical properties of soil such as soil organic carbon, total nitrogen, modulus of rupture and aggregate stability were improved by HA amendment. HA increased soil EC and aggregate stability during the incubation period. Soil modulus of rupture was the most dramatically affected by the HA application. The use of HA may contribute to enhancing the level of organic carbon and nitrogen in soil. According to the results, HA (K-Humate) has potential to be used as an effective conversation and management tool for sustainability of the soil environment. Ferreras, L., Gomez, E., Toresani, S., Firpo, I., and Rotondo, R.: Effect of organic amendments on some physical, chemical and biological properties in a horticultural soil, Bioresource Technol., 97, 635–640, 2006. Garcıa-Gill, J. C., Plaza, C., Senesi, N., Brunetti, G., and Polo, A.: Effects of sewage sludge amendment on humic acids and micro- biological properties of a semiarid Mediterranean soil, Biol. Fert. Soils, 39, 320–328, 2004. Glaser, B., Lehmann, J., and Zech, W.: Ameliorating physical and chemical properties of highly weathered soils in the tropics with charcoal: a review, Biol. Fert. Soils, 35, 219–230, 2002. Imbufe, A. U., Patti, A. F., Surapeneni, A., Jakson, R., and Webb, A. J.: Effects of brown coal derived materials on pH and electri- cal conductivity of an acidic vineyard soil, SuperSoil 2004: 3rd Australian New Zealand Soils Conference, 5–9 December 2004, University of Sydney, Australia, 2004. Edited by: A. Cerdà Edited by: A. Cerdà Imbufe, A. U., Patti, A. F., Burrow, D., Surapaneni, A., Jackson, W. R., and Milner, A. D.: Effects of potassium humate on aggregate stability of two soils from Victoria, Australia, Geoderma, 125, 321–330, 2005. 3.4 Effects of different rates of humic acid (HA) applications on soil organic carbon (SOC) Yılmaz (2011) re- ported that biological and physicochemical properties of or- ganic materials (especially C / N, decomposition and miner- alization level) can play roles in mineralization of nitrogen from organic materials during incubation periods. Bryan, R.: The inﬂuence of some soil conditioners on soil proper- ties: laboratory tests Kenyan soil samples, Soil Technol., 5, 225– 247, 1992. Chan, K. Y., Heenan, D. P., and So, H. B.: Sequestration of carbon and changes in soil quality under conservation tillage on light- textured soils in Australia: a review, Aust J. Exp. Agr., 43, 325– 334, 2003. Day, P. R.: Particle fractionation and particle-size analysis, In: Methods of Soil Analysis, Part I, (Ed Black, C.A.), 545–566, American Society of Agronomy, Madison, WI, 1965. 3.4 Effects of different rates of humic acid (HA) applications on soil organic carbon (SOC) The effects of HA on total nitrogen values of the soil are given in Fig. 5. As illustrated in Fig. 5, the total nitrogen val- ues signiﬁcantly increased with respect to elevated HA ap- plication. According to investigation at 21, 42 and 62 days, the applications (2 and 4 %) signiﬁcantly increased and has the strongest effects on the total nitrogen, except for 0.5, 1 and 2 %. However, amendments rates of doses 2 and 4 % are similar. Generally, total nitrogen content of soil increased The effects of HA on SOC values of the soil are given in Fig. 4. As illustrated in Fig. 4, the SOC values signiﬁcantly increased with respect to elevated HA application. Accord- ing to investigation at 21 days, the applications signiﬁcantly increased the SOC, except for 0.5 %. Investigation performed at 42 and 62 days revealed that soil SOC linearly increased Solid Earth, 6, 1231–1236, 2015 www.solid-earth.net/6/1231/2015/ ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applicatio d e e d d d c c c b b b a a a 0.00 0.50 1.00 1.50 2.00 2.50 3.00 21 day 42 day 62 day Organic C % control 0.5% 1% 2% 4% Figure 4. Effects of different rates of HA applications on soil or- ganic carbon. 1235 I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applicatio d e e d d d c c c b b b a a a 0.00 0.50 1.00 1.50 2.00 2.50 3.00 21 day 42 day 62 day Organic C % control 0.5% 1% 2% 4% Figure 4. Effects of different rates of HA applications on soil or- ganic carbon. c b c c b c c b c b a b a a a 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 21 day 42 day 62 day Total N (%) control 0.5% 1% 2% 4% control 0.5% 1% 2% 4% Figure 4. Effects of different rates of HA applications on soil or- ganic carbon. Figure 4. Effects of different rates of HA applications on soil or- ganic carbon. Figure 5. Effects of different rates of HA applications on total ni- trogen. with increasing amendment rates of HA. ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications 1236 Kemper, W. D.: Aggregate stability, in: Methods of soil analysis, Part I, edited by: Black, C. A., American Society of Agronomy, Madison, WI, 511–519, 1965. Shanmuganathan, R. T. and Oades, J. M.: Inﬂuence of anions on dis- persion and physical properties of the A horizon of a red-brown earth, Geoderma, 29, 257–259, 1983. Shukla, M. R., Lal, R., and Ebinger, M.: Determining soil quality indicators by factor analysis, Soil Till. Res., 87, 194–204, 2006. Kütük, C., Çaycı, G., Baran, A., and Ba¸skan, O.: Effect of humic acid on some soil properties. In: Proceedings of the International Symposium on Desertiﬁcation, Konya, Turkey, 324–328, 2000. Solera-Mataix, J., Cerdà, A., Jordàn, A., and Zavala, L. M.: Fire effects on soil aggregation: a review, Earth-Sci. Rev., 109, 44– 66, 2011. LECO Corporation: Truspec carbon/nitrogen determinator, Leco Corporation 3000, Lakeview Avenue, St Jeseph, M1 49085- 2396, USA, 2003. Snedecor, G. W. and Cochron, W. G.: Statistical methods (7th Edn.), Iowa State, University Press, Ames Iowa, 1980. Lewandowski, A. and Zumwinkle, M.: Assessing the soil system: a review of soil quality literature, Minnesota Department of Agri- culture Energy and Sustainable Agriculture Program, 1999. ¸Seker, C.: Effects of selected amendments on soil properties and emergence of wheat seedlings, Can. J. Soil Sci., 83, 615–621, 2003. Martinez-Blanco, J., Munoz, P., Anton, A., and Rieradevall, J.: As- sesment of tomato Mediterranean production in open-ﬁlled and standard multi-tunnel greenhouse, with compost or mineral fer- tilizers, from an agricultural and environmental standpoint, J. Clean. Prod., 19, 985–997, 2011. ¸Seker, C. and Karakaplan, S.: Konya Ovasında Toprak Özellikleri ile Kırılma De˘gerleri Arasındaki ˙Ili¸skiler, Tr. J. of Agriculture and Forestry, 23, 183–190, 1999. Tarchitzky, J., Chen, Y., and Banin, A.: Humic substances and pH effects on sodium and calcium montmorillonite ﬂocculation of soil structure, Soil Sci. Soc. Am. J., 57, 367–372, 1993. Minitab Inc.: Minitab reference manual (Release7.1), Minitab Inc., State Coll. PA, 16801, USA, 1995. Vaconcellos, R. L. F., Bonﬁm, J. A., Baretta, D., and Cardoso, E. J. B. N.: Arbuscular mycorrhizal fungi and glomalin- releated soil protein as potential indicators of soil quality in a recupera- tion gadient of the Atlantic Forest in Brazil, Land Degrad. Dev., doi:10.1002/Idr.2228, 2013. Moncada, M. P., Gabriels, D., Cornelis, W., and Lobo, D.: Compar- ing aggregate stability tests for soil physical quality indicators, Land Degrad. Dev., doi:10.1002/Idr.2225, 2013. Özdemir, N.: Toprak ve su koruma, OMÜ Zir. Fak. Ders Kitabı, No: 22 Samsun, 2002. References Alagöz, Z. and Erdem, Y.: Effects of different sources of organic matter on soil aggregate formation and stability: a laboratory study on a Lithic Rhodoxeralf from Turkey, Soil Till. Res., 103419–103424, 2009. Jackson, M. C.: Soil chemical analyses, Prentice Hall of India Pri- vate Limited, New Delhi, India, 1967. Kavdır, Y. and Killi, D.: Inﬂuence of olive oil solid waste appli- cations on soil pH, electrical conductivity, soil nitrogen trans- formations, carbon content and aggregate stability, Bioresource Technol., 99, 2326–2332, 2008. Bal, L., ¸Seker, C., and Gümü¸s Ersoy, ˙I.: Kaymak tabakasıolu¸sumuna ﬁziko-kimyasal faktörlerin etkileri, Selçuk Tarım ve Gıda Bilim- leri Dergisi, 25, 96–103, 2011. Keesstra, S. D., Geissen, V., Mosse, K., Piiranen, S., Scudiero, E., Leistra, M., and Van Schaik, L.: Soil as a ﬁlter for groundwater quality, Current Opinion in Environmental Sustainability, 281, 507–516, 2012. Brevik, E. C., Cerdà, A., Mataix-Solera, J., Pereg, L., Quinton, J. N., Six, J., and Van Oost, K.: The interdisciplinary natur of soil, Soil, 1, 117–129, 2015. Solid Earth, 6, 1231–1236, 2015 www.solid-earth.net/6/1231/2015/ Solid Earth, 6, 1231–1236, 2015 ˙I. Gümü¸s and C. ¸Seker: Inﬂuence of humic acid applications Verhulst, N., Govaerts, B., Verachtert, E., Castellanos-Navarrete, A., Mezzalama, M., Wall, P., Chocobar, A., Deckers, J., and Sayre, K.: Conversation Agriculture, Improving Soil Quality for Sustainable Production Systems, in: Advances in Soil Science: Food Security and Soil Quality, CRC Press, Boca Raton, FL, USA, 137–208, 2010. Ozgöz, E., Günal, H., Acır, N., Gökmen, F., Birol, M., and Bu- dak, M.: Soil quality and spatil variability assessment of land use effects in a Typic Haplustoll, Land Degrad. Dev., 24, 277–286, 2013. Paz-Ferreiro, J. and Fu, S.: Biological indices for soil quality evaluation: perspectives and limitations, Land Degrad. Dev., doi:10.1002/Idr.2262, 2013. Yılmaz, E.: Changes of some soil properties by agricultural process- ing waste (soybean pulp) amendment, J. Food Agric. Environ., 8, 1057–1060, 2010. Peters, D. B.: Water availability, in: Methods of Soil Analysis, Part I, edited by: Black, C. A., 279–285, American Society of Agron- omy, Madison, Wl, 1965. Yılmaz, E.: Effects of different sources of organic matter on some soil fertility properties: A laboratory study on a Lithic Rhodoxer- alf from Turkey, Communication in Soil Science and Plant Anal- ysis, 42, 962–970, 2011. Piccolo, A. and Mbagwu, J. S. C.:Effects of different organic waste amendments on soil microaggregates stability and molecular- sizes of humic subtances, Plant Soil, 123, 27–37, 1990. Piccolo, A., Pietramellara, G., and Mbagwu, J. S. C.: Use of hu- mic substances as soil conditioners to increase aggregate stabil- ity, Geoderma, 75, 267–277, 1997. Zhang, K., Zheng, H., Chen, F. L., Ouyang, Z. Y., Wang, Y., Wu, Y. F., Lan, J., Fu, M., and Xiang, X. W.: Changes in soil quality af- ter converting Pinus to Eucalyptus plantations in southern China, Solid Earth, 6, 115–123, doi:10.5194/se-6-115-2015, 2015. Reeve, R. C.: Modulus of rupture, In: Method of soil analysis, Part 1, edited by: Black, C. A., 446–471, American Society of Agron- omy, Madison, WI, 1965. Zhao, X., Wu, P., Gao, X., and Persaud, N.: Soil quality indicators in relation to land use and topography in a small catchment on the loess plateau of China, Land Degrad. Dev., 26, 54–61, 2015. Saygın, D. S., Erpul, G., and Ba¸saran, M.: Comparision of aggregate stability measurement methods for clay-rich soils in Asartepe catchment of Turkey, Land Degrad. Dev., doi:10.1002/Idr.2383, 2015. Solid Earth, 6, 1231–1236, 2015 www.solid-earth.net/6/1231/2015/ www.solid-earth.net/6/1231/2015/
https://openalex.org/W3000039898	https://europepmc.org/articles/pmc6965183?pdf=render	English	null	The impact of adjusting for baseline in pharmacogenomic genome-wide association studies of quantitative change	npj genomic medicine	2,020	cc-by	6,730	1Department of Clinical Pharmacy, University of California, San Francisco, CA 94143, USA. 2Institute for Human Genetics, University of California, San Francisco, CA 94143, USA. 3Division of Research, Kaiser Permanente Northern California, Oakland, CA 94612, USA. 4Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA. 5Department of Medicine, University of California, San Francisco, CA 94143, USA. 6Department of Epidemiology and Biostatistics, University of California, San Francisco, CA 94158, USA. 7These authors contributed equally: Neil Risch, Thomas J. Hoffmann. email: Akinyemi.Oni-Orisan@ucsf.edu www.nature.com/npjgenmed INTRODUCTION Pharmacogenomic studies of continuous (quantitative) pheno- types most commonly identify genetic determinants of the change between pretreatment (baseline) and on-treatment levels from the administration of a therapeutic drug intervention. This approach has improved statistical power in detecting a genetic effect over using dichotomous outcomes (i.e., case-control design), especially when the dichotomous outcome is rare.1 Pharmacogenomic studies of continuous (quantitative) pheno- types most commonly identify genetic determinants of the change between pretreatment (baseline) and on-treatment levels from the administration of a therapeutic drug intervention. This approach has improved statistical power in detecting a genetic effect over using dichotomous outcomes (i.e., case-control design), especially when the dichotomous outcome is rare.1 ARTICLE OPEN The impact of adjusting for baseline in pharmacogenomic genome-wide association studies of quantitative change Akinyemi Oni-Orisan 1,2, Tanushree Haldar2, Dilrini K. Ranatunga3, Marisa W. Medina 4, Catherine Schaefer 3, Ronald M. Krauss4,5, Carlos Iribarren3, Neil Risch2,3,6,7 and Thomas J. Hoffmann 2,6,7 In pharmacogenomic studies of quantitative change, any association between genetic variants and the pretreatment (baseline) measurement can bias the estimate of effect between those variants and drug response. A putative solution is to adjust for baseline. We conducted a series of genome-wide association studies (GWASs) for low-density lipoprotein cholesterol (LDL-C) response to statin therapy in 34,874 participants of the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort as a case study to investigate the impact of baseline adjustment on results generated from pharmacogenomic studies of quantitative change. Across phenotypes of statin-induced LDL-C change, baseline adjustment identiﬁed variants from six loci meeting genome- wide signiﬁcance (SORT/CELSR2/PSRC1, LPA, SLCO1B1, APOE, APOB, and SMARCA4/LDLR). In contrast, baseline-unadjusted analyses yielded variants from three loci meeting the criteria for genome-wide signiﬁcance (LPA, APOE, and SLCO1B1). A genome-wide heterogeneity test of baseline versus statin on-treatment LDL-C levels was performed as the deﬁnitive test for the true effect of genetic variants on statin-induced LDL-C change. These ﬁndings were generally consistent with the models not adjusting for baseline signifying that genome-wide signiﬁcant hits generated only from baseline-adjusted analyses (SORT/CELSR2/PSRC1, APOB, SMARCA4/LDLR) were likely biased. We then comprehensively reviewed published GWASs of drug-induced quantitative change and discovered that more than half (59%) inappropriately adjusted for baseline. Altogether, we demonstrate that (1) baseline adjustment introduces bias in pharmacogenomic studies of quantitative change and (2) this erroneous methodology is highly prevalent. We conclude that it is critical to avoid this common statistical approach in future pharmacogenomic studies of quantitative change. npj Genomic Medicine (2020) 5:1 ; https://doi.org/10.1038/s41525-019-0109-4 npj Genomic Medicine (2020) 5:1 ; https://doi.org/10.1038/s41525-019-0109-4 comprehensive literature search to determine the prevalence of adjusting for baseline in pharmacogenomic studies. Published in partnership with CEGMR, King Abdulaziz University RESULTS Population and genetic characteristics A total of 34,874 statin users from the GERA cohort met the criteria for inclusion. The study population was multiethnic (Supplemen- tary Table 1). The median percent reduction in LDL-C from statin treatment was 35% (interquartile range (IQR), 24−45%). There were 17,708,023, 16,329,859, 8,984,232, and 10,307,784 variants that remained for analyses in White/European, Black/African, East Asian, and Hispanic/Latino participants, respectively. Of these, 13,250,765 variants were shared between two or more race/ ethnicity groups: these were carried forward for the GWASs. There was no evidence of genomic inﬂation7 based on a genomic inﬂation factor (λ) value of 0.997 in the combined population (for baseline-unadjusted percent change in LDL-C from statin therapy). Likewise, the genomic inﬂation factor within each race/ethnicity group was 1.001, 1.001, 1.005, and 0.983 for White/European, Black/African, East Asian, and Hispanic/Latino participants, respectively. A critical objective of this type of analysis is to identify genetic effects on drug-induced changes independent of the baseline value, especially when it is known that the baseline value itself has a strong genetic component. Any association between genetic variants and the baseline measurement can produce a false- positive association between that variant and the drug response phenotype. A putative solution is to add the baseline value as a covariate to the linear regression model. However, it has been documented in multiple studies of statistics and epidemiology that this analytical approach (adjusting for baseline) may introduce the bias that it seeks to prevent.2–6 Here, we present the results for a series of statin-induced low- density lipoprotein cholesterol (LDL-C) response genome-wide association studies (GWASs) as a case example illustrating that the bias introduced when adjusting for baseline also applies to pharmacogenomic analyses. We also report the results of a Published in partnership with CEGMR, King Abdulaziz University A. Oni-Orisan et al. 2 -log(10)P Chromosome b LPA SLCO1B1 APOE - P ) 0 1 (g ol -log(10)P -log(10)P - P ) 0 1 (g ol Chromosome Chromosome Chromosome Chromosome a c b d LPA SLCO1B1 APOE LPA SLCO1B1 APOE LPA SLCO1B1 APOE SORT/ CELSR2/ PSRC1 APOB SMARCA4/LDLR LPA SLCO1B1 APOE SORT/ CELSR2/ PSRC1 APOB SMARCA4/LDLR Fig. 1 Manhattan plots for a series of genome-wide association studies (GWAS) of statin-induced low-density lipoprotein cholesterol (LDL-C) response, highlighting key chromosomal regions. RESULTS A GWAS for baseline-adjusted difference of natural log-transformed LDL-C levels yielded six signiﬁcant loci (a), whereas a GWAS of baseline-unadjusted statin-induced percent LDL-C lowering yielded three loci that met genome-wide signiﬁcance (b). Consistent with the impact of baseline-adjustment on results, baseline-unadjusted difference of natural log- transformed LDL-C levels (c) and baseline-adjusted statin-induced percent LDL-C lowering (d) yielded three and six genome-wide signiﬁcant loci, respectively. All tests were two-sided. - P ) 0 1 (g ol - P ) 0 1 (g ol Chromosome Chromosome a c LPA SLCO1B1 APOE LPA SLCO1B1 APOE SORT/ CELSR2/ PSRC1 APOB SMARCA4/LDLR APOE -log(10)P Chromosome Chromosome d LPA SLCO1B1 APOE SORT/ CELSR2/ PSRC1 APOB SMARCA4/LDLR d Chromosome Chromosome Fig. 1 Manhattan plots for a series of genome-wide association studies (GWAS) of statin-induced low-density lipoprotein cholesterol (LDL-C) response, highlighting key chromosomal regions. A GWAS for baseline-adjusted difference of natural log-transformed LDL-C levels yielded six signiﬁcant loci (a), whereas a GWAS of baseline-unadjusted statin-induced percent LDL-C lowering yielded three loci that met genome-wide signiﬁcance (b). Consistent with the impact of baseline-adjustment on results, baseline-unadjusted difference of natural log- transformed LDL-C levels (c) and baseline-adjusted statin-induced percent LDL-C lowering (d) yielded three and six genome-wide signiﬁcant loci, respectively. All tests were two-sided. The impact of phenotype and baseline-adjustment in GWASs of statin LDL-C response The impact of phenotype and baseline-adjustment in GWASs of statin LDL-C response consistent with the baseline-unadjusted models: variants from LPA and APOE met genome-wide signiﬁcance and variants from SLCO1B1 met suggestive signiﬁcance (Fig. 2, Table 2). The GWAS of statin LDL-C response using the Postmus et al. deﬁnition (the difference of natural log-transformed baseline and on-treatment LDL-C levels adjusted for the natural log- transformed baseline level) revealed variants from six loci that met genome-wide signiﬁcance (SORT/CELSR2/PSRC1, LPA, SLCO1B1, APOE, APOB, and SMARCA4/LDLR; Fig. 1a, Table 1). In contrast, the GWAS of statin LDL-C response using our previous deﬁnition (statin-induced LDL-C percent lowering without adjust- ment for baseline LDL-C) yielded only three loci that met genome- wide signiﬁcance: LPA, SLCO1B1, and APOE (Fig. 1b, Table 1). It was unknown if the phenotype itself, baseline-adjustment, or both were impacting this discrepancy in results (i.e., the discrepant count of signiﬁcant genome-wide loci). When we altered the Postmus et al. npj Genomic Medicine (2020) 1 RESULTS deﬁnition so that baseline was not included as a covariate (the difference of natural log-transformed baseline and on-treatment LDL-C levels without adjustment for baseline), we identiﬁed only the three same loci as statin-induced percent LDL-C lowering without baseline adjustment (LPA, SLCO1B1, and APOE; Fig. 1c). This suggested that baseline-adjustment was impacting the number of signiﬁcant genome-wide loci. The GWAS of statin- induced percent LDL-C lowering with adjustment for baseline LDL-C identiﬁed the same six loci as the Postmus et al. deﬁnition (SORT/CELSR2/PSRC1, LPA, SLCO1B1, APOE, APOB, SMARCA4/LDLR; Fig. 1d), further conﬁrming the impact of baseline-adjustment on results. Taken together, when baseline LDL-C was included as a covariate in the GWAS regression model, six loci met genome- wide signiﬁcance; when baseline was not included as a covariate, three loci met the threshold of genome-wide signiﬁcance. Effect size magnitude and direction did not vary signiﬁcantly by race/ ethnicity (Table 1, Supplementary Tables 2–5). Separate GWASs of baseline and on-treatment LDL-C levels each revealed multiple genome-wide associations as expected (Supplementary Figs. 1 and 2). Notably, among the three loci meeting genome-wide signiﬁcance only when baseline was adjusted for (and not in the analyses of baseline-unadjusted phenotypes), all were also signiﬁcantly associated with baseline LDL-C levels at the genome-wide level: SORT/CELSR2/PSRC1, APOB, SMARCA4/LDLR (Table 1). In contrast, among the three loci identiﬁed in the unadjusted analyses (LPA, SLCO1B1, APOE), only APOE was associated with baseline LDL-C levels (Table 1). Baseline adjustment in previous genome-wide pharmacogenomic studies of quantitative change Among GWAS studies in the NHGRI-EBI GWAS Catalog, 59 included ≥1 quantitative drug response phenotype (using base- line and on-treatment measures) where covariates were added to the linear regression model (Supplementary Table 6). These studies investigated drug response to a variety of disease biomarkers including asthma, diabetes, dyslipidemia, hyperten- sion, schizophrenia, depression, and others. Among the 59, 35 (59%) adjusted the drug-induced change phenotype for baseline values. At the time of the literature search, the year of publication for these studies ranged from 2009 to 2018. A majority of the studies (21 of 35; 60%) were published within the last 3 years from the time of literature search (2016 to 2018). Published in partnership with CEGMR, King Abdulaziz University DISCUSSION An important source of bias in studies of quantitative change is the potential impact of the baseline measurement on the change. In this report, we extend the work of previous studies on this topic to the ﬁeld of pharmacogenomics through a series of genome- wide analyses. We demonstrate that the number of signiﬁcant We then conducted an interaction test, a genome-wide heterogeneity test of statin baseline versus on-treatment LDL-C levels, as the deﬁnitive test for the true effect of genetic variants on statin-induced LDL-C change. Findings were generally Published in partnership with CEGMR, King Abdulaziz University A. Oni-Orisan et al. 3 Table 1. All six lead variants from the genome-wide signiﬁcant meta-analysis loci for baseline-adjusted difference of natural log-transformed statin on- and baseline low-density lipoprotein cholesterol levels by statin response variable in combined race/ethnicity groups (N = 34,874). Gene SNP CHR BP Minor allele MAF Statin response variablea Beta (SE)b P value Cochrane’s Q statistic P valuec I^2 heterogeneity index (0–100)c SORT1 rs7528419 1 109817192 G 0.198 Baseline-adjusted −0.019 (0.002) 9.55E-18 0.944 0 Baseline-unadjusted −0.046 (0.010) 3.59E-06d 0.496 0 Interaction 0.009d Baseline only −0.035 (0.002) 1.57E-54 0.352 8 APOB rs1713222 2 21271323 A 0.145 Baseline-adjusted −0.013 (0.002) 4.68E-08 0.255 26 Baseline-unadjusted −0.031 (0.011) 5.05E-03d 0.455 0 Interaction 0.176d Baseline only −0.027 (0.003) 1.09E-26 0.670 0 LPA rs10455872 6 161010118 G 0.068 Baseline-adjusted 0.042 (0.003) 1.01E-33 0.128 51 Baseline-unadjusted 0.179 (0.016) 4.24E-30 0.160 45 Interaction 1.21E-11 Baseline only 0.008 (0.003) 0.019d 0.593 0 SLC01B1 rs58310495 12 21357711 T 0.179 Baseline-adjusted 0.015 (0.002) 4.58E-11 0.225 31 Baseline-unadjusted 0.069 (0.010) 7.48E-12 0.247 28 Interaction 5.83E-04d Baseline only −0.001 (0.002) 0.622d 0.489 0 SMARCA4/ LDLR rs67337506 19 11207982 C 0.250 Baseline-adjusted −0.012 (0.002) 3.08E-08 0.602 0 Baseline-unadjusted −0.033 (0.010) 8.41E-04d 0.609 0 Interaction 0.043d Baseline only −0.017 (0.002) 1.26E-13 0.162 42 APOE rs7412 19 45412079 T 0.070 Baseline-adjusted −0.068 (0.004) 1.08E-78 0.35 5 Baseline-unadjusted −0.202 (0.016) 1.46E-36 0.229 32 Interaction 7.89E-19 Baseline only −0.086 (0.004) 1.67E-118 0.068 63 BP base pair, CHR chromosome, MAF minor allele frequency, SE standard error, SNP single nucleotide polymorphism aBaseline-adjusted difference of natural log-transformed statin on- and baseline low-density lipoprotein cholesterol levels, baseline-unadjusted statin-induced percent low-density lipoprotein cholesterol lowering, interaction of natural log-transformed statin on- versus baseline low-density lipoprotein cholesterol levels, and natural log-transformed baseline low-density lipoprotein cholesterol level bFixed effects calculated with respect to the minor allele. DISCUSSION A negative value indicates more intense statin LDL-C lowering cRefers to the variation on results between race/ethnicity groups dNot reaching genome-wide signiﬁcance (P < 5E-08) Table 1. All six lead variants from the genome-wide signiﬁcant meta-analysis loci for baseline-adjusted difference of natural log-transformed statin on- and baseline low-density lipoprotein cholesterol levels by statin response variable in combined race/ethnicity groups (N = 34,874). BP base pair, CHR chromosome, MAF minor allele frequency, SE standard error, SNP single nucleotide polymorphism aBaseline-adjusted difference of natural log-transformed statin on- and baseline low-density lipoprotein cholesterol levels, baseline-unadjusted statin-induced percent low-density lipoprotein cholesterol lowering, interaction of natural log-transformed statin on- versus baseline low-density lipoprotein cholesterol levels, and natural log-transformed baseline low-density lipoprotein cholesterol level bFixed effects calculated with respect to the minor allele. A negative value indicates more intense statin LDL-C lowering cRefers to the variation on results between race/ethnicity groups dNot reaching genome-wide signiﬁcance (P < 5E-08) It turns out that a likely source of this bias is the “regression toward the mean” phenomenon.2,3 Brieﬂy, baseline values should not necessarily impact the quantitative change. However, any measurement error in the baseline value will produce a false statistical correlation between the baseline and change.6 This is because any baseline measurement error will be negatively correlated with an observed change (e.g., baseline error in the positive direction from the true baseline measurement value will show an observed change in the negative direction: enhanced reduction if the true change causes a reduction in value or attenuated increase if the true change increases the value). The error in the second measurement will not produce changes of a great enough magnitude to balance out changes from errors in the baseline measurement. Accordingly, any association between a covariate in the model and the apparent baseline value will also falsely correlate with the quantitative change, since there is now an artiﬁcial statistical relationship between baseline and change. Thus the “regression toward the mean” in this case of quantitative change is related to the error for the second measurement, which is likely to be less extreme than the original baseline measurement error. This is the case whether the baseline error is at a positive or negative extreme. Importantly, measurement error must be present for this biasing to occur. However, all laboratory values associations can be strongly inﬂuenced by baseline adjustment. Published in partnership with CEGMR, King Abdulaziz University Published in partnership with CEGMR, King Abdulaziz University DISCUSSION Gene SNP CHR BP Minor allele MAF Baseline betaa On-treatment betaa P value I^2 heterogeneity index (0–100)b LPA rs10455872 6 161010118 G 0.068 0.008 (0.004) 0.048 (0.004) 1.21E-11 98 SLC01B1 rs73079476 12 21343833 C 0.152 0.0004 (0.004) 0.016 (0.004) 4.49E-07c 96 APOE rs7412 19 45412079 T 0.070 −0.086 (0.003) −0.128 (0.003) 7.89E-19 99 BP base pair, CHR chromosome, MAF minor allele frequency, SNP single nucleotide polymorphism aFixed effects calculated with respect to the minor allele bRefers to the variation on results between baseline versus statin on-treatment low-density lipoprotein cholesterol levels cSuggestive of genome-wide signiﬁcance (P < 1E-05) To demonstrate the impact of baseline adjustment, we used two regression models (either adjusting for baseline or not) for each of two statin-induced LDL-C change phenotypes. We found that adjusting for baseline led to more genome-wide associations compared to unadjusted models (6 versus 3 hits). Unlike the effect of baseline adjustment in the regression model, the speciﬁc phenotype (statin-induced percent LDL-C lowering vs. the difference of natural-log-transformed LDL-C levels) had no impact on results. We then performed a genome-wide heterogeneity study investigating the interaction of statin treatment on genetic variant LDL-C effects. These results mirrored our baseline- unadjusted ﬁndings conﬁrming the appropriateness of not adjusting for baseline in our regression models. We believe that the extra genome-wide hits (SORT1, APOB, SMARCA4/LDLR) from the baseline-adjusted analyses are false-positives. All three of these loci were found to be strong predictors of baseline LDL-C in our current report as well as in previous studies.10 inherently have some degree of analytical error. Thus, adjusting for baseline has the potential to bias data regardless of study, disease state, or phenotype. , p yp In regards to the current study, we previously estimated overall measurement error in LDL-C values from the GERA cohort to be 34%.10 Since statin therapy generally produces large reductions in LDL-C levels, any error in baseline LDL-C levels in a positive direction (for example) from the true LDL-C value will tend to be associated with falsely larger reductions in LDL-C than in actuality. An error in the statin on-treatment LDL-C level that is also in the positive direction would have an opposite correlation on LDL-C change (compared to the aforementioned baseline LDL-C measurement error in the positive direction). DISCUSSION We also suggest, through the results of a systematic literature search, that confusion exists surrounding baseline adjustment in recent pharmacogenomic studies of quantitative change. An excellent paper that touched on this topic was published in 2008 (online) by McArdle and Whitcomb.8 In this publication, the authors used simulations with blood pressure measurements of the HAPI Heart Study (the mean, distribution, and measurement error of the blood pressures were simulated; observed measure- ments from the HAPI Heart Study were used to ensure the measurements were biologically plausible) and genotype data for loci in an area of chromosome 2q to demonstrate the bias introduced from adjusting for baseline in genetic association studies. However, at the time of that publication, there were less than a dozen pharmacogenomic GWASs published with only a few investigating phenotypes of quantitative change.9 In the current study, we use full-genome and real-world (not simulated) data to report the false-positives that appear as a result of baseline-adjustment in pharmacogenomic studies. Furthermore, now that a decade has passed since the McArdle and Whitcomb article (allowing time for the number of published pharmacoge- nomic GWASs to accumulate), we also report the prevalence of analyses performing this improper baseline-adjustment approach. Published in partnership with CEGMR, King Abdulaziz University npj Genomic Medicine (2020) 1 A. Oni-Orisan et al. Chromosome -log(10)P LPA APOE SLCO1B1 Fig. 2 Manhattan plot for the genome-wide heterogeneity test of baseline versus statin on-treatment low-density lipoprotein cholesterol (LDL-C) levels. Results analyzing the interaction of genetic variants on statin LDL-C response revealed variants from two loci that met genome-wide signiﬁcance and variants from one loci that met suggestive statistical signiﬁcance. A Cochran’s Q test comparing baseline versus on-treatment betas was performed to test the gene−drug interaction of each variant. All tests were two-sided. 4 Chromosome -log(10)P LPA APOE SLCO1B1 APOE Fig. 2 Manhattan plot for the genome-wide heterogeneity test of baseline versus statin on-treatment low-density lipoprotein cholesterol (LDL-C) levels. Results analyzing the interaction of genetic variants on statin LDL-C response revealed variants from two loci that met genome-wide signiﬁcance and variants from one loci that met suggestive statistical signiﬁcance. A Cochran’s Q test comparing baseline versus on-treatment betas was performed to test the gene−drug interaction of each variant. All tests were two-sided. Table 2. Lead variants of the signiﬁcant loci from the genome-wide heterogeneity test of baseline versus statin on-treatment low-density lipoprotein cholesterol levels in combined race/ethnicity groups (N = 34,874). npj Genomic Medicine (2020) 1 Published in partnership with CEGMR, King Abdulaziz University Phenotype We used a pair of lipid measurements from each participant: the most recent pretreatment level (designated as the baseline) and earliest on- treatment LDL-C from the EHRs before and after statin initiation, respectively. On-treatment LDL-C values had to have been measured within a time frame starting 3 weeks after statin initiation and ending 3 weeks after the initial statin ﬁll days’ supply. Lipid measurements within the time frame of a non-statin LDL-C therapy (bile acid sequestrants, ezetimibe, ﬁbrates, prescription niacin, and prescription omega-3 fatty acids) were excluded. All lipid measurement data collected from the EHRs were fasting levels, were obtained in the course of patient care, and were assayed in Kaiser Permanente laboratories, as previously described.10,23 From the pair of LDL-C measurements per participant, we calculated statin LDL-C response using two formulas. The ﬁrst formula was based on the deﬁnition of Postmus et al.15 as the difference between the natural log- transformed baseline LDL-C (X) and on-treatment LDL-C (Y) values adjusted for the natural log-transformed baseline value: ln(Y) −ln(X) adjusted for ln (X). The second formula was a percent change in LDL-C from statin therapy that we used previously.23 Brieﬂy, response was expressed as (Y −X)/X. Both phenotypes were adjusted for the following prespeciﬁed covariates (at the time of statin initiation) within self-reported race/ethnicity groups for each participant: age, sex, body mass index (BMI), statin type, statin dose, hypertension, diabetes, and cigarette smoking (current/former). Each race/ethnicity group (White/European, Black/African, East Asian, and Hispanic/Latino) was analyzed separately. Participants who did not self- identify within these four race/ethnicity groups were excluded. Statin dose adjustment was based on a revised deﬁned daily dose equivalency table as previously described.23 Genetic ancestry eigenvectors previously gener- ated from principal component analyses within the race/ethnicity groups were also included as covariates.21 In particular, the ﬁrst ten eigenvectors for White/Europeans and the ﬁrst six eigenvectors for Black/Africans, East Asians, and Hispanic/Latinos were included as covariates. The additional eigenvectors in White/Europeans were to ensure robustness against any potential minute population structure variation that might be detected within this larger race/ethnicity group. To investigate the impact of adjusting for baseline LDL-C values, both calculations of statin LDL-C response were performed with and without baseline adjustment. This resulted in four phenotypes (regression models) within each race/ethnicity group for interrogation of genome-wide variants: ln(Y) −ln(X) adjusted for ln(X), (Y −X)/X, ln(Y) −ln(X), and (Y −X)/X adjusted for X. DISCUSSION But as stated above, extreme error in baseline levels is not balanced out by the error from the second measurement of LDL-C due to “regression toward the mean”. This imbalance results in a false correlation between baseline LDL-C levels and statin LDL-C response (i.e., a more positive apparent baseline level is correlated with larger statin LDL-C reduction and vice versa). Accordingly, the addition of baseline LDL-C to a regression model of statin LDL-C change will produce false correlations between any covariate that is truly associated with baseline (e.g., genetic variants) and the quantita- tive change. It should be mentioned that regardless of whether the response variable is in units of percent reduction from baseline (%) or absolute difference (mg/dL), this principle holds as our data shows (Supplementary Table 7). Ultimately, we identiﬁed three unconfounded genome-wide signiﬁcant loci of statin-induced LDL-C response: LPA, APOE, SLCO1B1. These loci have been previously identiﬁed among the six past GWASs of statin LDL-C response.11–16 Speciﬁcally, multiple GWASs (including baseline-unadjusted analyses) have shown associations between LPA and APOE with statin response. Only one previous study (Postmus et al.) identiﬁed SLCO1B1 as a genome-wide hit.15 As a meta-analysis (19 observational and RCT studies covering 40,914 European participants), this is the only previous GWAS that investigates genetic predictors of statin LDL-C lowering with a sample size that is comparable to the current report; other studies may not have had the statistical power to identify SLCO1B1 at the genome-wide level. y In addition to the aforementioned large measurement error of LDL-C levels, it is established that untreated (baseline) LDL-C is highly heritable with several genome-wide predictors.10 Both features of LDL-C make GWASs of statin-induced LDL-C response a good case study to illustrate the impact of baseline adjustment in pharmacogenomic studies of quantitative change. Along with LPA, APOE, and SLCO1B1, Postmus et al. also reported SORT1 as a genome-wide predictor of statin LDL-C A. Oni-Orisan et al. 5 response.15 However, a baseline-adjusted phenotype was used to perform this GWAS. Of note, though the inclusion of baseline in the regression increases the chances of false-positives, it does not warrant a complete dismissal of all results generated with this approach (some of which may be true positives). That being stated, there are multiple lines of evidence suggesting that SORT1 may not be a true genetic predictor of statin-induced LDL-C change. First, in a post-hoc interaction study, Postmus et al. Data source and study population We used electronic health records (EHRs) from the Kaiser Permanente Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort as previously described.21–23 Lipid panel measurements, statin dispensing records, and other medical records from individuals who initiated statin therapy between 1996 and early 2018 were extracted for the study. We used the same methods as previously described to deﬁne a new statin user and classify individuals by cardiovascular risk factor status (hypertension, diabetes, cigarette smoking status).23 Brieﬂy, participants had to have ≥2 dispensing records of a statin (to exclude potentially nonadherent participants) as well as ≥1 lipid measurement before and after statin initiation (to determine statin LDL-C response). Approval was obtained from Kaiser Permanente and University of California Institutional Review Boards. Participants gave written informed consent. To assess how commonly baseline adjustment had been conducted in pharmacogenomic studies, we systematically reviewed GWASs of drug-induced quantitative change. We determined that the majority of studies performed ﬂawed baseline-adjusted analyses. Furthermore, we found that most of the studies were published in recent years. Clearly, this type of error is widespread in the pharmacogenomics community and persists today, though it is unknown how many false-positive associations have been dissemi- nated due to this practice. As the use of quantitative biomarkers for drug efﬁcacy and safety continues to grow in pharmacogenomics research, it is important that this erroneous approach is avoided in future studies to prevent any further reporting of false-positive genetic associations in the literature. DISCUSSION demonstrated that among their four genome-wide signiﬁcant hits, while LPA, APOE, and SLCO1B1 showed a signiﬁcant interaction (or suggestive interaction) with statin effect at the genome-wide level, the interaction P value for the SORT1 hit was far from genome-wide signiﬁcant (P = 0.047). Second, our interaction results of the current study were similar to the above interaction results (top hit for SORT1 [rs7528419] interaction P value = 0.009). Third, Postmus et al. showed that while adjusting for measure- ment error at LPA, APOE, and SLCO1B1 only modestly attenuated the apparent genetic effect with statin LDL-C response, the measurement error-adjusted effect for SORT1 was dramatically attenuated (by 65 to 68%). And ﬁnally our own baseline-adjusted results of the current study replicated all 4 hits reported by Postmus et al. providing a positive control that SORT1 is only associated with LDL-C response to statins when baseline LDL-C levels are inappropriately added to the model as a covariate. Further studies are necessary investigating the role of SORT1 variants in statin-induced LDL-C lowering. reduced measurement error, (b) perform baseline-unadjusted analyses on change response variables, and (c) perform interaction analyses (e.g., baseline vs. on-treatment) to conﬁrm the true genetic effects on drug response. Our results support that of not adjusting for baseline in GWASs of quantitative change. A concerted effort from the scientiﬁc community to implement appropriate statistical approaches in pharmacogenomic studies will improve the reporting of true genetic determinants of drug response in the literature and ultimately improve the translation of pharmacogenomic discovery into clinical application. Published in partnership with CEGMR, King Abdulaziz University Literature search We conducted a comprehensive review of previous GWASs from an online resource maintained by the National Human Genome Research Institute (NHGRI-EBI GWAS Catalog; accessed 02/07/19)27 and determined how often drug response studies included quantitative change phenotypes that adjusted for baseline. We accomplished this in three stages. In stage 1, we used four search terms (“pharm”, “drug”, “treat”, “response”) to select for GWASs of drug response phenotypes. Studies that included ≥1 of these search terms in its title or disease/trait description were carried forward for further review. In stage 2, the studies selected from stage 1 were manually reviewed to: (1) conﬁrm that the studies included a phenotype of a pharmacologic intervention in humans, (2) select only studies that included ≥1 phenotype(s) of change in a quantitative measurement (as a response to a pharmacologic intervention), and (3) select only studies that included clinical or demographic covariates in the linear regression model. Phenotypes of vaccine effects, antibody titer response, drug-treated cell lines, and pharmacokinetic metrics were not considered phenotypes of drug response for the purposes of this study. We also excluded drug−gene interaction studies not involving quantitative change phenotypes (i.e., baseline versus on-treatment measurement values), abstracts, and sensitivity analyses of an exploratory nature. Moreover, studies using the ANCOVA approach were inherently excluded, as GWASs of quantitative change are primarily regression analyses. In stage 3, among the ﬁnal set of studies selected from stage 2, we determined the proportion of studies that included baseline as a covariate in the regression model. 13. Deshmukh, H. A. et al. Genome-wide association study of genetic determinants of LDL-c response to atorvastatin therapy: importance of Lp(a). J. Lipid Res. 53, 1000–1011 (2012). 14. Hopewell, J. C. et al. Impact of common genetic variation on response to sim- vastatin therapy among 18 705 participants in the Heart Protection Study. Eur. Heart J. 34, 982–992 (2013). 15. Postmus, I. et al. Pharmacogenetic meta-analysis of genome-wide association studies of LDL cholesterol response to statins. Nat. Commun. 5, 5068 (2014). 16. Thompson, J. F. et al. Comprehensive whole-genome and candidate gene ana- lysis for response to statin therapy in the Treating to New Targets (TNT) cohort. Circ. Cardiovasc. Genet. 2, 173–181 (2009). 17. Van Breukelen, G. J. ANCOVA versus change from baseline: more power in ran- domized studies, more bias in nonrandomized studies [corrected]. J. Clin. Epi- demiol. 59, 920–925 (2006). 18. Blance, A., Tu, Y. K. & Gilthorpe, M. S. Statistical analysis Residuals derived from the above regression models (four response formulas for each of four race/ethnicity groups) were evaluated as a function of genotype using multiple linear regression in an additive model of inheritance. For imputed variants, the phenotypes were evaluated as a function of allelic dosage.25 A ﬁxed-effects meta-analysis of the combined race/ethnicity groups was then conducted to generate the ﬁnal genome- wide association results (for each of the four phenotypes). We also conducted repeat GWASs with untransformed residuals for the (Y −X)/X phenotype to approximate an effect size (beta) of the associations in the units of percent change from baseline. Finally, we performed an interaction analysis to determine the effect of statin on baseline versus on-treatment LDL-C values. For this, we ﬁrst ran regression analyses on the natural log- transformed baseline and on-treatment LDL-C values to generate residuals for ln(X) and ln(Y), respectively. For ln(Y), we adjusted for the following covariates analogous to the statin LDL-C response phenotypes described above: age, sex, BMI, statin type, statin dose, hypertension, diabetes, cigarette smoking (current/former) and genetic ancestry eigenvectors in population-stratiﬁed analyses. A similar regression model was used for ln (X) except statin type and statin dose were not added to the model. The beta of each variant was then generated for baseline and on-treatment LDL-C values by conducting GWAS of the ln(X) and ln(Y) residuals, respectively. A Cochran’s Q test (genome-wide heterogeneity test) comparing baseline versus on-treatment betas was performed to test the gene−drug interaction of each variant. For all genome-wide analyses, P < 5 × 10−8 was considered to meet genome-wide signiﬁcance and P < 1 × 10−5 was considered to be suggestive of an association. Signiﬁcant/ suggestive variants >0.5 Mb from each other were considered to be from independent loci. Statistical analyses were conducted with R (R Foundation for Statistical Computing, version 3.5.1, https://www.R-project.org/) and PLINK (version 1.07, http://pngu.mgh.harvard.edu/purcell/plink/).26 All statistical tests were two-sided. Received: 23 July 2019; Accepted: 5 December 2019; Genotype Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article. Study participants were previously genotyped on one of four Affymetrix Axiom arrays (ranging from 674,518 to 893,631 variants) based on self- reported race/ethnicity.21,22,24 Imputation was performed to the 1000 Genomes Project (Phase I integrated release, March 2012, with August 2012 chromosome update),10 as has been described. Only common variants were included in the analysis. For Black/Africans, East Asians, and Hispanic/Latinos, minor allele frequencies >1% were considered common variants. Due to a larger sample size, a more liberal minor allele frequency threshold (>0.05%) was used to deﬁne common variants in White/ Europeans. These minor allele frequency thresholds ensured a minimum minor allele count of ten within each race/ethnicity group. REFERENCES 1. Bush, W. S. & Moore, J. H. Chapter 11: Genome-wide association studies. PLoS Comput. Biol. 8, e1002822 (2012). 2. Blomqvist, N. On the bias caused by regression toward the mean in studying the relation between change and initial value. J. Clin. Periodontol. 14, 34–37 (1987). 3. Glymour, M. M., Weuve, J., Berkman, L. F., Kawachi, I. & Robins, J. M. When is baseline adjustment useful in analyses of change? An example with education and cognitive change. Am. J. Epidemiol. 162, 267–278 (2005). 4. Oldham, P. D. A note on the analysis of repeated measurements of the same subjects. J. Chronic Dis. 15, 969–977 (1962). 5. Tu, Y. K. & Gilthorpe, M. S. Revisiting the relation between change and initial value: a review and evaluation. Stat. Med. 26, 443–457 (2007). 6. Yanez, N. D. 3rd, Kronmal, R. A. & Shemanski, L. R. The effects of measurement error in response variables and tests of association of explanatory variables in change models. Stat. Med. 17, 2597–2606 (1998). 7. Devlin, B. & Roeder, K. Genomic control for association studies. Biometrics 55, 997–1004 (1999). 8. McArdle, P. F. & Whitcomb, B. W. Improper adjustment for baseline in genetic association studies of change in phenotype. Hum. Hered. 67, 176–182 (2009). 9. Crowley, J. J., Sullivan, P. F. & McLeod, H. L. Pharmacogenomic genome-wide association studies: lessons learned thus far. Pharmacogenomics 10, 161–163 (2009). 10. Hoffmann, T. J. et al. A large electronic-health-record-based genome-wide study of serum lipids. Nat. Genet. 50, 401–413 (2018). 11. Barber, M. J. et al. Genome-wide association of lipid-lowering response to statins in combined study populations. PLoS ONE 5, e9763 (2010). 12. Chasman, D. I. et al. Genetic determinants of statin-induced low-density lipo- protein cholesterol reduction: the Justiﬁcation for the Use of Statins in Preven- tion: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial. Circ. Cardiovasc. Genet. 5, 257–264 (2012). Phenotype Residuals were rank-normalized for the two phenotypes that were not already trans- formed: (Y −X)/X and (Y −X)/X adjusted for X. Approaches beyond using baseline as a regression covariate include matching, stratifying, and excluding patients by baseline value. These methods generate the same bias as the baseline covariate.3 Another approach is to use the baseline-adjusted on- treatment level alone (rather than quantitative change) as the response variable. This method has high statistical power and performs well in randomized controlled trials, but induces large biases with more heterogeneous observational data.17 We conﬁrmed this bias in a GWAS of statin on-treatment LDL-C adjusted for baseline LDL-C (Supplementary Table 8), which also produced false-positive genome-wide hits similar to other baseline-adjusted analyses in the current report. One may also adjust for baseline measurement error variance.2 However, this approach requires an estimate of measurement error variance (e.g., through repeat measures), which many not be feasible to do. Furthermore, the impact of regression to the mean may not be adequately removed through this approach.18 These consequences of baseline-adjusted analyses also apply to the pharmacogenetic candidate gene approach with phenotypes of quantitative change. This is because a common method in candidate gene studies is to compare mean change between groups deﬁned by genotype or haplotype using analysis of covariance (ANCOVA) to adjust for baseline. Past studies in statistics show that the “regression toward the mean” principle described in this current work occurs for ANCOVA.19,20 Evidently, no options are free of limitations. We generally recommend that researchers performing pharmacogenomic studies of quantitative change observe the following best practices: (a) use multiple baseline measures if available to Published in partnership with CEGMR, King Abdulaziz University npj Genomic Medicine (2020) 1 A. Oni-Orisan et al. 6 Reporting summary DATA AVAILABILITY Genotype data are available on approximately 78% of GERA participants from the database of Genotypes and Phenotypes (dbGaP) under accession phs000674. v1.p1. This includes individuals who consented to having their data shared with dbGaP. The complete GERA data are available upon application to the Kaiser Permanente Research Bank Portal (https://researchbank.kaiserpermanente.org/for-researchers/). Summary statistics will be made publicly available from the National Human Genome Research Institute-European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog, (https://www.ebi.ac.uk/gwas/downloads/summary-statistics). npj Genomic Medicine (2020) 1 ACKNOWLEDGEMENTS This work was supported by grants RC2 AG036607, K01 HL143109, and P50 GM115318 from the National Institutes of Health (NIH). The development of the Research Program on Genes, Environment, and Health was supported by grants from the Robert Wood Johnson Foundation, the Wayne and Gladys Valley Foundation, the Ellison Medical Foundation, and Kaiser Permanente Community Beneﬁt Programs. We also thank Maria Glymour for her valuable advice regarding baseline-adjusted analyses. Finally, we thank the Kaiser Permanente Northern California study participants who have generously agreed to participate in the Kaiser Permanente Research Program on Genes, Environment, and Health. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. COMPETING INTERESTS The authors declare no competing interests. 24. Hoffmann, T. J. et al. Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/eth- nicity using imputation and a novel hybrid SNP selection algorithm. Genomics 98, 422–430 (2011). A. Oni-Orisan et al. 7 S., R.M.K., C.I., N.R. and T.J.H. interpreted the results of analysis. A.O.-O., M.W.M., C.S., R. M.K., N.R. and T.J.H. contributed to the drafting and critical review of the manuscript. 22. Kvale, M. N. et al. Genotyping informatics and quality control for 100,000 subjects in the genetic epidemiology research on adult health and aging (GERA) cohort. Genetics 200, 1051–1060 (2015). 23. Oni-Orisan, A. et al. Characterization of statin low-density lipoprotein cholesterol dose−response using electronic health records in a large population-based cohort. Circ. Genom. Precis. Med. 11, e002043 (2018). Reprints and permission information is available at http://www.nature.com/ reprints 27. Buniello, A. et al. The NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays and summary statistics 2019. Nucleic Acids Res. 47, D1005–D1012 (2019). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. ADDITIONAL INFORMATION Supplementary information is available for this paper at https://doi.org/10.1038/ s41525-019-0109-4. 25. Zheng, J., Li, Y., Abecasis, G. R. & Scheet, P. A comparison of approaches to account for uncertainty in analysis of imputed genotypes. Genet. Epidemiol. 35, 102–110 (2011). Correspondence and requests for materials should be addressed to A.O.-O. 26. Purcell, S. et al. PLINK: a tool set for whole-genome association and population- based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007). Reprints and permission information is available at http://www.nature.com/ reprints Published in partnership with CEGMR, King Abdulaziz University Literature search A multilevel modelling solution to math- ematical coupling. Stat. Methods Med. Res. 14, 553–565 (2005). 19. Carroll, R. J., Gallo, P. & Gleser, L. J. Comparison of least squares and errors-in- variables regression, with special reference to randomized analysis of covariance. J. Am. Stat. Assoc. 80, 929–932 (1985). 20. Lord, F. M. A paradox in the interpretation of group comparisons. Psychol. Bull. 68, 304–305 (1967). 21. Banda, Y. et al. Characterizing race/ethnicity and genetic ancestry for 100,000 subjects in the genetic epidemiology research on adult health and aging (GERA) cohort. Genetics 200, 1285–1295 (2015). Published in partnership with CEGMR, King Abdulaziz University npj Genomic Medicine (2020) 1 Published in partnership with CEGMR, King Abdulaziz University AUTHOR CONTRIBUTIONS A.O.-O., M.W.M., C.S., R.M.K., C.I., N.R. and T.J.H. conceived and designed the study. D.K. R., in collaboration with C.I., C.S., N.R. and T.J.H., extracted phenotype data from the EHRs. A.O.-O., N.R. and T.J.H. performed the statistical analysis. A.O.-O., T.H., M.W.M., C. © The Author(s) 2020 npj Genomic Medicine (2020) 1
https://openalex.org/W4365133797	https://dergipark.org.tr/tr/download/article-file/2584957	Turkish	null	Broyler (Etlik Piliç) Üretiminde Maliyet ve Gelir Analizi: Doğu Marmara Bölgesi Örneği	DergiPark (Istanbul University)	2,022	cc-by	3,963	Research Article Araştırma Makalesi doi: 10.5152/JASE.2023.1159190 Research Article Araştırma Makalesi doi: 10.5152/JASE.2023.1159190 doi: 10.5152/JASE.2023.1159190 ÖZ Bu çalışmada, Doğu Marmara bölgesinde broyler üretiminin en yoğun olarak yapıldığı Bolu, Düzce, Kocaeli ve Sakarya illerinde bulunan 122 adet broyler üretim dalının üretim maliyeti ve gelir durumunun analizi amaçlanmıştır. Çalışmada üretim dalları dört kapasite grubuna ayrılarak yürütülmüştür. Broyler işletmelerin kapasite kullanım oranı ortalama olarak %97,61, ölüm oranı yaz devresinde %3,80, kış devresinde %5,95 ve besi süresi 42 gün olarak hesaplanmıştır. Broyler işletmelerinde işgücü ortalaması 3,92 EİB olarak tespit edilmiştir. Broyler işletmelerinde ortalama üretim masraflarının %82,76’sını yem masrafı oluşturmuştur. Birim canlı ağırlık maliyeti 2,20 ₺ / kg olarak hesaplanmıştır. Ortalama brüt marj 10 802,80 ₺, net gelir ise 1 371,38 ₺ bulunmuştur. Kapasite büyüklüğü arttıkça brüt marj ve net gelir değerinin de arttığı belirlenmiştir. Küçük kapa- siteli üretim dallarının gelirlerini artırabilmeleri için üretim kapasitelerini artırmaları önerilmiştir. 1Artvin İl Tarım ve Orman Müdürlüğü, Artvin, Türkiye 2Atatürk Üniversitesi Ziraat Fakültesi Tarım Ekonomisi Bölümü, Erzurum, Türkiye Anahtar Kelimeler: Broyler, maliyet, brüt gelir, net gelir Broyler (Etlik Piliç) Üretiminde Maliyet ve Gelir Analizi: Doğu Marmara Bölgesi Örneği Cost and Income Analysis in Broiler Production: The Example of the East Marmara Region ABSTRACT In this survey, it was aimed to analyze the production cost and income status of 122 broiler pro- duction branches in Bolu, Düzce, Kocaeli, and Sakarya, where broiler production is most intense in the Eastern Marmara region. In the study, production branches were divided into four capac- ity groups. The average capacity utilization rate of broiler farms was 97.61%, mortality rate was 3.80% in the summer period, 5.95% in the winter period, and the fattening period was 42 days. The average labor force in broiler enterprises was determined as 3.92 MBU. Feed cost consti- tuted 82.76% of the average production costs in the broiler business. The unit live weight cost was calculated as 2.20 ₺/kg. Average gross margin was found to be 10,802.80 ₺ and net income was 1371.38 ₺. It has been determined that as the size of the capacity increases, the gross margin and net income value also increase. It has been suggested that small-capacity production branches should increase their production capacity in order to increase their income. Keywords: Broiler, cost, gross income, net income Çalışma Atatürk Üniversitesi Fen Bilimler Enstitüsü doktora tez (2012) çalışmasından türetilmiştir. Geliş Tarihi/Received: 08.08.2022 Kabul Tarihi/Accepted: 05.11.2022 Yayın Tarihi/Publication Date: 24.03.2023 Sorumlu Yazar/Corresponding Author: Vedat DAĞDEMİR E-mail: dagdemir@atauni.edu.tr Cite this article as: Yeni A, Dağdemir V. (2023). Cost and income analysis in broiler production: The example of the east Marmara region. Journal of Animal Science and Economics, 2(1), 7-12. Content of this journal is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License Çalışma Atatürk Üniversitesi Fen Bilimler Enstitüsü doktora tez (2012) çalışmasından türetilmiştir. Çalışma Atatürk Üniversitesi Fen Bilimler Enstitüsü doktora tez (2012) çalışmasından türetilmiştir. Geliş Tarihi/Received: 08.08.2022 Kabul Tarihi/Accepted: 05.11.2022 Yayın Tarihi/Publication Date: 24.03.2023 1Artvin İl Tarım ve Orman Müdürlüğü, Artvin, Türkiye 2Atatürk Üniversitesi Ziraat Fakültesi Tarım Ekonomisi Bölümü, Erzurum, Türkiye Şekil 1. Şekil 1. Türkiye’de Düzey 1 Bölgeleri Haritası (Taşkan, 2006). Bunun en önemli nedeni ise toplam maliyetin %70,00’ini oluş- turan hayvan yemi, özellikle de soya ve mısırın önemli ölçüde yurt dışından tedarik edilmesidir. İç pazar fiyatlarının dış pazar fiyatlarından daha pahalı seyretmesi maliyetlere yansımakta ve diğer ülkelere kıyasla üretici fiyatlarının yüksek olmasına neden olmaktadır. gerçekleştirildiği Doğu Marmara Bölgesinde (Kocaeli, Sakarya, Bolu, Düzce ve Yalova) faaliyet gösteren işletmelerde yapılan anketler yoluyla sağlanan verilerden elde edilmiştir. Söz konusu işletmelere ait kayıtlar Bolu, Düzce, Kocaeli, Adapazarı İl Gıda, Tarım ve Hayvancılık müdürlüklerinden temin edilmiştir. Çalışmanın ikincil verileri literatüre dayalı veriler olup Tarım ve Köyişleri Bakanlığı, Türkiye İstatistik Kurumu (TÜİK), Gıda ve Tarım Örgütü (FAO), Beyaz Et Sanayicileri Birliği (Besd-Bir)’nden sağ- lanan konu ile ilgili yapılmış çalışmalar, yerli ve yabancı yayınlar, konu üzerinde daha önce yapılmış olan araştırma sonuçları ve ilgili web sayfaları kullanılarak elde edilmiştir. Sektörün önemli diğer sorunlarından biri de damızlık materya- lin ithalat yoluyla temin edilmesidir. Üretim faktörlerinin opti- mum düzeyde kullanılması broyler işletmelerinin hedefi olmalıdır. Bundan dolayı broyler üretim maliyetinin hesaplanması ön plana çıkmaktadır. Üretim maliyet analizinin işletmelerin ekonomik etkinliklerinin ölçülmesine yardımcı olması beklenmektedir Bunun yanında üreticilerin teknolojik yenilikleri rasyonel olarak uygulayabilmesinde üretim faaliyetlerinden elde edilen gelir, üre- tim maliyeti ve maliyeti oluşturan masrafların toplam maliyet içerisindeki payının bilinmesine ihtiyaç vardır. Nitekim gelir ve maliyet analizleri ile genel olarak üreticilerin uyguladıkları yetiş- tirme teknikleri, üretimde kullanılan fiziki girdilerin miktarları ve değerleri belirlenmektedir (Özkan & Kuzgun, 1997). Maliyet ana- lizleri işletmede kullanılan üretim faktörlerinin rasyonel kullanı- lıp kullanılmadığını ortaya koymakta, işletmelerin etkinliklerinin değerlendirmesine temel oluşturmakta ve böylece üretim faali- yetleriyle ilgili sağlıklı veri tabanları oluşturulabilmektedir. Tarım- sal üretim maliyeti, üreticilerin yetiştirecekleri ürün seçiminde dikkate aldıkları kriterlerden olup tarım politikasını oluşturucu- ları ve araştırıcılar yönünden de oldukça önemlidir (Özkan ve ark., 2002). 1 Türkiye’de İstatistiki Bölge Birimleri Sınıflandırması 2002 yılında Bakanlar Kurulu Kararı ile yürürlüğe girmiş ve bölge birimlerinin gruplandırılması sonucu 12 adet Düzey 1 (Nuts 1) bölgesi oluşturulmuştur. Alan Çalışmasından Elde Edilen Verilerin Toplanmasında İzlenen Yöntem Türkiye’de broyler yetiştiriciliği yapılan işletme sayısı 9028 adettir (Anonim, 2008). Düzey II (26 alt bölgeden oluşmakta) istatistikî bölge sınıflandırmasına göre işletmelerin gruplandırması yapıla- rak yüzde dağılımları belirlenmiştir. Türkiye’deki toplam broyler işletme varlığının %53,65’inin (4844 adet) Doğu Marmara Böl- gesinde bulunduğu tespit edilmiştir. Bu bölgenin broyler sektö- rünü en iyi şekilde temsil edeceği düşünüldüğünden çalışmada kullanılacak birincil veriler işletmelerden anketler yardımıyla elde edilmiştir. Düzey II bölge sınıflandırmasına göre işletmelerin illere göre yüzde dağılımı Tablo 1’de verilmiştir. Tablo 1. Doğu Marmara Bölgesinde Bulunan Broyler İşletmelerinin İllere Göre Dağılımı Doğu Marmaam Bölgesi İşletme Sayısı Yüzde (%) Dağılım TR41 Bursa 163 3,37 Eskişehir 223 4,60 Bilecik 33 0,68 TR42 Kocaeli 448 9,25 Sakarya 997 20,58 Bolu 2520 52,02 Düzce 458 9,46 Yalova 2 0,04 Toplam 4844 100,00 Türkiye ekonomisi için büyük öneme sahip broyler sektörünün rantabl çalışıp çalışmadığının, kıt ve sınırlı kaynakların etkin ve rasyonel kullanılıp kullanılmadığının ortaya konması önemlidir. Bu çalışmanın temel amacı Türkiye broyler üretiminin en yoğun ola- rak yapıldığı ve Türkiye’yi temsil eden Doğu Marmara Bölgesinde faaliyet gösteren broyler üretim dallarında yıllık faaliyet sonuçla- rını ortaya koyarak üretim maliyetlerinin hesaplanmasıdır. Giriş Türkiye ekonomisinde önemli bir yere sahip olan tarım sektörünün lokomotif üretim dallarından birisi broyler (etlik piliç) sektörüdür. 1970’li yıllarda aile işletmeleri şeklinde üretime başlanılan broyler sek- törü yıllık cirosu yaklaşık 4,5 milyar dolar olan ve aileleri ile birlikte 2 milyon kişinin geçimini temin ettiği endüstriyel bir faaliyet alanı haline gelmiştir (Anonim, 2011). Türkiye’de kanatlı et üretiminin %97,00’si, dünyada ise %87,00’si tavuk etinden karşılandığı için kanatlı sektörü ve tavukçuluk sektörü kavramları iç içe geçmiştir. FAO 2009 yılı verilerine göre Türkiye dünya tavuk eti üretiminde 12. sırada- dır. Sektörün son 20–25 yıllık gelişimi temel alındığında üretimin ve ihracatın önemli oranda artacağı düşünülmektedir. Cite this article as: Yeni A, Dağdemir V. (2023). Cost and income analysis in broiler production: The example of the east Marmara region. Journal of Animal Science and Economics, 2(1), 7-12. Broyler sektöründeki önemli gelişmeler yanında Türk kanatlı sektörünün önemli sorunları da bulun- maktadır. Beyaz et üretim maliyetinin diğer ülkelere kıyasla yüksek olması sektörün en önemli sorun- larından biridir. Bu durum özellikle dış satım sektörün rekabet gücünü zayıflatmaktadır. FAO 2009 yılı verilerine göre Türkiye’deki tavuk eti üretici fiyatları, ABD ve Brezilya’ya kıyasla yaklaşık 2 kat fazladır. Content of this journal is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License NonCommercial 4.0 International License 8 Şekil 1. Türkiye’de Düzey 1 Bölgeleri Haritası (Taşkan, 2006). Materyal ve Yöntem Türkiye’de İstatistikî Bölge Birimleri Sınıflandırmasına1 göre oluş- turulan Düzey 1 bölge sınıflandırmasında 12 bölge bulunmakta- dır (Şekil 1). Çalışma materyalini oluşturan birincil veriler, Düzey 1 bölgesinde bulunan ve broyler üretiminin en yoğun olarak 9 Tablo 2. Kümes Kapasitelerine Göre Üretim Dallarının Gruplandırılması Ve Örnek Sayısı Tabakalar Frekans % Örnek Büyüklüğü %20 yedek Anket Sayısı I 711 19,52 20 4 24 II 1268 34,82 36 7 43 III 1410 38,71 39 8 47 IV 253 6,95 7 1 8 Toplam 3642 100,00 102 20 122 Tablo 3. Anket Sayısının İllere Göre Dağılımı (Adet) Tabakalar Bolu Düzce Kocaeli Sakarya Anket Sayısı I 18 5 0 1 24 II 21 7 5 10 43 III 13 8 9 17 47 IV 2 1 1 4 8 Toplam 54 21 15 32 122 Örnek büyüklüğü, verilerin derlendiği tarihte faal durumda olan 4068 adet işletme dikkate alınarak tespit edilmiştir. Ancak 50000 adet üzeri kapasiteye sahip işletmeler ortalamayı temsil etmediği için ihmal edilmiş ve toplam işletme sayısının %89,52 sini temsil eden 3642 adet işletme dikkate alınmıştır. Bölgede toplam kapa- site 52 666 215 adet olup çalışmada dikkate alınan kapasite mik- tarı toplam kapasitenin %94,33’ünü oluşturan 49 881 915 adettir. Araştırma yöresi olarak seçilen ve Türkiye broyler (etlik piliç) işletme varlığının %53,65’ini oluşturan Doğu Marmara Bölgesini oluşturan sekiz il arasından zaman ve maddi kısıtlamalar gözö- nüne alınarak broyler üretiminin en yoğun olarak gerçekleştirildiği dört ilde (Bolu, Düzce, Sakarya, Kocaeli) bulunan broyler üretim dallarına yer veren işletmeler dikkate alınmıştır. Tablo 3. Anket Sayısının İllere Göre Dağılımı (Adet) Tabakalar Bolu Düzce Kocaeli Sakarya Anket Sayısı I 18 5 0 1 24 II 21 7 5 10 43 III 13 8 9 17 47 IV 2 1 1 4 8 Toplam 54 21 15 32 122 Araştırmada popülasyondaki farklı kapasitelere sahip işletmeleri temsil edebilmesi için tabakalı örnekleme metodu kullanılmıştır. Örneğe girecek işletme sayısı belirlenirken oransal tabakalı örnek- leme yöntemi dikkate alınmış olup hesaplamada Eşitlik 1 kullanıl- mıştır (Yamane, 1967). n N Nh Sh N D Nh Sh * * * 2 2 2 2 (1) n N Nh Sh N D Nh Sh * * * * 2 2 2 2 (1) Eşitlikte; Eşitlikte; n = Örnek hacmi N = Ana kitledeki birim sayısını Nh = h'nci tabadaki birim sayısını Sh² = h'nci tabakadaki varyansı D2 = d2/z2 ((607,1)2/(1,96)² = 95942) Araştırma yöresinde bulunan üretim dalları dört kapasite grubuna ayrılarak analize tabi tutulmuşlardır. 2 EİB: Erkek İş Birimidir. 15–49 yaş arası erkek =1, 15–49 yaş arası kadın =0,75, >50 yaş erkek =0,75, >50 yaş kadın=0,50 ve 7–14 yaş arası çocuk =0,50 erkek iş birimi sayılmaktadır (Dağ- demir, 2004). Materyal ve Yöntem Sosyo–ekonomik özellikler değerlendirilirken yapılan hesaplama ve işlemlerin standartlaştırı labilmesi açısından aile işgücü potansiyeli “Erkek İşgücü Birimine (EİB)2 dönüştürülerek hesaplamalar yapılmıştır. D2 = d2/z2 ((607,1)2/(1,96)² = 95942) d = Ana kitle ortalamasından izin verilen hata miktarını (121420,05 = 607,1) Entansif bir üretim dalı olan broyler (etlik piliç) üretimi, doğal koşul- lara pek bağımlı değildir. Gün içerisinde sürekli olarak ve istenilen saatlerde hayvanlarla ilgilenmek olasıdır. Ayrıca broyler yetiştirici- liğinde işleri aksatmak ve hatta gün içerisinde yapılması gereken bir işi rutin saatinde yapmamak bile üretimde kayıplara yol aça- cağından aile işgücü kullanımı yıllık 365 gün olarak hesaplanmış ve sabit masraflar olarak değerlendirilmiştir. Bu yüzden araştırma bölgesinde kullanılan erkek, kadın ve çocuk işgücü birimleri 365 ile çarpılarak “Erkek İş Günü (EİG)” rakamları elde edilmiştir. z = İzin verilen güvenlik sınırının t dağılım tablosundaki değerini ifade etmektedir. n 3642 36486880446 13264164 95942 36486880446 102 * n 3642 36486880446 13264164 95942 36486880446 102 * * n 3642 36486880446 13264164 95942 36486880446 102 * * Tahmini örnek büyüklüğü %5 hata payı ve %95 güven aralığı ile çalışıldığında 102 olarak hesaplanmıştır. Tabakalara göre örnek sayısının dağılımı ise n1=(Nh/N)*n oranlı dağıtım formülü kullanı- larak yapılmıştır (Çiçek & Erkan, 1996). Brüt Üretim Değeri (BÜD); broyler üretim dalında bir yıl içerisinde yapılan üretim faaliyeti sonucu elde elden ana ürün ve yan (tali) ürün değerlerinin toplamından oluşmaktadır. Brüt Üretim Değeri- nin hesaplanmasında; canlı piliç satışından elde edilen gelir, çuval satışından elde edilen gelir ve gübre satışından elde edilen gelir dikkate alınmış, ancak araştırma yöresinde tavuk gübresinin eko- nomik değeri bulunmadığı için çalışmada dikkate alınmamıştır. Araştırmada dikkate alınan broyler üretim dalları, kapasiteleri dik- kate alınarak dört tabakaya ayrılarak incelenmiştir. Bunlar; Tabaka Adet I → 1000–5000 II → 5001–10000 III → 10001–25000 IV → 25001–50000 Değişken masraflar olarak; yem giderleri, geçici işçi ücretleri, veteriner, ilaç ve aşı masrafları, akaryakıt giderleri, elektrik ve su giderleri, ısıtma, altlık, temizlik/dezenfektan, alet-makine tamir-bakım masrafları, muhasebe ücreti dikkate alınmıştır. Sabit masraf unsurları ise işletmeci ve ailesi işgücü ücret karşılığı, daimi işçi ücretleri, amortismanlar (bina ve alet-makine amortisman- ları), bina tamir-bakım masrafları, bina ve makine sigorta masraf- ları, borç faizleri, vergiler ve harç giderleridir. Anketlerde yanlışlık veya eksiklik olabileceği göz önünde bulun- durularak hesaplanan örnek büyüklüğünün %20’si kadar yedek anket doldurulması ile 122 anket yapılması uygun bulunmuştur (Tablo 2). Anket sayısının illere göre dağılımı Tablo 3’de verilmiştir. Broyler Üretim Dallarında Devreler İtibariyle Ölüm Oranları Üretim masrafları değişken ve sabit masrafların toplamından oluşmaktadır. Etlik piliç birim canlı ağırlık maliyeti, toplam üretim masraflarından yan ürün geliri çıkarılmış olup toplam canlı ağır- lığa bölünmesiyle elde edilmiştir. Genel idare giderleri masraflar toplamının %3,00’ı alınmış (Açıl, 1974) ve masraflar toplamına uygulanmıştır (Dağdemir, 1998). T.C. Ziraat Bankasının hayvancılık kredilerine uygulamış olduğu %11,25 faiz oranı (Devlet tarafından uygulanan %25,00 sübvansiyon desteği faiz oranından düşülmüş- tür) (Anonim, 2012) dikkate alınmıştır. Söz konusu faiz oranı beş üretim devresinde devre başına düşen miktarı dikkate alınarak hesaplama yapılmıştır. Broyler üretim kümeslerinde hayvan kayıpları yaz ve kış dönemine rastlayan devreler itibariyle farklılık göstermektedir. Yetiştiriciler besi süresinin başladığı ilk haftalarda kayıpların %1–3 dolayında olduğunu, fakat olağanüstü durumlarda bu oranın arttığını ifade etmişlerdir. Doğu Marmara Bölgesinde ölüm oranı yaz devresinde %3,80 iken bu oran kış devresinde %5,95’e çıkmaktadır (Tablo 4). Kış devresinde yaz devresine göre hayvan ölüm oranlarının art- tığı görülmektedir Kış devrelerinde ölüm oranlarının artmasında, kümese konulan hayvan sayısının artmasının ve hava sıcaklığının azalmasının etkili olduğu düşünülmektedir. Broyler Üretim Dallarında Üretim Masrafları ve Canlı Ağırlık Maliyeti İşletmelerin ekonomik analizlerinin yapılmasında brüt marj yön- temi kullanılmıştır. BÜD’den değişken masrafların çıkarılmasıyla brüt marj, BÜD’den üretim masraflarının çıkarılmasıyla net gelir elde edilmiştir (Karagölge, 1996). Doğu Marmara Bölgesinde üretim masrafları ve yüzde dağılımları Tablo’5 te gösterilmiştir. Broyler üretimi sözleşmeli olduğundan işletmeler civciv için para ödememekte, civciv masrafları söz- leşme yapılan firma tarafından karşılanmaktadır. Kümes kapasi- tesi arttıkça üretim masrafları toplamının arttığı belirlenmiştir. Ortalama üretim masrafı içerisinde en büyük payı %82,76 ile yem masrafı oluşturmuştur. Yem masrafını sırasıyla işçilik masrafları, ısıtma masrafları ve veteriner-aşı-ilaç masrafları takip etmektedir (Tablo 5). Uygulanan Metot Broyler üretim dallarının üretim maliyeti analizinin yapılmasında aşağıda kısaca özetlenen yöntemler kullanılmıştır. 10 Tablo 4. Broyler Üretim Kümeslerinde Devreler İtibariyle Ölüm Oranları (%) Gruplar Bölge Ortalaması Yaz Devresi Kış Devresi I 3,95 6,09 II 3,51 5,92 III 3,69 5,75 IV 4,06 6,06 Ortalama 3,80 5,95 Yem masrafı hesaplanırken, firmanın verdiği kg fiyat dikkate alınmıştır. Veteriner, ilaç ve aşı, akaryakıt, elektrik, ısıtma gider- lerinin tespitinde üretici beyanı esas alınmıştır. Borç faizleri üreticilerin beyan ettikleri faiz oranı ile kullandıkları kredi mik- tarının çarpılmasıyla bulunmuştur. Aile işgücü ücret karşılığı hesaplanırken yörede geçerli olan işçi ücretler esas alınmıştır. Broyler yetiştiriciliğinde civcivler işletmede bir yıl tutulmadığı, 42–45 günde kesime gönderildiği için hayvan amortismanı hesaplanmamıştır. Yem masrafı hesaplanırken, firmanın verdiği kg fiyat dikkate alınmıştır. Veteriner, ilaç ve aşı, akaryakıt, elektrik, ısıtma gider- lerinin tespitinde üretici beyanı esas alınmıştır. Borç faizleri üreticilerin beyan ettikleri faiz oranı ile kullandıkları kredi mik- tarının çarpılmasıyla bulunmuştur. Aile işgücü ücret karşılığı hesaplanırken yörede geçerli olan işçi ücretler esas alınmıştır. Broyler yetiştiriciliğinde civcivler işletmede bir yıl tutulmadığı, 42–45 günde kesime gönderildiği için hayvan amortismanı hesaplanmamıştır. Amortismanlar, Doğru Hat Metodu ile hesaplanmış ve eşitlik 2’de verilmiştir (Aras, 1988; Dağdemir, 1998; Gülten, 1994). (2) olduğu 42 gündür. Araştırmaya konu olan broyler üretim dalla- rında yılda 5 devre halinde üretim faaliyeti gerçekleştirilmektedir. Demirbaşın Değeri Hurda Değeri Amortisman Ekonomik Ömür − = (2) Journal of Animal Science and Economics l 2023 2(1): 7-12 l doi: 10.5152/JASE.2023.1159190 Broyler Üretim Dallarında İşgücü Varlığı, Kapasite Kullanım Oranları ve Besi Süreleri Doğu Marmara Bölgesinde bulunan üretim dallarında dört kapa- site grubuna ait erkek işgücü sırasıyla, 3,86, 4,01, 4,27 ve 3,52 EİB olup işgücü ortalaması 3,92 EİB olarak hesaplanmıştır. Doğu Marmara Bölgesinde broyler üretim dallarına ait birim canlı ağırlık maliyetleri Tablo 5’te gösterilmiştir. Bölgenin birim canlı ağırlık maliyetleri dört ilin ortalamalarından oluşmaktadır. Birim canlı ağırlık maliyetinin bölge ortalaması 2,20 ₺/kg’dır. Üretim dallarının kapasiteleri arttıkça birim canlı ağırlık maliyetlerinin azaldığı görülmektedir (Tablo 5). Broyler üretim dalları daha büyük kapasitelerle çalıştıklarında birim maliyetlerinin azaldığı, daha karlı çalıştıkları, aynı miktarda ürünü daha az masrafla üreterek daha etkin çalıştıkları tespit edilmiştir. Broyler üretim dallarında grupların kapasite kullanım oranları sırasıyla %98,33, %98,04, %97,48 ve %97,25 olup bölge ortalaması %97,61 bulunmuştur. Bulunan oranlar bölgede broyler üretim dal- larının tam kapasiteye yakın çalıştıklarını göstermektedir. Genotipe bağlı olarak değişmekle beraber, etlik piliçlerin 30–42 günde gelişimlerini tamamlayarak kesim ağırlığına ulaştıkları bil- dirilmektedir (Sarıca, 1996; Şenköylü, 1996). Nitekim bazı çalış- malarda broyler yetiştiriciliğinde ortalama besi süresi yaklaşık 41 gün olarak ortaya konulmuştur (Dağdemir ve ark., 2007). Günü- müzde tavuk etini işleyebilen veya entegre tesislerle üretim yapa- bilen işletmelerin 45. günde kesim yaparak daha fazla gelir elde edebilecekleri belirtilmektedir (Konak ve ark., 1999). Ancak yetiş- tiriciler, besi süresinin uzamasının yem maliyetlerinin artmasına neden olduğunu ifade etmişlerdir. Broyler Üretim Dallarında Brüt Marj ve Net Gelir Broyler Üretim Dallarında Brüt Marj ve Net Gelir Doğu Marmara Bölgesinde broyler üretim dallarında brüt ve net gelir hesabı yapılmış ve Tablo 6’da gösterilmiştir. Broyler üretim dallarında üretim kümeslerinin kapasitesi arttıkça brüt üretim değerinin arttığı görülmektedir. Bu da büyük kapasiteli üretim dallarının daha karlı çalıştığını göstermektedir (Tablo 6). Araştırmaya dahil olan Doğu Marmara bölgesindeki illerde bulu- nan broyler yetiştiricileri, hayvanların kesim yaşına karar vermede, bağlı oldukları sözleşmeci firmanın söz sahibi olduğunu, firmanın da gerek piyasadaki fiyatlara gerekse alıcının talep ettiği piliç ağır- lığına, cinsiyete ve teslim zamanını dikkate alarak kesim zamanını belirlediğini ifade etmişlerdir. Broyler üretim dallarında ortalama besi süresi, hayvanların beslenme durumu, iklimsel koşullar, depo- lama olanaklarının yetersizliği gibi nedenlerle firmanın belirlemiş Doğu Marmara Bölgesinde dört kapasite grubundaki üretim kollarında brüt marj değerleri pozitif olarak hesaplanmaktadır. Bunun nedeni brüt marjın hesaplanmasında sabit masrafların dikkate alınmaması ve daha kesin sonuçlara ulaşılmasıdır. Kapa- site büyüklüğü arttıkça brüt marj değerinin de arttığı görül- mektedir (Tablo 6). Bu durum broyler üretim kümeslerinde kapasite büyüklüğü arttıkça, bir başka ifadeyle ölçek büyüklüğü 11 Tablo 5. Doğu Marmara Bölgesinde Üretim Masrafları Ve Yüzde Dağılımı Gider Unsurları I. Grup II. Grup III. Grup IV. Grup Ortalama ₺ % ₺ % ₺ % ₺ % ₺ % 1.Civciv Değeri – – – – – – – – – – 2.Yem Masrafları 18602,42 70,80 37860,00 80,32 83791,77 85,06 107402,5 84,35 61914,17 82,76 3. Veteriner, İlaç ve Aşı Masrafı 671,54 2,56 779,89 1,65 1070,06 1,09 1911,25 1,50 1108,19 1,48 4. İşçilik Masrafları 2989,23 11,38 2945,95 6,25 3742,06 3,80 5278,68 4,15 3738,98 5,00 -Aile İşgücü Karşılığı 2989,23 11,38 2947,95 6,25 2733,41 2,77 2098,08 1,65 2692,17 3,60 -Daimi İşçi Ücretleri 0 0,00 0 0,00 1008,65 1,02 3180,6 2,50 1047,31 1,40 5.Isıtma Masrafları 618,37 2,35 1083,53 2,30 1608,26 1,63 2406,25 1,89 1429,10 1,91 6. Aydınlatma Masrafları 307 1,17 411,63 0,87 741,82 0,75 864,38 0,68 581,21 0,78 7. Su Masrafları 0 0,00 9,38 0,02 0 0,00 0 0,00 2,35 0,00 8.Akaryakıt masrafları 99,63 0,38 118,31 0,25 313,13 0,32 287,5 0,23 204,64 0,27 9. Altlık Masrafları 313,59 1,19 380,05 0,81 493,38 0,50 620,63 0,49 451,91 0,60 10.Temizlik Masrafı 139,3 0,53 213,99 0,45 323,66 0,33 618,75 0,49 323,93 0,43 11.Tamir-Bakım Masrafı 432,4 1,65 150,11 0,32 198,23 0,20 288,75 0,23 267,37 0,36 -Alet-Makine Masrafı 19,81 0,08 19,9 0,04 27,61 0,03 181,25 0,14 62,14 0,08 -Bina Masrafı 412,59 1,57 130,21 0,28 170,62 0,17 107,5 0,08 205,23 0,27 12. Broyler Üretim Dallarında Brüt Marj ve Net Gelir Vergi-Sigorta Masrafı 0 0,00 0 0,00 0 0,00 0 0,00 0,00 0,00 13.Amortismanlar 711,42 2,71 751,49 1,59 1157,46 1,18 1155,5 0,91 943,97 1,26 -Alet-Makine Amortismanı 385,31 1,47 452,88 0,96 668,32 0,68 635,09 0,50 535,40 0,72 -Bina Amortismanı 326,11 1,24 298,51 0,63 489,14 0,50 520,42 0,41 408,55 0,55 14.Muhasebe Ücreti 80 0,30 80 0,17 150 0,15 150 0,12 115,00 0,15 15. Masraflar Toplamı 24964,90 95,01 44784,33 95,01 93589,83 95,01 120984,19 95,01 71080,81 95,01 16.Genel İdare Giderleri (%3) 748,95 2,85 1343,53 2,85 2807,69 2,85 3629,53 2,85 2132,42 2,85 17.Masraflar Top. Faizi (%2,25) 561,71 2,14 1007,647 2,14 2105,77 2,14 2722,14 2,14 1599,32 2,14 18. Üretim Masraflar Toplamı 26275,56 100,00 47135,51 100,00 98503,30 100,00 127335,86 100,00 74812,56 100,00 19.Toplam Canlı Ağırlık (kg) 10 701,00 21 500,25 46 768,63 61 752,38 35 180,56 20. Birim Canlı Ağırlık Maliyeti (₺/kg) (18/19) 2,46 2,19 2,11 2,06 2,20 12 Tablo 6. Doğu Marmara Bölgesinde Broyler Üretim Dallarında Brüt ve Net Gelir Hesabı (₺) I. Grup II. Grup III. Grup IV. Grup Ortalama 1. Brüt Üretim Değeri 22 368,65 47 221,89 102 434,56 132 710,66 76 183,94 2. Değişken Masraflar 20 711,46 40 431,89 87 717,48 112 663,75 65 381,14 3. Toplam Masrafları 26 275,56 47 135,51 98 503,30 127 335,86 74 812,56 Brüt Marj (1–2) 1657,19 6780,00 14 717,08 20 046,91 10 802,80 Net Gelir (1–3) -3906,91 86,38 3931,26 5374,80 1371,38 arttıkça, üretim faaliyetinde rekabet güçlerinin de arttığını göstermektedir. Anonim. (2011). Tarımsal Ekonomi ve Politika Geliştirme Enstitüsü. Kanatlı Sektörü Raporu. Retrieved from [CrossRef] Anonim. (2012). T.C. Ziraat Bankası Kayıtları. Bölgede ortalama net gelir değeri 1 371,38 ₺ olarak hesaplan- mıştır. Broyler üretim faaliyetinde küçük kapasitelerle çalışan işletmeler yüksek masraflarla çalışmakta ve masraflarını karşı- layacak gelir elde edememektedirler. I. grupta bulunan üretim dalları negatif net gelire sahip olup zarar etmektedirler. Fakat küçük üretim dalları zarar etmelerine rağmen üretim faaliyetle- rine devam etmektedirler. Çünkü üretim masraflarının büyük bir kısmını itibari masraflar (işletmeci ve ailesi işgücü ücret karşılığı, amortismanlar) oluşturmaktadır ve gerçekte karlı çalıştıklarını düşünmektedirler. Kapasite büyüklüğü arttıkça net gelir değerleri de artmaktadır (Tablo 6). Bu da küçük kapasiteli üretim dallarının üretim faaliyetlerinden kar elde edebilmeleri için üretim kapasite- lerinin artırılması gerektiğini göstermektedir. Aras, A. (1988). Tarım muhasebesi. Ege Üniversitesi, Ziraat Fakültesi ders kitapları. Aras, A. (1988). Tarım muhasebesi. Ege Üniversitesi, Ziraat Fakültesi ders kitapları. Çiçek, A., & Erkan, O. (1996). Tarım ekonomisinde araştırma ve örnekleme yöntemleri. Gaziosmanpaşa üniversitesi, Ziraat Fakültesi Yayınları. Dağdemir, V. (1998). Broyler Üretim Dallarında Brüt Marj ve Net Gelir Kuzeydoğu Anadolu Bölgesi’nde süt ürünlerinin imalat maliyeti ve pazarlama şekli üzerine bir araştırma [Doktora Tezi (Yayınlanmamış)]. Atatürk Üniversitesi. Dağdemir, V. (2004). Bayburt ili kop ve Burnaz Dere havzalarında arıcılık yapan işletmelerin genel durumu ve kooperatifleşmeye bakış açısı. Kooperatifçilik Dergisi, 146, 102–111. Dagdemir, V., Demir, O., & Macit, M. (2007). Estimation of optimum fat- tening period in broylers. Journal of Applied Animal Research, 3L, 159–160. Dagdemir, V., Demir, O., & Macit, M. (2007). Estimation of optimum fat- tening period in broylers. Journal of Applied Animal Research, 3L, 159–160. FOA. (2009). Retrieved from https://www.fao.org/faostat ( ) p g Gülten, Ş. (1994). Kıymet takdiri. Atatürk Üniversitesi Yayınları Gülten, Ş. (1994). Kıymet takdiri. Atatürk Üniversitesi Yayınları. Hakem Değerlendirmesi: Dış bağımsız. Hakem Değerlendirmesi: Dış bağımsız. Karagölge, C. (1996). Tarımsal işletmecilik. Tarım kümeslerinin analiz ve planlaması. Atatürk Üniversitesi Yayınları. Karagölge, C. (1996). Tarımsal işletmecilik. Tarım kümeslerinin analiz ve planlaması. Atatürk Üniversitesi Yayınları. Çıkar Çatışması: Yazarlar herhangi bir çıkar çatışması olmadığını beyan ederler. Çıkar Çatışması: Yazarlar herhangi bir çıkar çatışması olmadığını beyan ederler. Konak, K., Çobanoğlu, F., & Bozkurt, M. (1999). Cinsiyete göre yemlenen etlik piliçlerde bitiş yeminin besi performansı üzerine etkilerinin ekonomik analizi. Uluslararası hayvancılık Kongresi. Ege Üniversitesi. Finansal Destek: Yazarlar bu çalışma için finansal destek almadıklarını beyan etmişlerdir. Finansal Destek: Yazarlar bu çalışma için finansal destek almadıklarını beyan etmişlerdir. Özkan, B., Akçaöz, H. V., & Karadeniz, C. F. (2002). Antalya ilinde turunçgil üretim maliyeti ve geliri. Akdeniz Üniversitesi Ziraat Fakültesi Dergisi, 1(15), 1–7. Peer-review: Externally peer-reviewed. Declaration of Interests: The authors declare that they have no compet- ing interest. Declaration of Interests: The authors declare that they have no compet- ing interest. Özkan, B., & Kuzgun, M. (1997). Ana ve ikinci ürün susam üretim maliyeti ve geliri. Akdeniz Üniversitesi Ziraat Fakültesi Dergisi, 10, 45–60. Funding: The authors declared that this study has received no financial support. Funding: The authors declared that this study has received no financial support. Sarıca, M. (1996). Etlik piliçlerde besi süresinin uzatılmasının verim özel- liklerine etkisi ve ekonomik değerlendirmesi. Ondokuz Mayıs Üniver- sitesi Ziraat Fakültesi Dergisi, 11. Kaynaklar Şenköylü, N. (1996). Türkiye’deki tavukçuluğun temel sorunları ve çözüm önerileri. Hayvancılık Kongresi, 1. Açıl, A. F. (1974). Tarımsal ürün maliyetlerinin hesaplanması ve memle- ketimiz tarımsal ürün maliyetlerindeki gelişmeleri. Ankara üniversi- tesi Ziraat Fakültesi Yayınları. Taşkan, P. (2006). Retrieved from [CrossRef] Yamane, T. (1967). Statistics. An introductory analysis (2nd ed.). Harper and Row. Anonim. (2008). Tarım ve Köyişleri bakanlığı, koruma ve kontrol genel Müdürlüğü internet kayıtları. Anonim. (2008). Tarım ve Köyişleri bakanlığı, koruma ve kontrol genel Müdürlüğü internet kayıtları.
https://openalex.org/W2589089780	http://www.scielo.br/pdf/ref/v25n1/1806-9584-ref-25-01-00215.pdf	Portuguese	null	Como famílias de baixa renda em São Paulo conciliam trabalho e família?	Revista Estudos Feministas	2,017	cc-by	11,978	Como famílias de baixa renda em Como famílias de baixa renda em Como famílias de baixa renda em Como famílias de baixa renda em Como famílias de baixa renda em São São São São São P P P P Paulo conciliam trabalho e aulo conciliam trabalho e aulo conciliam trabalho e aulo conciliam trabalho e aulo conciliam trabalho e família? família? família? família? família? Resumo: Resumo: Resumo: Resumo: Resumo: Embora a participação das mulheres no mercado de trabalho tenha aumentado, a comparação entre a carga de responsabilidade familiar entre homens e mulheres e seus diferentes impactos na vida pessoal e profissional de pais e mães ainda é bastante desigual. Partindo de uma pesquisa representativa de 700 mães e pais com crianças pequenas e residentes em bairros de baixa renda em São Paulo, esse artigo analisa a diferença de gênero no mercado de trabalho, sua relação com as responsabilidades familiares e acesso a creches e pré- escolas para seus filhos nesta classe social. A análise desses dados permite concluir que os impactos do conflito trabalho-família são desproporcionais para as mães, independente de preferirem ou não permanecer no mercado de trabalho. Políticas de coparticipação do Estado e dos pais no cuidado são indicadas para redução desse conflito. Palavras-chave: Palavras-chave: Palavras-chave: Palavras-chave: Palavras-chave: família; mercado de trabalho; gênero; famílias de baixa renda http://dx.doi.org/10.1590/1806-9584.2017v25n1p215 http://dx.doi.org/10.1590/1806-9584.2017v25n1p215 Regina Madalozzo Instituto de Ensino Superior, São Paulo, SP, Brasil g Instituto de Ensino Superior, São Paulo, SP, Brasil Merike Blofield University of Miami, Miami, Flórida, EUA Como famílias de baixa renda em Como famílias de baixa renda em Como famílias de baixa renda em Como famílias de baixa renda em Como famílias de baixa renda em São São São São São P P P P Paulo conciliam trabalho e aulo conciliam trabalho e aulo conciliam trabalho e aulo conciliam trabalho e aulo conciliam trabalho e família? família? família? família? família? Merike Blofield University of Miami, Miami, Flórida, EUA Estudos Feministas, Florianópolis, 25(1): 422, janeiro-abril/2017 215 Introdução Introdução Introdução Introdução Introdução As questões de gênero1 na participação da força de trabalho, bem como o papel das relações culturais e de poder que perpetuam a diferença entre homens e mulheres, até mesmo na questão de trabalhos domésticos e de cuidado, são discutidas por diversas áreas de estudo. O foco na economia é, geralmente, direcionado para prêmios no salário para os homens que têm filhos e penalidade para as mulheres que os têm. Na sociologia, as escolhas entre trabalho remunerado ou não (incluindo as esferas de cuidado) colocam em disputa as questões de preferências pessoais em contraponto com as restrições que mantêm as mulheres como responsáveis pelo cuidado na família. Em REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD e mulher. Uma discussão mais profunda sobre esse assunto pode ser encontrada em Guedes (1995). e mulher. Uma discussão mais profunda sobre esse assunto pode ser encontrada em Guedes (1995). ambas as disciplinas, o impacto de políticas públicas no comportamento das pessoas é discutido. Nesse sentido, uma maior disponibilidade de creches e pré-escolas (com o objetivo de permitir uma escolha profissional para as mulheres que assim o quiserem) pode ter um impacto bastante relevante nas diferenças entre homens e mulheres. Em um país com tamanhas desigualdades sociais e de gênero (Waldemir ROSA, 2009), focar na questão das responsabilidades familiares e seus efeitos no mercado de trabalho é muito relevante. A literatura internacional foca majoritariamente em estudos sobre países industrializados. Esse estudo, em direção oposta, analisa o caso do Brasil, mais especificamente da cidade de São Paulo, com uma pesquisa inédita com 700 indivíduos (pais e mães). A seleção amostral foi direcionada para a população de renda mais baixa (classes C, D e E), pois são para essas famílias que as oportunidades são menores e menos frequentes e, portanto, as tensões entre trabalho e família tendem a ser mais elevadas (Jody HEYMANN, 2006; Bila SORJ, 2013). Com base em um conjunto de dados original, que consiste em um levantamento representativo de 700 pais de crianças com menos de 6 anos de idade em bairros de baixa renda de São Paulo, este estudo avalia a diferença de gênero no mercado de trabalho, sua relação com as responsabilidades familiares e acesso a creches e pré-escolas para seus filhos nesta classe social. A análise desses dados permite concluir que os impactos do conflito trabalho-família são desproporcionais para as mães, cuja participação no mercado de trabalho sofre com a falta de acesso a alternativas com relação a creches e pré-escolas e direta discriminação por parte dos empregadores. Também é possível perceber que, enquanto os pais que moram com seus filhos estão muito envolvidos em cuidar dessas crianças, a maioria dos pais não residentes não apoia seus filhos financeiramente e de forma regular, e menos de 5% deles participam no cuidado direto de seus filhos pelo menos uma vez pela semana. A partir dessas conclusões, entende-se que uma maior responsabilização por parte dos pais e uma corresponsabilização por parte do estado são fatores essenciais para o aumento do bem-estar das mães e das crianças. Esta obra está sob licença Creative Commons. Esta obra está sob licença Creative Commons. Esta obra está sob licença Creative Commons. 1 Por gênero entende-se “qualquer agrupamento de indivíduos, obje- tos, idéias, que tenham caracteres comuns” (FERREIRA, 1986, p. 844). Entretanto, mais do que uma defi- nição de grupamentos, a definição de gênero também inclui as diferenças culturais, de relação de poder, entre outras que definem as relações de feminino, masculino e transgênero, e, não, somente, de sexo biológico, como homem 215 Estudos Feministas, Florianópolis, 25(1): 422, janeiro-abril/2017 e mulher. Uma discussão mais profunda sobre esse assunto pode ser encontrada em Guedes (1995). 216 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? compara com as bases do referencial teórico. Por fim, a seção 5 (cinco) conclui o estudo. compara com as bases do referencial teórico. Por fim, a seção 5 (cinco) conclui o estudo. REGINA MADALOZZO E MERIKE BLOFIELD A promoção da igualdade de gêneros, além de reduzir a pobreza e desigualdade, faria com que o capital humano no Brasil fosse mais bem utilizado. O presente artigo conta com 5 (cinco) seções. A seção 2 (dois) apresenta o referencial teórico que baseou a construção do questionário e motivou a pesquisa conduzida. A seção seguinte apresenta a criação e condução da pesquisa de campo. A seção 4 (quatro) apresenta a descrição dos resultados encontrados na pesquisa e os 216 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 217 REGINA MADALOZZO E MERIKE BLOFIELD 73%. Mulheres de alta renda, bem como homens de baixa renda, têm mais do que o dobro da probabilidade de estar na força de trabalho – e empregados – do que as mulheres de baixa renda, refletindo a interação entre classes sociais e desigualdades de gênero. O fator-chave para a diferença entre os gêneros é a desigual divisão do trabalho entre homens e mulheres. Concomitante a isso, o fator mais importante na dramática diferença entre classes no emprego das mulheres diz respeito à relativa facilidade com a qual as famílias de alta renda terceirizam cuidados e trabalho doméstico ao setor privado (Helena HIRATA; Nadya Araujo GUIMARÃES, 2012). Com mais de seis milhões, ou 16% da força de trabalho feminina urba- na, o Brasil tem o maior número de mulheres empregadas no serviço doméstico do que qualquer país do mundo (DIEESE, 2013). De fato, o serviço doméstico é o modo domi-nante para resolução dos conflitos com os cuidados e sua prevalência no Brasil (bem como no resto da América Latina) está diretamente ligada às desigualdades socioeconô- micas (Merike BLOFIELD, 2012). Como resultado, as profissio- nais mulheres – especialmente o coorte mais jovem, onde muitas delas têm maior probabilidade de ter ensino superior completo do que os homens da mesma faixa etária – têm sido relativamente bem-sucedidas em entrar no mercado de trabalho e alcançar posições mais altas hierarquicamente. E quanto à grande maioria das brasileiras que não tem essa opção, e muitas das quais trabalham no serviço doméstico elas mesmas? Conforme dados da PNAD 2013 (IBGE, 2013), a diferença de emprego entre homens e mulheres no quintil de renda mais baixa, de 49%, é, também, grande. Essa pesquisa examina como os conflitos entre trabalho e família são resolvidos nesses quintis de renda. Conclui-se que, entre as mães, a falta de acesso a creches e pré-escolas, bem como a discriminação por parte dos empregadores, reduz a possibilidade dessas mulheres em participar do mercado de trabalho, causando ainda mais estresse para essas famílias. Pais que residem com seus filhos, por outro lado, têm um ajuste mais suave para equilibrar as demandas de trabalho e as familiares. A seguir, será descrita a metodologia e, em seguida, serão apresentadas as estatísticas descritivas do resultado da pesquisa. Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero Os valores correspondentes para o caso dos homens são de 91% e 217 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 217 REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero Referencial teórico: o hiato de gênero É bastante conhecido o resultado de que, enquanto a paternidade tende a ser boa para a carreira dos homens, a maternidade prejudica a carreira das mulheres (Michelle BUDIG; Paula ENGLAND, 2009; Kimberly KELLY; Linda GRANT, 2012; Markus GANGLE; Andrea ZIEFLE, 2009; Alexandra KILLEWALD, 2013). Isso funciona nos dois sentidos: os empregadores tendem a preferir pais sobre as mães e, por uma variedade de fatores, muitas mulheres saem da força de trabalho ou reduzem suas horas de trabalho, especialmente se elas têm um parceiro estável que participa da força de trabalho. De fato, um estudo usando amostras de áreas metropolitanas no Brasil constatou que a presença de crianças no domicílio reduz a inatividade de trabalho para os homens, mas aumenta a mesma para as mulheres (Pedro Rodrigues DE OLIVEIRA; Luiz Guilherme SCORZAFAVE; Elaine Toldo PAZELLO, 2009). Duas significativas teorias sociológicas apresentam diferentes interpretações a respeito da redução da participação de mães no mercado de trabalho: a teoria das preferências e a teoria do papel do conflito. A primeira afirma que as mães preferem ficar em casa, com seus filhos, enquanto a segunda coloca seu foco nas restrições: as mães têm a responsabilidade do cuidado para com as crianças, independente de suas preferências pessoais, fazendo com que a presença de crianças no domicílio seja mais uma força impeditiva do trabalho remunerado, algo que é “virtualmente ausente da vida dos homens” (Michèle Ernst STÄHLI; René Levy LE GOFF; Eric WIDMER, 2009). No Brasil, as taxas de participação das mulheres na força de trabalho aumentaram de pouco menos de 50%, em 1990, para 59,4%, em 2013 (Banco Mundial), e para 72% para as mulheres em idade fértil (UNDP/ILO, 2009; IBGE, 2013). Ao mesmo tempo, enquanto a distribuição de renda tornou-se um pouco menos desigual ao longo da última década (Marcelo Cortes NERI, 2011), o Brasil continua sendo um dos países com maior desigualdade social no mundo (João ARNOLD; Jen JALLES, 2014), com um índice de Gini de 0,55 em comparação com 0,30 para a União Europeia (Banco Mundial). ( ) Segundo dados da PNAD 2013 (IBGE, 2013), a taxa de ocupação das mulheres brasileiras em idade fértil a partir do quintil mais elevado é de 80%, enquanto ela se revela de apenas 36% para o quintil de menor renda. 3 Para os grupos focais, contratou- se a empresa Ética, Pesquisa de Mercado e Opinião. Já a pesquisa quantitativa teve seu campo conduzido pela empresa Radar Representações e Pesquisas. 2 Além do critério renda, utilizou- se uma extratificação dos bairros via Critério Brasil de classe social da ABEP – Associação Brasileira das Empresas de Pesquisa – para, efetivamente, se ter acesso mais focado a famílias das classes C-, D e E. 4 Antes de ser aplicado em campo, o questionário foi testado em um piloto (pré-teste) nos dias 10 e 11 de agosto de 2012. COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? graves, uma vez que as crianças necessitam de cuidados mais intensivos nessa faixa etária e os pais de baixa renda têm menos recursos financeiros para terceirizar esse cuidado. A pesquisa permitiu a formação de uma base de dados original com 700 respondentes entre mães e pais que residem em bairros de baixa renda da cidade de São Paulo e que tinham, ao menos, uma criança com menos de 6 anos de idade vivendo no domicílio. A definição do critério de baixa renda foi estabelecida para bairros com uma renda familiar média de R$1.500 mensais ou menos.2 A fase de entrevistas foi realizada entre agosto e outubro de 2012 e teve financiamento da FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo). Para a confecção do questionário e antes mesmo da definição dos bairros que fariam parte da amostra a ser coletada, a pesquisa contou com quatro grupos focais. Dois grupos de mães com, ao menos, um filho menor de 6 anos e um grupo com pais, também com crianças nessa faixa etária. Os grupos focais se reuniram no mês de abril de 2012 e as entrevistas foram conduzidas por uma mesma especialista para os quatro grupos:3 dois com mulheres que tinham um companheiro residindo em seu domicílio, um com mulheres solteiras, separadas, divorciadas ou viúvas, e um grupo com homens que residiam com uma companheira. Essas entrevistas em grupos focais tinham duração de 2 horas, aproximadamente, e o roteiro prévio incluía a discussão dos principais tópicos da pesquisa: como é a vida de uma mãe/ pai com filhos pequenos, a questão da escola e vagas em escolas públicas, a distribuição de responsabilidades dentro da família (trabalho doméstico, trabalho remunerado e cuidado com crianças), desejos para melhorar a qualidade de vida, conflitos com empregadores e análise geral de políticas públicas ou serviços públicos (como transporte e saúde, por exemplo). A partir dos resultados coletados nos grupos focais, montou-se um questionário a ser aplicado em campo.4 Esse questionário contava com 65 questões divididas nos seguintes blocos: – Questões sobre composição familiar e renda. – Características demográficas do entrevistado (mãe e pai, um em cada momento do tempo). – Informações sobre todos os filhos, independente da idade ou se residem com o pai e/ou a mãe. Construção da pesquisa Construção da pesquisa Construção da pesquisa Construção da pesquisa Construção da pesquisa A presente pesquisa, denominada “Conciliando trabalho e família”, teve foco em pais e mães de crianças com menos de seis anos de idade (abaixo da idade escolar obrigatória em 2012), pois é neste grupo que os conflitos entre trabalho remunerado e não remunerado são mais 218 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 219 REGINA MADALOZZO E MERIKE BLOFIELD Do ponto de vista de análise, não conside- ramos, no momento da pesquisa, a necessidade de dividir essas categorias, pois o nosso interesse principal era a análise da situação das famílias com crianças em ida- de pré-escolar e suas necessida- des de serviços públicos (vagas em creches, por exemplo) ou políticas públicas (com relação à proteção contra discriminação de gênero no trabalho, para citar uma delas). No caso das famílias onde o casal residia no domicílio, entrevistamos separadamente ambos os pais;5 no caso das famílias monoparentais, a entrevista foi sempre realizada com a mãe residente.6 Os entrevistadores retornaram aos domicílios entrevistados tantas vezes quantas foram necessárias para garantir a entrevista de ambos os pais. No total, a pesquisa quantitativa incluiu 399 famílias: 300 de mães casadas e seus cônjuges – i.e., 300 pais, que residiam com a mãe de, pelo menos, um de seus filhos – e 99 mães não casadas.7 Assim, as famílias chefiadas por mulheres sem parceiro representam 25% da amostra, face à média nacional de 27% das famílias com crianças (PNAD 2013). REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD – Questionário sobre trabalho doméstico e cuidado com crianças (itens separados): número de horas, tipo de trabalho, contribuição própria e do cônjuge (quando existente). ) – Perguntas sobre participação do(a) parceiro(a) na educação dos filhos e sobre pensão alimentícia no caso de pais separados ou divorciados. – Características do trabalho do(a) entrevistado(a). – Perguntas sobre divisão do poder com relação ao dinheiro e compras da casa. – Renda individual e familiar. – Renda individual e familiar. – Dados de classificação econômica de acordo com – Renda individual e familiar. – Dados de classificação econômica de acordo com a ABEP. A coleta de dados foi focada em bairros com famílias que, na média, tivessem renda familiar inferior a R$1.500. Foram selecionados, para a amostra, os seguintes bairros: Brasilândia, Campo Grande, Capão Redondo, Casa Verde, Cidade Ademar, Cidade Dutra, Cidade Tiradentes, Ermelino Matarazo, Grajaú, Guaianazes, Iguatemi, Itaim Paulista, Jaraguá, Jardim Ângela, Jardim Helena, Jardim São Luis, José Bonifácio, Lajeado, Parelheiros, Parque São Domingos, República, São Domingos, São Mateus, Sacomã, Sapopemba, Tiradentes, Tremembé, Vila Matilde, Vila Jacui, Vila Andrade. Ao chegar ao bairro, o entrevistador se dirigia a um primeiro endereço sorteado e iniciava a pesquisa perguntando se, no domicílio, residia ao menos uma criança com menos de 6 anos de idade. Em caso negativo, a entrevista era concluída e ele se dirigia ao domicílio vizinho. Em caso positivo, ele prosseguia com a entrevista desde que o pai e/ou a mãe da criança residissem no mesmo domicílio. 5 Os entrevistadores seguiram a recomendação de entrevistá-los separadamente para que as respostas não fossem viesadas ou que pais e mães não se sentissem constrangidos em responder perguntas, especialmente às ligadas ao comportamento do companheiro(a). 6 Em nossa amostra não é possível analisar o caso de famílias mono- parentais com somente pai resi- dente por não termos encontrado respondentes com esse perfil. p p 7 Ao longo do artigo, utilizaremos a expressão “mães não casadas” para incluir as mulheres solteiras, viúvas, separadas, divorciadas e desquitadas. As “mães casadas” são aquelas casadas formalmente ou que residem com um compa- nheiro. Nesse sentido, por termos unificado, no questionário, os ca- sais casados formalmente e os coabitantes sem registro formal, o texto utilizará um mesmo nome para ambas as situações. COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? – Questões sobre vagas e matrículas em escolas particulares e públicas; avaliação das preferências do entrevistado sobre matricular seu(sua) filho(a) em creches e pré-escolas ou permitir que a criança permaneça fora da escola por mais tempo. Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 219 8 A média de filhos dos homens (pais) é maior do que a média de filhos das mães casadas, pois existem homens (pais) entrevista- dos que têm filhos de relações anteriores à atual. 1. Características sociodemográficas e 1. Características sociodemográficas e 1. Características sociodemográficas e 1. Características sociodemográficas e 1. Características sociodemográficas e do mercado de trabalho da amostra do mercado de trabalho da amostra do mercado de trabalho da amostra do mercado de trabalho da amostra do mercado de trabalho da amostra A tabela 1 tabela 1 tabela 1 tabela 1 tabela 1 apresenta as estatísticas descritivas da amostra coletada na pesquisa. As mães não casadas são 220 220 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 11 ECEC é a sigla utilizada pela OCDE (Organização para Coope- ração e Desenvolvimento Econô- mico) para designar Early Child- hood Education and Care, livre- mente traduzida por Educação e Cuidado para Primeira Infância. Utilizar-se-á, ao longo do texto, a sigla original ECEC. 12 Uma emenda constitucional aprovada em 2009 torna, a partir de 2016, a frequência escolar obrigatória para crianças com 4 e 5 anos anos de idade. REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD Os dados da pesquisa demonstram, sem surpresa, que os pais casados têm a maior taxa de emprego (de 90%), enquanto somente 60% das mães não casadas e 47,3% das mães casadas encontram-se empregadas. Também é interessante o fato de que os pais são os mais propensos a serem registrados pelo INSS, isto é, no mercado de trabalho formal; de todos os pais que estavam empregados, 70% eram registrados pelo INSS, enquanto que a cifra era de 53% para as mães não casadas e 63% para as mães casadas. Cerca de metade das mães não casadas que trabalham e pouco menos de um terço de todas as mães que trabalham não têm direito à licença-maternidade.9 9 Também questionamos as mães se elas estavam ativas no merca- do de trabalho quando o filho mais novo nasceu e se recebe- ram licença maternidade: 58% das mães casadas e 40% das mães não casadas receberam licença maternidade, o que significa que o benefício é inferior à sua eligibilidade. COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? ligeiramente mais jovens (27 anos, em média) do que as mulheres casadas (média de 29 anos de idade), e as mulheres são, em média, mais jovens do que os homens (média de 32 anos de idade). As mães não casadas têm, em média, 1,8 filhos, enquanto as mães e pais casados têm 2.1 e 2.2 filhos,8 respectivamente, próximos à taxa de fecundidade nacional de 1,8 filhos por mulher adulta. Com relação a aspectos de autoidentificação racial, mais de metade da amostra coletada se identifica como pertencente às raças “preta” ou “parda”, enquando a segunda maior proporção de autoidenficação se dá com a raça “branca” (39% para mulheres não casadas, 46% para mulheres casadas e 35% para os homens ). TTTTTabela 1: abela 1: abela 1: abela 1: abela 1: Estatísticas descritivas da amostra Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Nota: Os valores entre parênteses indicam o desvio padrão. TTTTTabela 1: abela 1: abela 1: abela 1: abela 1: Estatísticas descritivas da amostra Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Nota: Os valores entre parênteses indicam o desvio padrão. Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Nota: Os valores entre parênteses indicam o desvio padrão. As mães casadas da amostra apresentam níveis mais elevados de educação, com quase metade delas tendo concluído o Ensino Médio em comparação com 45% dos pais e 37% das mães não casadas. No entanto, é importante salientar que 20% das mães não casadas e não empregadas relatam ainda estudar. Por fim, a renda mensal familiar das mulheres não casadas é, em média, R$1.168, representando 77% do valor da renda familiar média para as mulheres casadas, que é R$1.526. Um fato interessante a ressaltar é que os pais casados reportam uma renda familiar média de R$1.688, valor superior ao declarado por suas esposas, mas, como ambos os dados são percepções individuais, é natural certa discrepância. 221 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 221 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? à necessidade de um dia integral de trabalho (tipicamente, de 8 horas diárias). Dentre as mães que trabalham fora, metade tem seus filhos com idade entre 0-3 anos em creches, enquanto que menos de um terço das mães que não trabalham os têm. A grande maioria das crianças está em escolas públicas. Claramente, os números não apresentam as razões que estão por detrás desta baixa cobertura de inclusão escolar. Entretanto, a razão preponderante para explicar o motivo pelo qual as crianças não estavam matriculadas em creches públicas é a não existência de vagas. Dos entrevistados que não têm filhos em ECECs públicos (51% das famílias), ao menos metade tem suas crianças em lista de espera, o que confirma uma grande demanda não atendida por este serviço. O maior percentual de mães com filhos na lista de espera por ECECs (65%) está entre mães não casadas que trabalham. Quando se perguntou aos pais sobre qual o motivo pelo qual seus filhos não estavam matriculados em ECECs privados, sem surpresa, o principal fator foi o custo. Apenas 7% das famílias da amostra tinham um filho matriculado em ECEC privado, e, mesmo para essas famílias, a razão predominante para esta escolha em todos os tipos de família foi a falta de vagas em ECECs públicos. Para os pais cujos filhos estavam matriculados em creche pública, perguntou-se sobre a preferência por horário mais amplo de atendimento. A figura 1 figura 1 figura 1 figura 1 figura 1 indica que entre 45 a 60% de pais que trabalham fora ou que não trabalham gostariam de escolas abertas e disponíveis por um período mais amplo de atendimento. Para as mães que não trabalham, a possibilidade de procurar um emprego é a razão mais importante, enquanto que, para os pais e mães casados e que trabalham fora, o principal motivo é não ter de pagar ou depender de outras formas de cuidado com a criança, pois dependem, muitas vezes, de um cuidado informal (por exemplo, uma pessoa que leve e/ou busque a criança na escola). Os cuidados informais envolvem familiares e vizinhos e podem ou não ser remunerados. Para um quinto dos pais, a principal razão para querer mais horas de creche é para que sua esposa possa trabalhar. 1.1 Acesso a creches e pré-escolas 1.1 Acesso a creches e pré-escolas 1.1 Acesso a creches e pré-escolas 1.1 Acesso a creches e pré-escolas 1.1 Acesso a creches e pré-escolas Estudos empíricos têm demonstrado que países ou regiões que disponibilizam acesso a creches a preços acessíveis têm taxas de participação da força de trabalho muito maior entre mães.10 A própria dupla jornada das mulheres, que será discutida mais profundamente nas seções a seguir, pode ser amenizada com uma maior rede de serviços públicos que complementem o cuidado individual com crianças (Cristiane SOARES e Ana Lucia SABOIA, 2007). A literatura sobre a América Latina mostra resultados semelhantes. Tanto para o Chile como para a Argentina, estudos concluíram que o acesso à educação pré-escolar e a creches de baixo custo (ECEC11) ou escolas em tempo integral tem efeito positivo sobre as taxas de participação na força de trabalho das mães (Samuel BERLINSKI; Sebastian GALLIANI e Patrick McEWAN, 2008; Dante CONTRERAS; Paulina SEPULVEDA; Soledad CABRERA, 2009; Patricia MEDRANO, 2009). Outros estudos mostram que o acesso a ECECs formais permite às mães alterarem a natureza de sua participação no mercado de trabalho de tempo parcial para integral (BERLINSKI; GALLIANI e McEWAN, 2008, na Argentina; Kelly HALLMAN; Agnes R. QUISUMBING; Marie RUEL; Benedicte de LA BRIÈRE, 2005; Agnes R. QUISUMBING, 2003 sobre Guatemala; Mirela CARVALHO et al., citado por Laura CHIODA, 2010, para Brasil) e mudar de informal para formal o seu contrato de trabalho (CHIODA, 2011). É 10 A respeito, ver: Contreras et al. (2010) para uma visão geral da literatura econômica e Esping- Andersen (2009) para uma visão geral da literatura científica em sociologia e política. É um direito constitucional, no Brasil, a disponibilidade de ECECs gratuita a partir de quatro meses de idade da criança, o que coloca o país entre os mais progressistas do mundo com relação a esse direito. Para a faixa etária de 4 e 5 anos de idade, as taxas de cobertura são cerca de 80%, refletindo o objetivo do Brasil de cobertura universal para esta faixa etária a partir de 2016.12 Mesmo assim, no que tange à necessidade de cuidado com essas crianças, o dia médio de escola tem duração de 4 horas, significativamente inferior 222 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 224 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre mães e pais mães e pais mães e pais mães e pais mães e pais Pesquisas para diversos países mostram que, dentro da família, as mães ainda são as maiores responsáveis por cuidar das crianças, embora o envolvimento dos pais tenha apresentado um aumento sensível, especialmente quando suas esposas trabalham fora de casa (Gøsta ESPING- 223 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 223 REGINA MADALOZZO E MERIKE BLOFIELD Figura 1: Figura 1: Figura 1: Figura 1: Figura 1: Razões para desejar que a escola funcione por mais horas durante a semana, por tipo de família Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) ANDERSEN, 2009). No entanto, esta tendência varia muito, dependendo do país (mais uma vez, os países nórdicos têm os pais mais envolvidos) e por nível sócio-econômico. Nos países industrializados, os pais de famílias de baixa renda e escolaridade são os que se dedicam menos ao cuidado com as crianças. Esping-Andersen (2009) também mostra que a diferença entre a participação de mães e pais é menor para o trabalho desejável de cuidar das crianças do que para o trabalho tedioso de manutenção do lar. Outros sociólogos afirmam que, apesar do aumento da atenção para o papel do pai, “a função central da paternidade continua a ser a prestação de apoio econômico” (William MARSIGLIO; Paul R. AMATO; Randal D. DAY; Michael E. LAMB, 2000; Shawn CHRISTIANSEN; Rob PALKOVITZ, 2001) e os papéis tradicionais de gênero permanecem fortes (STÄHLI; LE GOFF; WIDMER, 2009). A literatura sobre o Brasil encontra uma dinâmica semelhante. Em um destes estudos, mulheres casadas que trabalham em tempo integral (entre 40 e 44 horas semanais) dedicam, em média, 20 horas semanais para trabalhos domésticos, enquanto os homens que trabalhavam o mesmo número de horas no mercado de ANDERSEN, 2009). No entanto, esta tendência varia muito, dependendo do país (mais uma vez, os países nórdicos têm os pais mais envolvidos) e por nível sócio-econômico. Nos países industrializados, os pais de famílias de baixa renda e escolaridade são os que se dedicam menos ao cuidado com as crianças. Esping-Andersen (2009) também mostra que a diferença entre a participação de mães e pais é menor para o trabalho desejável de cuidar das crianças do que para o trabalho tedioso de manutenção do lar. Outros sociólogos afirmam que, apesar do aumento da atenção para o papel do pai, “a função central da paternidade continua a ser a prestação de apoio econômico” (William MARSIGLIO; Paul R. AMATO; Randal D. COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? trabalho contribuíam com apenas 5 horas semanais para o mesmo fim (Regina MADALOZZO; Sérgio Ricardo MARTINS; Ludmila SHIRATORI, 2010). Os dados da pesquisa permitem comparar as estimativas dessa divisão de trabalho do ponto de vista do entrevistado, e, também, sob a perspectiva de seu cônjuge.13 Diferente da forma como a questão do trabalho doméstico e o cuidado com as crianças é abordada pelo Instituto Brasileiro de Geografia e Estatística (IBGE) na PNAD – com a união do tempo gasto em afazeres domésticos e cuidados com as crianças –, a presente pesquisa diferenciou o tempo gasto com as tarefas domésticas (limpar a casa,14 cuidar da cozinha, cuidar das roupas, cuidar do automóvel ou moto, cuidar das plantas ou animais domésticos, consertar objetos da casa) das atividades de cuidado com crianças (dar banho, trocar fraldas/roupas, fazer dormir, dar comida, brincar, levar e/ou buscar na escola, levar ao médico/ dentista/etc.). 13 É interessante perceber que as diferentes perspectivas sobre o trabalho e a responsabilidade en- tre cônjuges leva em considera- ção diferentes perspectivas tam- bém nas relações de poder dentro da família (ou quem é o responsá- vel pelo cuidado e quem “ajuda” o responsável). Nesse sentido, Hirata (2016) contribui para o en- tendimento dessas relações – inclusive com a perspectiva de classe e raça – na questão da “interseccionalidade” na interde- pendência das relações. 14 As categorias expostas acima forarm as categorias consideradas “trabalho doméstico” tanto pelos participantes dos grupos focais como no pré-teste, onde as ques- tões eram colocadas de forma aberta. Para facilitar o entendi- mento do entrevistado, o entrevis- tador tanto perguntava o número de horas dedicados ao trabalho doméstico como, também, usan- do um cartão, pedia para o(a) entrevistado(a) apontar a ordem da tarefa em que o(a) mesmo(a) mais gastava seu tempo até aquela que ele(a) gastava menos tempo. Para o caso de tarefas de cuidado com as crianças, usamos o mesmo procedimento. Maria Cristina Aranha BRUSCHINI e Arlene Martínez RICOLDI (2009) apresentam uma análise bastante profunda da situação do trabalho doméstico e do cuidado com crianças utilizando tanto os dados da PNAD 2002 como, também, dados coletados com grupos focais. Embora ambas as tarefas sejam de “cuidado”, como ressaltado em Bruschini e Ricoldi (2009), a tarefa de cuidar dos filhos/ crianças é considerada, usualmente, de maior prestígio ou valor, até mesmo pela cultura de valorização da educação e do cuidado. 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre 1.2 A divisão sexual do trabalho entre mães e pais mães e pais mães e pais mães e pais mães e pais DAY; Michael E. LAMB, 2000; Shawn CHRISTIANSEN; Rob PALKOVITZ, 2001) e os papéis tradicionais de gênero permanecem fortes (STÄHLI; LE GOFF; WIDMER, 2009). A literatura sobre o Brasil encontra uma dinâmica semelhante. Em um destes estudos, mulheres casadas que trabalham em tempo integral (entre 40 e 44 horas semanais) dedicam, em média, 20 horas semanais para trabalhos domésticos, enquanto os homens que trabalhavam o mesmo número de horas no mercado de 224 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 225 226 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? Por envolver afeto, segundo a amostragem dessas pesquisadoras, as tarefas de cuidado não deveriam ser consideradas “tarefas domésticas”. Seguindo essa linha de raciocínio, dividimos o tempo gasto com essas tarefas em nosso questionário e, assim, apresentamos os dados separadamente. As figuras 2 e 3 apresentam a quantidade de tempo dedicado ao cuidado de crianças e ao trabalho doméstico por sexo, situação de emprego e tipo de família. Ambas as figuras apresentam as estimativas próprias (das esposas e maridos) e de seus cônjuges para tempo dedicado ao cuidado das crianças e tempo dedicado ao trabalho doméstico. Para as mães não casadas, a declaração é somente das estimativas próprias. Diversos fatores se destacam. Em primeiro lugar, como a figura 2 figura 2 figura 2 figura 2 figura 2 indica, a diferença entre os autorrelatos e as percepções do cônjuge é praticamente inexistente para casais onde pais e mães participam do mercado de trabalho. Para os casais em que a mãe não está ocupada, existe uma diferença mais proeminente com relação ao papel do pai: as mães percebem a participação dos pais em aproximadamente 4 horas semanais a menos no 225 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 225 REGINA MADALOZZO E MERIKE BLOFIELD COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? explicação para essa diferença: o número de horas dispendido nos estudos. Não medimos o número de horas por semana dedicadas ao estudo em geral, mas nossos dados mostram que 20% das mães não casadas que não estão trabalhando se encontram estudando, enquanto 10% das mães casadas que não trabalham o fazem. 15 Parte das horas reportadas no cuidado com as crianças, possivel- mente, é feita conjuntamente, i.e., quando ambos os pais estão em contato com seus filhos. q Um terceiro fator interessante que a figura 2 aponta é que a participação dos pais casados no cuidado com as crianças está longe de ser insignificante: em média eles dedicam 27 horas por semana para esse fim, independen- temente de suas esposas trabalharem fora de casa. Embora um fator interessante: ele também indica que os pais não parecem adaptar seu comportamento e aumentar a sua parcela de responsabilidade no cuidado das crianças quando suas mulheres trabalham, o que implica que o tempo gasto com as crianças é mais uma preferência por parte dos pais do que uma questão de responsabilidade parental – é a opção por passar o tempo com seus filhos, mas não, necessariamente, assumir a responsabilidade adicional no caso de suas esposas dedicarem tempo ao mercado de trabalho. Os grupos focais dão apoio a essa interpretação, pois tanto pais como mães percebiam elas – mães – como principais responsáveis pelo cuidado com as crianças, enquanto os pais têm a possibilidade de escolher “ajudar” as suas mulheres com esta responsabilidade quando se sentem inclinados a isso.15 Em suma, para casais onde as mães trabalham fora, elas gastam 50% mais de horas no cuidado com seus filhos do que os pais e, para aqueles casais onde as mulheres não trabalham fora, as mães dedicam o dobro de horas do que os pais no cuidado com as crianças. Por fim, filhos de mães não casadas simplesmente recebem menos tempo de atenção parental total. A figura 3 figura 3 figura 3 figura 3 figura 3 aponta o tempo semanal gasto em tarefas domésticas, que podem ou não se sobrepor ao tempo de cuidado com as crianças. Aqui, em primeiro lugar, é possível observar a existência de uma lacuna clara nas percepções dos casais. Os homens têm uma percepção de mais tempo gasto em tarefas domésticas tanto desempenhadas por eles como por elas. 15 Parte das horas reportadas no cuidado com as crianças, possivel- mente, é feita conjuntamente, i.e., quando ambos os pais estão em contato com seus filhos. REGINA MADALOZZO E MERIKE BLOFIELD cuidado com os filhos do que os pais afirmam fazer. O contrário – percepção dos pais com relação às horas que as mães dedicam aos filhos – apresenta uma diferença de apenas uma hora semanal. Figura 2: Figura 2: Figura 2: Figura 2: Figura 2: Horas por semana dedicadas ao cuidado com as crianças, por tipo de família Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Focando a partir de agora na percepção própria quanto à dedicação aos filhos, percebe-se que as mães que trabalham fora gastam menos tempo no cuidado para com seus filhos do que as mães que não trabalham. Essa diferença é, em média, 10 horas semanais a menos (o que não é, contudo, próximo à carga de trabalho semanal por período integral). Mães casadas que não participam do mercado de trabalho gastam mais tempo cuidando de seus filhos, 53,7 horas por semana, enquanto que mães não casadas que não trabalham gastam 49,7 horas semanais no cuidado com as crianças, representando uma diferença de 4 horas por semana. Mães casadas e que trabalham fora de casa gastam 38,9 horas por semana no cuidado com as crianças, enquanto as mães não casadas que trabalham gastam 36,9 horas por semana cuidando de seus filhos. Em geral, mães não casadas, seja trabalhando ou não, gastam um pouco menos de tempo cuidando de seus filhos do que as mães casadas. Aqui é importante ressaltar que pode existir uma variável não existente e com potencial 226 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 227 228 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 16 Lembrando que a PNAD inclui, no trabalho doméstico, as horas dedicadas ao cuidado com as crianças. COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? Mulheres casadas que trabalham fora têm a percepção de que trabalham 2 horas a menos por semana do que os maridos percebem e que eles trabalham 1,5 hora a menos do que declaram. Mulheres casadas que não trabalham fora têm uma visão ainda mais dissonante do que a de seus maridos: elas declaram gastar 10 horas a menos em trabalho doméstico por semana do que eles percebem e que eles dedicam 3 horas a menos do que declaram. Em segundo lugar, as autodeclarações, entre as mães casadas, tanto aquelas que trabalham fora de casa como as que não trabalham fora, é a de que gastam praticamente 227 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 227 REGINA MADALOZZO E MERIKE BLOFIELD Figura 3: Figura 3: Figura 3: Figura 3: Figura 3: Horas por semana dedicadas ao trabalho doméstico, por tipo de família Figura 3: Figura 3: Figura 3: Figura 3: Figura 3: Horas por semana dedicadas ao trabalho doméstico, por tipo de família Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) a mesma quantidade de tempo por semana em tarefas domésticas: cerca de 30 horas semanais. As mães não casadas, por outro lado, dedicam consideravelmente menos tempo a essas tarefas: 25,3 horas se não trabalham fora de casa e 22,9 horas se elas trabalham fora de casa. Isso dá suporte a um estudo anterior, baseado em dados do Brasil, que constatou que essas mães com filhos menores de 14 anos de idade gastam 2 horas adicionais por semana em tarefas domésticas se um homem estiver residindo com a família do que se um homem não estiver presente (Gary BARKER, Juan Manuel CONTRERAS, Brian HEILMAN, Ajay SINGH; Ravi VERMA, 2011, p. 36). Em terceiro lugar, os pais com as mulheres que não trabalham reportam gastar 10,7 horas por semana em tarefas domésticas, enquanto os pais com as esposas que trabalham fora gastam, em média, 13,9 horas semanais. Por outro lado, a diferença de gênero entre mães e pais no trabalho doméstico é mais ampla: quando a mãe não trabalha, ela gasta 2,8 vezes mais horas no trabalho doméstico do que o marido; mesmo quando trabalha, ela ainda gasta 2,2 vezes mais horas no trabalho doméstico do que seu marido. 228 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 REGINA MADALOZZO E MERIKE BLOFIELD homens. Comparando com o presente estudo, as médias, somente dessas duas tarefas nos dados reportados na figura 4, são bastante superiores. Entretanto, como a pergunta feita pelo IBGE agrega os dois tipos de trabalho (doméstico e de cuidado), é bastante plausível que as pessoas, ao reportar o número de horas dedicadas aos mesmos, esqueçam-se de períodos de sobreposição. Por exemplo, quando perguntadas pelas horas de trabalho doméstico, as pessoas podem pensar no número de horas que estiveram cozinhando, limpando a casa etc. e reportam esse número. Ao serem perguntadas sobre o número de horas que dedicaram ao cuidado com as crianças, elas reportam o mesmo e, em diversos períodos, elas estavam fazendo as duas coisas concomitantemente. Ao empregar a técnica utilizada pelo IBGE, a contagem dupla se desfaz. Ao nosso entender, por serem trabalhos distintos – e até mesmo com diferentes status, como apontam Bruschini e Ricoldi (2012) – é mais adequado medi-los separadamente, mesmo quando sobrepostos. Outro ponto interessante da pesquisa é a forma como os casais tomam decisões financeiras. Perguntou-se aos entrevistados quem era responsável pela administração do dinheiro em suas famílias. Para mulheres que trabalhavam fora de casa, 17,8% responderam que era o marido/ companheiro ou outra pessoa que não ela, enquanto para aquelas que não trabalhavam fora, esse percentual chegou a 41,6%. Esses números indicam que o poder decisório na questão financeira e, possivelmente, o poder de barganha envolvido em outras negociações que são baseadas em decisões financeiras é muito superior para mulheres que “ganham seu próprio dinheiro” do que para aquelas que dependem financeiramente de outras pessoas. COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? A figura 4 figura 4 figura 4 figura 4 figura 4 mostra a quantidade semanal total de tempo gasto, via autodeclaração, no trabalho remunerado (para pais e mães que trabalham), transporte (também para os que trabalham fora de casa), cuidado com as crianças e trabalho doméstico. Figura 4: Figura 4: Figura 4: Figura 4: Figura 4: Horas por semana dedicadas ao trabalho doméstico, cuidado com crianças, trabalho remunerado e transporte, por gênero e tipo de família Figura 4: Figura 4: Figura 4: Figura 4: Figura 4: Horas por semana dedicadas ao trabalho doméstico, cuidado com crianças, trabalho remunerado e transporte, por gênero e tipo de família Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Fonte: Pesquisa “Conciliando Trabalho e Família” (2012) Quando todas as horas são computadas em conjunto, incluindo o transporte, a maior carga de trabalho se revela entre as mães casadas que também trabalham fora de casa. Em segundo lugar, para mães não casadas que trabalham. Isso confirma que a “dupla carga” para as mães que trabalham existe. Para os pais, o tempo total de trabalho é praticamente o mesmo se suas esposas trabalham fora de casa ou não, mais uma vez apoiando a hipótese de que os pais não alteram seu comportamento em resposta a suas esposas que trabalharem fora de casa. Bruschini (2006) criticava o status quo onde o trabalho doméstico é considerado inatividade econômica. Em seu artigo, a autora apontava a dissonância entre as médias semanais de trabalho doméstico entre homens e mulheres usando dados da PNAD 2002:16 27,2 horas para as mulheres e 10,6 para os 229 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 229 REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD 230 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 17 Considerando restrições de financiamento e o tempo gasto com os questionários, somente foram questionadas as mães nesse quesito. 1.3 As mães não casadas e os “pais 1.3 As mães não casadas e os “pais 1.3 As mães não casadas e os “pais 1.3 As mães não casadas e os “pais 1.3 As mães não casadas e os “pais ausentes” ausentes” ausentes” ausentes” ausentes” No caso de mães não casadas, os pais das crianças no Brasil são obrigados, por lei, a pagar uma parte de sua renda em apoio à criança (e muitos podem desejar fazê-lo, independentemente da lei). A falta de pagamento, quando reportada à Justiça, pode resultar em prisão. Ao todo, 37,5% das famílias em nossa pesquisa têm crianças na residência cujos pais não vivem com eles (25% são filhos de mães não casadas e outros 12,5% são crianças de famílias com presença de um casal residente, mas cujo pai não é o homem residente com a família). ) Das mulheres entrevistadas, apenas cerca de um quarto (27%) dos pais não residentes têm um acordo legal e, destes, 80% contribui ao menos ocasionalmente com o sustento de seus filhos. Por outro lado, 57% dos pais que não 230 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? têm um acordo legal nunca paga pensão alimentícia. No total, metade de todos os pais não residentes nunca fornece apoio financeiro para seus filhos, enquanto pouco mais de um terço (35%) os apoia com regularidade. O restante (15%) contribui de forma eventual. Esses dados indicam que, entre mães e pais que não vivem juntos, existe uma lacuna de gênero substancial com relação ao apoio financeiro para seus filhos. Também se questionou as mães se os pais não residentes passam algum tempo – ao menos uma vez por semana – com seus filhos. Aqui, os resultados são gritantes: menos de 5% dos pais não residentes veem seus filhos pelo menos uma vez por semana. Isto evidencia uma lacuna de gênero dramática no tempo dedicado ao cuidado das crianças entre casais não coabitantes. Estes resultados, em conjunto, indicam que, para muitos pais, a renda e o tempo que dedicam à sua prole é mínimo quando não coabitam na mesma residência. Como resultado, as mães não casadas têm o desafio de liderar uma família com renda menor, maior necessidade de trabalho no mercado e, também, são dependentes de outras instâncias de arranjos com os cuidados para com seus filhos tanto formal como informalmente. Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 231 REGINA MADALOZZO E MERIKE BLOFIELD mercado de trabalho e entrariam na categoria de mães não casadas que não trabalham fora. Pouco menos de 70% das mães não casadas que não trabalham – muitas das quais ainda estudam – recebem ajuda informal por parte de outras mulheres. Já a ajuda informal por parte de homens é bem menos comum, variando de 23% entre mães casadas que não trabalham fora até 38% para as mães não casadas que trabalham fora. Para as mulheres não casadas que não trabalham fora, toda a ajuda informal recebida é não remunerada, provavelmente relacionada ao fato de que a baixa renda familiar impede a contratação desse serviço. Entre mães não casadas que trabalham fora, um quarto da ajuda informal vinda por parte de homens é remunerada, enquanto se observa que, para as mães casadas, a ajuda remunerada é insignificante. 1.4 Redes de cuidados informais 1.4 Redes de cuidados informais 1.4 Redes de cuidados informais 1.4 Redes de cuidados informais 1.4 Redes de cuidados informais Dada a situação financeira dessas famílias, combi- nada com a evidente falta de vagas em creches e pré-escolas, se faz importante o exame da disponibilidade de redes informais de cuidado. Ou seja, pessoas que possam auxiliar mães e pais no cuidado diário com as crianças.17 Perguntou- se qual o tipo de ajuda informal que a família recebia semanalmente para cuidar das crianças pequenas, inclusive se essa ajuda era proveniente dos filhos mais velhos, avós maternas e paternas, avôs maternos e paternos, outros parentes, amigos, vizinhos ou trabalhadores domésticos (babás, em especial). Em seguida, agruparam-se as instâncias de ajuda recebidas em “ajuda vinda de um homem” e “ajuda vinda de uma mulher”. As respostas à pesquisa apontam claramente a proeminência de ajuda informal por parte de outras mulheres. Entre as famílias com ambos os pais presentes ou somente com as mães residentes, independente dessas mães trabalharem ou não fora de casa, 70% das famílias recebem ajuda de outras mulheres. A grande maioria é não remunerada. Entre as mães não casadas que trabalham, cada uma delas recebe algum tipo de ajuda informal por parte de outra mulher; na verdade, é provável que, sem essa ajuda, as mães não fossem capazes de se envolver no 231 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 231 REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD 232 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 18 Esse percenual inclui um pai que se declara incapaz para o trabalho e dois pais aposentados. 19 Embora seja necessário ressaltar que esse percentual inclui somente 2 (duas) observações. 1.5 Preferências vs restrições em conciliar 1.5 Preferências vs restrições em conciliar 1.5 Preferências vs restrições em conciliar 1.5 Preferências vs restrições em conciliar 1.5 Preferências vs restrições em conciliar vida profissional e familiar vida profissional e familiar vida profissional e familiar vida profissional e familiar vida profissional e familiar Com a finalidade de testar as teorias que tratam das preferências pessoais ou com relação ao conflito de papéis, duas questões se colocam: em primeiro lugar, pedimos às mães e pais que não trabalham o motivo pelo qual eles não estavam empregados. Em segundo lugar, examinou-se a autoavaliação das mudanças de relações com o mercado de trabalho após o nascimento do(s) filhos(s). p ( ) ( ) Agregou-se as respostas para a primeira pergunta em 3 diferentes categorias: aqueles que não trabalham por escolha própria, isto é, por preferências pessoais ou familiares; aqueles que estão procurando, mas não conseguem encontrar trabalho; e, por fim, aqueles que não trabalham por não ter acesso a creches. Os dois últimos foram considerados como “restrições”, enquanto que, na categoria de preferência, foram agregadas as seguintes razões: preferência pessoal por ficar em casa com as crianças; um dos cônjuges prefere que a mãe (ou pai) fique em casa com as crianças; frequência à escola que impede o trabalho concomitante; salários disponíveis abaixo do nível desejado; problemas de saúde ou incapacidade física; licença-maternidade e aposentadoria. Enquanto alguns desses motivos (por exemplo, salários abaixo do nível desejado, preferência do cônjuge e problemas de saúde) possam ser considerados como restrições, a opção foi por mensurar quando os pais e mães estão ativamente empenhados em participar da força de trabalho ou não e, dessa forma, interpretar as restrições de forma mais focada. Segundo os dados coletados, no mínimo 20% das mães casadas e não casadas não trabalham fora de casa 232 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? porque elas não têm quem cuide de seus filhos. Já os pais – sempre analisando os pais casados – não têm esse problema. 35% das mães não casadas, 18% das mães casadas e 50% dos pais casados – e não empregados – estão à procura de trabalho, mas não conseguem encontrá- lo. Finalmente, apenas 43% das mães não casadas – e não empregadas – não se encontram empregadas por escolha própria de permanecer fora do mercado de trabalho, enquanto o percentual é 50% superior para as mães casadas. Dos homens que não estavam trabalhando durante a pesquisa, 50% dos pais18 se enquadram nesta categoria. Das mães casadas cujas respostas são agrupadas na categoria de preferência ou opção por não trabalhar, 56% preferem ficar em casa com seus filhos, enquanto que, para 15,% delas, foi seu marido quem manifestou a preferência de que ela ficasse em casa com as crianças e 2% ainda estão estudando. Para as mães não casadas, entre as que se encaixam na categoria de preferência própria, 53% preferem ficar em casa com seus filhos, enquanto 24% estão estudando. Nenhuma mãe não casada optou por ficar em casa porque os salários são muito baixos, enquanto que 2% das mães casadas se encontram nesta categoria.19 Vários pontos são dignos de nota. Em primeiro lugar, a diferença de empregabilidade global entre mães casadas e não casadas é quase inteiramente composta devido à escolha das mães casadas em ficar de fora do mercado de trabalho, em grande parte impulsionada pela preferência de seus maridos. Presumivelmente, as mães não casadas, com suas rendas familiares significativamente inferiores às famílias com mães e pais residentes, não têm a mesma opção. Em segundo lugar, os níveis mais elevados de emprego entre as mães não casadas, comparados às mães casadas são devidos ao engajamento em trabalhos informais ao invés da participação do mercado formal de trabalho. Se considerarmos esta uma proxy para a qualidade do emprego, parece que as mães não casadas se sujeitam a aceitar empregos de menor qualidade do que as mães casadas. Finalmente e mais impressionante, mais de metade das mães não casadas que não trabalham (58%) e quase metade das mães casadas que também não trabalham (38%) estão desocupadas por duas restrições: elas estão procurando, mas não conseguem encontrar trabalho ou não têm acesso a cuidados com seus filhos e, por isso, não podem trabalhar. Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 233 REGINA MADALOZZO E MERIKE BLOFIELD (quer trabalhassem ou não), as mães também percebiam o conflito entre família e trabalho de forma mais intensa; os grupos focais formados por mulheres gastavam grande parte do tempo das entrevistas na discussão a respeito do conforto de suas famílias, enquanto, entre os homens, as respostas mais enfáticas tinham como temática o trabalho. Os homens declaravam a percepção de que a responsabilidade com as crianças era das mães, eles somente “ajudavam”. Um dos entrevitados declarou, inclusive, concordar que sua esposa trabalhasse fora de casa desde que ela também conseguisse manter o gerenciamento das responsabilidades com a casa e a família. 20 Para garantir a confiabilidade da codificação, as respostas foram codificadas individualmente por duas pesquisadoras e a correlação de 95% foi auferida. Em seguida, as respostas não similares foram reanalisadas e categorizadas via consenso. Na pesquisa, uma pergunta com resposta aberta foi incluída para avaliar se o nascimento dos filhos tinha afetado a relação do pai/mãe no mercado de trabalho e, em caso positivo, de qual forma. Independentemente de estarem ou não casadas ou empregadas, mais da metade das mães (55%) responderam que sua relação com o mercado de trabalho mudou depois de terem filhos, enquanto somente 21% dos pais disseram ter percebido essa mudança, sendo que essa resposta se refere somente aos pais residentes no domicílio.20 Alguns indivíduos se referem às tentativas de equilibrar as demandas do trabalho com as da família e não é possível perceber se a indicação é de aumento ou redução de um dos lados. Essas respostas foram classificadas como alterações neutras. Segundo os dados coletados, 7% dos pais (33% dos pais que perceberam mudança na relação com o trabalho após o nascimento dos filhos) e 6% das mães (10% das mães que perceberam alterações na relação trabalhista após o nascimento dos filhos) encontram- se nessa categoria. As afirmações vão desde a percepção de que o trabalho exige um tempo de dedicação que poderia ser dado aos filhos, mas que é compensado pelo retorno financeiro, até declarações de que a escolha de local de trabalho foi afetada. Uma segunda categoria é daqueles indivíduos que perceberam alterações negativas na relação trabalhista. Novamente, 7% dos homens (33% dos que perceberam mudanças no mercado de trabalho) encaixam-se nessa definição. Já, entre as mulheres, 48% delas (o que representa 88% das que perceberam alterações na relação de trabalho) estão nessa categoria. 20 Para garantir a confiabilidade da codificação, as respostas foram codificadas individualmente por duas pesquisadoras e a correlação de 95% foi auferida. Em seguida, as respostas não similares foram reanalisadas e categorizadas via consenso. COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? é Um segundo ponto interessante é o exame da autoavaliação com relação às mudanças do mercado de trabalho após o nascimento do(s) filho(s). Desde a condução dos grupos focais foi possível perceber que, mesmo que trabalhar fora de casa fosse algo valorizado por muitas mães 233 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 233 REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD 234 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? Por fim, uma parte dos entrevistados percebe altera- ções positivas no mercado de trabalho: 7% dos homens e 1% das mulheres afirmam que a relação trabalhista melhorou após o nascimento dos filhos. Os homens, de forma geral, afirmam que a paternidade os fez mais responsáveis e que os patrões perceberam e os recompensam por isso. Já as 4 mulheres que apontam melhoras no mercado de trabalho falam sobre a motivação extra que os filhos dão para que elas permaneçam no mercado e tenham melhora financeira para a família. REGINA MADALOZZO E MERIKE BLOFIELD As afirmações dos homens estão mais frequentemente relacionadas ao fato de que se preocupam com o bem-estar dos filhos enquanto no trabalho. Já as mulheres também declaram essa preocupação como efeito negativo na relação trabalhista, mas, em maior número, fazem afirmações a respeito de tratamento discriminatório com relação aos empregadores que temem contratar ou manter empregadas com filhos pequenos. 234 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 235 REGINA MADALOZZO E MERIKE BLOFIELD os empregadores supõem que essas mulheres incorrerão em maiores taxas de absenteísmo do que o restante da popula- ção. Já para os pais, o efeito é inverso: alguns empregadores preferem contratar pais por considerá-los mais responsáveis. A dificuldade enfrentada por essas mães para participar do mercado de rabalho tem impacto direto na renda familiar e, consequentemente, na pobreza, pois uma família com dois provedores de renda tem menor probabi- lidade de ser pobre do que com somente um (CEPAL, 2009). Além disso, a dificuldade para garantir o cuidado para as crianças e ser capaz de gerar renda aumenta o custo em termos de estresse e qualidade de vida para as mães, algo que afeta muito menos os pais. A prescrição de política pública mais imediata que surge desse estudo é a necessi- dade de um aumento dramático da corresponsabilidade do Estado com a provisão de cuidados com crianças e aumento dessa provisão em tempo integral. Essa política, por si só, já teria um efeito na redução da discriminação por parte dos empregadores ao longo do tempo. os empregadores supõem que essas mulheres incorrerão em maiores taxas de absenteísmo do que o restante da popula- ção. Já para os pais, o efeito é inverso: alguns empregadores preferem contratar pais por considerá-los mais responsáveis. os empregadores supõem que essas mulheres incorrerão em maiores taxas de absenteísmo do que o restante da popula- ção. Já para os pais, o efeito é inverso: alguns empregadores preferem contratar pais por considerá-los mais responsáveis. A dificuldade enfrentada por essas mães para participar do mercado de rabalho tem impacto direto na renda familiar e, consequentemente, na pobreza, pois uma família com dois provedores de renda tem menor probabi- lidade de ser pobre do que com somente um (CEPAL, 2009). Além disso, a dificuldade para garantir o cuidado para as crianças e ser capaz de gerar renda aumenta o custo em termos de estresse e qualidade de vida para as mães, algo que afeta muito menos os pais. A prescrição de política pública mais imediata que surge desse estudo é a necessi- dade de um aumento dramático da corresponsabilidade do Estado com a provisão de cuidados com crianças e aumento dessa provisão em tempo integral. Essa política, por si só, já teria um efeito na redução da discriminação por parte dos empregadores ao longo do tempo. Agradecimentos Agradecimentos Agradecimentos Agradecimentos Agradecimentos As autoras agradecem o suporte financeiro da FAPESP que permitiu a coleta dos dados utilizados nessa pesquisa (processo 12/09609-5). Merike Blofield agradece o suporte financeiro através de bolsa para pesquisador estrangeiro, também concedida pela FAPESP (processo 2011/16171-3). Regina Madalozzo agradece a Bolsa de Produtividade de Pesquisa do CNPq (processo 302885/2012-9). Ambas as pesquisadoras agradecem a Matheus Hector Garcia pelo auxílio e disponibilidade na confecção dos gráficos. Conclusões Conclusões Conclusões Conclusões Conclusões A presente pesquisa é fruto de um trabalho de campo realizado em 2012 em bairros predominantemente de baixa renda em São Paulo. Com base teórica na teoria de preferências e dos conflitos, a pesquisa foi fundamentalmente focada em famílias com presença de crianças menores de 6 anos de idade e em questões que evidenciassem as escolhas com relação ao trabalho remunerado e não remunerado, bem como a divisão entre os gêneros das responsabilidades financeiras e familiares. Os resultados dessa pesquisa indicam que as mulhe- res, conforme esperado, ainda suportam um peso despropor- cinoal na responsabilidade pelo cuidado das crianças e, no caso das mães não casadas, também com relação ao sustento da família. As obrigações de cuidado são distri- buídas de forma desigual entre os pais, mesmo quando as mães trabalham fora de casa e essas obrigações acabam por restringir a possibilidade de trabalho delas, mesmo quando o desejam. A falta de de creches e pré-escolas com custo baixo ou pagas pelo governo tem um impacto direto na decisão das mães em participar (ou não) do mercado de trabalho. Nesse sentido, o presente estudo complementa o estudo de Sorj, Adriana FONTES e Danielle Carusi MACHADO (2007), que analisou dados da PNAD (IBGE) de 1992 a 2005 e constataram a dificuldade de acesso a serviços de apoio à participação de mulheres com filhos no mercado de trabalho. O conflito relatado por essas pesquisadoras é mais uma vez constatado, agora em uma amostra focada em bairros de baixa renda da cidade de São Paulo. Por ter sido uma pesquisa específica para o tema, foi possível, também, constatar que, além da dificuldade de conciliar as responsabilidades familiares com a participação no mercado de trabalho, que já havia sido levantadas por estudos anteriores, ainda existe uma discriminação anterior à contratação de mães de crianças pequenas que faz com que as taxas de desemprego para essa população específica seja ainda mais elevada, especialmente porque 235 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 235 REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD 236 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 Referências Referências Referências Referências Referências ARNOLD, Jens; JALLES, João. “Dividing the Pie in Brazil: Income Distribution, Social Policies and the New MiddleClass”. OECD Economics Department Working Papers, n. 1105, 2014. OECD Publishing. Disponível em: http://dx.doi.org/ 10.1787/5jzb6w1rt99p-en. BANCO Mundial. Databank, 2013. Disponível em: http:// data.worldbank.org/indicator/SL.TLF.CACT.FE.ZS?locations =BR. Acesso em: 05/12/2016. BARKER, Garry; CONTRERAS, Juan Manuel; HEILMAN, Brian; SINGH, Ajay; VERMA, Ravi. “Evolving Men: Initial Results from the International Men and Gender Equality Survey (IMAGES)”. Men, Boys and Gender Equality. Washington: International Center for Research on Women (ICRW); Rio de Janeiro: Instituto Promundo, jan. 2011. Disponível em: http:/ /menandboys.ids.ac.uk/files/evolving-men-initial-results- international-men-and-gender-equality-survey-images-0. 236 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? BERLINSKI, Samuel; GALLIANI, Sebastian e McEWAN, Patrick. “Preschool and Maternal Labor Market Outomces - Evidence form a Regression Discontinuity Design”. IFS Working Paper W9/5, Institute for Fiscal Studies, 2008. BLOFIELD, Merike. Care Work and Class: Domestic Workers’ Struggle for Equal Rights in Latin America. College Park: Pennsylvania State University Press, 2012. BRUSCHINI, Maria Cristina Aranha e RICOLDI, Arlene Martinez. “Família e Trabalho: difícil conciliação para mães traba- lhadoras de baixa renda”. Cadernos de Pesquisa, v. 39, n. 136, p. 93-123, 2009. BRUSCHINI, Cristina. “Trabalho doméstico: inatividade econô- mica ou trabalho não remunerado?”. Revista Brasileira de Estudos Populacionais, v. 23, n. 2, p. 331-353, 2006. BUDIG, Michelle; ENGLAND, Paula. “The Wage Penalty for Motherhood”. American Sociological Review, v. 66, p. 204- 225, 2002. CHIODA, Laura. Work & family: Latin America and Caribbean Women in search of new balance Conference Edition. Washington: World Bank, 2011. CHRISTIANSEN, Shawn L.; PALKOVITZ, Rob. “Why the ‘Good Provider’ Role Still Matters: Providing as a Form of Paternal Involvement”. Journal of Family Issues, v. 22, n. 1, p. 84- 106, 2001. CONTRERAS, Dante; SEPULVEDA, Paulina; CABRERA, Soledad. The Effects of Lengthening the School Day on Female Labor Supply: Evidence from a Quasi-Experiment in Chile. Departamento de Economía, Facultad Economía e Negocios, Universidad de Chile, 2010. (Serie Documento de Trabajo) DE OLIVEIRA, Pedro Rodrigues; SCORZAFAVE, Luiz Guilherme; PAZELLO, Elaine Toldo. “Desemprego e inatividade nas metropoles brasileiras: As diferencas entre homens e mulheres”. Nova Economia, v. 19, n. 2, p. 291-324, 2009. DIEESE. “O emprego doméstico no Brasil”. Estudos e Pesquisas, n. 68, 2013. Disponível em: http://www.dieese.org.br/ estudosetorial/2013/estPesq68empregoDomestico.pdf. Acesso em: 05/12/2016. ESPING-ANDERSEN, Gøsta. The Incomplete Revolution: Adapting to Women’s New Roles. Cambridge: The Polity Press, 2009. FERREIRA, Aurelio Buarque de Holanda. Novo Dicionário Aurelio da Língua Portuguesa, 2.ed. Rio de Janeiro: Nova Fronteira,1986. GANGLE, Markus; ZIEFLE, Andrea. “Motherhood, Labor Force Behavior, and Women’s Careers: An Empirical Assessment of the Wage Penalty for Motherhood in Britain, Germany, and the United States”. Demography, v. 46, p. 341-369, 2009. 237 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 237 REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD 238 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? COMO FAMÍLIAS DE BAIXA RENDA EM SÃO PAULO CONCILIAM TRABALHO E FAMÍLIA? Washington: International Food Policy Research Institute, 2003. ROSA, Waldemir. “Sexo e cor: categorias de controle social e reprodução das desigualdades socioeconômicas no Brasil”. Revista Estudos Feministas, v. 17, n. 3, p. 889-899, 2009. SOARES, Cristiane; SABOIA, Ana Lucia. Tempo, trabalho e afazeres domésticos: um estudo com base nos dados da Pesquisa Nacional por Amostra de Domicílios de 2001 e 2005. Texto para Discussão do IBGE – Diretoria de Pesquisas, n. 21. Ministério do Planejamento, Orçamento e Gestão. Instituto Brasileiro de Geografia e Estatística – IBGE. Diretoria de Pesquisas. Coordenação de População e Indicadores Sociais. Rio de Janeiro, 2007. Disponível em: http://www.ibge.com.br/home/estatistica/populacao/ tempo_trabalho_afdom_pnad2001_2005.pdf. Acesso em: 06/05/2016. SORJ, Bila. “Arenas de cuidado nas interseções entre gênero e classe social no Brasil”. Cadernos de Pesquisa, São Paulo, v. 43, n. 149, mai./ago. 2013. SORJ, Bila; FONTES, Adriana; MACHADO, Danielle Carusi. “Políticas e práticas de conciliação entre família e trabalho no Brasil”. Cadernos de Pesquisa, v. 37, n. 132, p. 573-594, 2007. STÄHLI, Michèle Ernst; LE GOFF, René Levy; WIDMER, Eric. “Wishes or Constraints? Mothers’ Labour Force Participation and its Motivation in Switzerland”. European Sociological Review, v. 25, n. 3, p. 333-348, 2009. UNITED Nations Development Programme and the International Labour Organization (PNUD-OIT). Trabajo y Família: Hacia nuevas formas de conciliación con corresponsabilidad social. Geneva: UNDP-ILO, 2009. [Recebido em 22/02/2015, reapresentado em 10/05/2016 e aceito para publicação em 23/06/2016] [Recebido em 22/02/2015, reapresentado em 10/05/2016 e aceito para publicação em 23/06/2016] REGINA MADALOZZO E MERIKE BLOFIELD REGINA MADALOZZO E MERIKE BLOFIELD GUEDES, Maria Eunice Figueiredo. “Gênero, o que é isso?”. Psicologia: Ciência e Profissão, v. 15, n. 1-3, p. 4-11, 1995. HALLMAN, Kelly; QUISUMBING, Agnes R.; RUEL, Marie; LA BRIÈRE, Benedicte de. “Mothers’ work and childcare: Findings from the urban slums of Guatemala City”. Economic Development and Cultural Change, v. 53, n. 4, p. 855- 885, 2005. HEYMANN, Jody. Forgotten Families Ending the Growing Crisis Confronting Children and Working Parents in the Global Economy. Oxford: Oxford University Press, 2006. HEYMANN, Jody. Forgotten Families Ending the Growing Crisis Confronting Children and Working Parents in the Global Economy. Oxford: Oxford University Press, 2006. HIRATA, Helena; GUIMARÃES, Nadya Araujo Guimarães (Eds.). Cuidado e cuidadoras: as várias faces do trabalho do Care. São Paulo: Atlas, 2012. HIRATA, Helena; GUIMARÃES, Nadya Araujo Guimarães (Eds.). Cuidado e cuidadoras: as várias faces do trabalho do Care. São Paulo: Atlas, 2012. HIRATA, Helena. “Gênero, classe e raça: intereseccionalidade e consubstancialidade das relações sociais”. Tempo Social, v. 26, n. 1, p. 61-73, 2016. IBGE. Instituto Brasileiro de Geografia e Estatística. Disponível em: www.ibge.gov.br. IBGE. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional por Amostra de Domicílios, 2013. Disponível em: www.ibge.gov.br. KELLY, Kimberly and GRANT, Linda. “Penalties and premiums: The impact of gender, marriage, and parenthood on faculty salaries in science, engineering and mathematics (SEM) and non-SEM fields”. Social Studies of Science, v. 42, Issue 6, p. 869-896, 2012. KILLERWALD, Alexandra. “A Reconsideration of the Fatherhood Premium: Marriage, Coresidence, Biology, and Fathers’ Wages”. American Sociological Review, v. 78, Issue 1, p. 96-116, 2013. MADALOZZO, Regina; MARTINS, Sérgio Ricardo; SHIRATORI, Ludmila. “Participação no Mercado de Trabalho e no Trabalho Doméstico: homens e mulheres têm condições iguais?”. Revista Estudos Feministas, v. 18, n. 2, p. 547- 566, 2010. MARSIGLIO, William; AMATO, Paul R.; DAY, Randal D.; LAMB, Michael E. “Scholarship on Fatherhood in the 1990s and Beyond”. Journal of Marriage and Family, v. 62, p. 1173- 1191, 2000. MEDRANO, Patricia. Public Day Care and Female Labor Force Participation: Evidence from Chile. Santiago de Chile: Facultad de Economía y Negocios Universidad de Chile, 2009. [Serie documentos de trabajo 306] NERI, Marcelo Cortes. Os emergentes dos emergentes: reflexões e ações para a nova classe média brasileira. Rio de Janeiro: Fundação Getulio Vargas, 2011. QUISUMBING, Agnes R. Household Decisions, Gender, and Development: A Synthesis of Recent Research. How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? How low-income families in São Paulo reconcile work and family? Abstract: Abstract: Abstract: Abstract: Abstract: While women’s labor force participation rates have increased in Brazil, unpaid care responsibilities remain unequally divided between mothers and fathers. Based on an original, representative survey of 700 mothers and fathers of young children in low-income neighborhoods in São Paulo, this article examines the gender differences in labor force participation, and its relationship to access to child care and family caregiving responsibilities. The analysis concludes that work-family conflicts and responsibility for children fall disproportionately on mothers, regardless of whether they prefer to remain active in the labor market or not. Policies that promote paternal and state co-responsibility are essential to reduce this conflict. Key words: Key words: Key words: Key words: Key words: family; labor market; gender; low-income families Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 239 240 Estudos Feministas, Florianópolis, 25(1): 215-240, janeiro-abril/2017 REGINA MADALOZZO E MERIKE BLOFIELD RRRRRegina Madalozzo egina Madalozzo egina Madalozzo egina Madalozzo egina Madalozzo (reginam@insper.edu.br). PhD em Economia pela University of Illinois at Urbana-Champaign. Professora Associada no Insper, onde atua desde 2002. Merike Blofield Merike Blofield Merike Blofield Merike Blofield Merike Blofield (m.blofield@miami.edu). University of Miami, Estados Unidos da América do Norte. É professora associada do departamento de Ciências Políticas da University of Miami. Nativa da Finlância, ela já residiu no Canadá, Chile, Brasil, Argentina e nos Estados Unidos.
https://openalex.org/W2560453537	https://figshare.com/ndownloader/files/7013777	English	null	The C-Reactive Protein to Albumin Ratio as a Predictor of Severe Side Effects of Adjuvant Chemotherapy in Stage III Colorectal Cancer Patients	PloS one	2,016	cc-by	83	Supplementary Table 1: Details of side effects greater than grade 3 Supplementary Table 1: Details of side effects greater than grade 3 1 Signs and symptoms Neutropenia 15 Anorexia 6 Diarrhea 5 Hyperbilirubinemia 2 Anaphylaxis 1 Perforation 1 Acute leukoencephalopathy 1 Liver dysfunction 1 Hand-foot syndrome 1 1 Signs and symptoms Neutropenia 15 Anorexia 6 Diarrhea 5 Hyperbilirubinemia 2 Anaphylaxis 1 Perforation 1 Acute leukoencephalopathy 1 Liver dysfunction 1 Hand-foot syndrome 1 Signs and symptoms General fatigue 1 Pneumonia 1 Pneumonia Pneumonia 2
https://openalex.org/W2102535343	https://www.animbiosci.org/upload/pdf/144.pdf	English	null	Effect of Dietary Vitamin E on Growth Performance and Immune Response of Breeder Chickens	Asian-Australasian journal of animal sciences	2,006	cc-by	6,130	* Corresponding Author: S. J. Chang. Department of Life Sciences, National Cheng Kung University, Tainan 70101, Taiwan, ROC. Tel: +886-6-2757575(65501), Fax: +886-6- 2742583, E-mail: sjchang@mail.ncku.edu.tw Received November 18, 2005; Accepted February 1, 2006 884 884 INTRODUCTION levels of vitamin E for breeder chickens are assessed ultimately in terms of reproductive performance, the allowance for optimal immunocompetence is not included. Dietary vitamin E was found to improve reproduction and antioxidant capability of breeder chickens (Lin et al., 2004; Lin et al., 2005a, b), but very few information is currently available on the effect of vitamin E supplement on the immune response. Considering vitamin E content in the ingredients, irrespective of degradation during storage, corn-soybean meal diet without supplemental vitamin E is supposed to meet or exceed the NRC (1994) recommended level of vitamin E. Egg production, egg fertility and hatchability are among the most important economic traits for chicken breeder farms. Failure of health maintenance may cause great economic loss. Although mass vaccination against Newcastle disease (ND), infectious bursal disease (IBD) and infectious bronchitis (IB) has been practiced throughout the commercial poultry industry, outbreaks of those diseases still occur occasionally. Hence, vaccination responses and enhancement of immune system are matters of great concern. Vitamin E is primarily known as an antioxidant in reducing cellular free radical damage. In addition, vitamin E may affect the development and maintenance of immunocompetence through multiple functions by acting directly on the immune cell or by indirectly altering metabolic and endocrine parameters, which in turn influence immune function (Gershwin et al., 1985). Vitamin E supplementation in mammals has been shown to increase production of interleukin (IL)-2, leading to enhanced proliferation of T cells, and reduce production of prostaglandin E, a T-cell suppressive factor, as a result of a decreased peroxynitrite formation (Meydani et al., 2005). Previous reports on effects of vitamin E on immune response of chickens were few and not consistent (Marsh et al., 1986; Ritcher et al., 1985, 1986; Lohakare et al., 2005). The purpose of this study, thus, was to evaluate whether long-term feeding of corn-soybean meal diet without supplemental vitamin E might impair immunity and whether supplementation of vitamin E enhances immunologic response to sheep red blood cell (SRBC) and different commercial vaccines in breeder chickens. Effect of Dietary Vitamin E on Growth Performance and Immune Response of Breeder Chickens Y. F. Lin and S. J. Chang* Y. F. Lin and S. J. Chang* Division of Technical Service, Livestock Research Institute, Council of Agriculture, Hsinhua, Tainan 71246, Taiwan, ROC ABSTRACT : The effect of dietary vitamin E supplementation on immune responses was studied in breeder chickens during the maturing period. In experiment 1, 17-week old female birds were fed corn-soybean meal based diets supplemented with either 0, 40, 80, 120, or 160 mg vitamin E (all-rac-α-tocopherol acetate)/kg diet for 19 weeks. In experiment 2, 23-week old male birds were fed the corn-soybean meal based diet supplemented with either 0, 20, 40, 80 or 160 mg vitamin E/kg diet for 8 weeks. The chickens were evaluated for growth performance, antibody titer to sheep red blood cell (SRBC), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV), and skin response to phytohemagglutinin-P (PHA-P). The results showed that supplemental vitamin E improved body weigh gain of laying pullets during peak-laying period but had no significant effect on growth performance of cockerels. For cockerels, addition of 20 mg vitamin E/kg diet significantly enhanced (p<0.05) immune response to SRBC compared to those added with 0, 80 and 160 mg vitamin E/kg diet; addition of 20 mg vitamin E/kg diet had higher (p<0.01) antibody titer to IBDV than those added with 40-160 mg vitamin E/kg diet. No significant effects on immune response were observed in laying pullets fed supplemental vitamin E. The findings suggest that moderate supplementation of vitamin E may enhance immune responses to selective antigens in cockerels but excessive vitamin E may depress specific immune response. (Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 6 : 884-891) Key Words : Vitamin E, Growth, Sheep Red Blood Cell, Vaccines, Antibody Titer, Immune Response, Breeder Chickens Vaccination program and antigen challenge Vaccinations were performed under standard vaccination programs implemented at the Livestock Research Institute for Taiwan native breeder chickens. All birds were vaccinated with Marek’s disease (MD), Newcastle disease (ND), infectious bursal disease (IBD) and infectious bronchitis (IB) vaccines (Fort Dodge Laboratories, Iowa 50616, USA) at different ages as described in Table 2. Killed vaccines were administered by intramuscular injection and live vaccines were administered by eye drop method. Vaccines were handled according to the recommendations of the manufacturer. All birds in the flock received the same dose of vaccine at the same time. Experimental animals The National Research Council (1994) recommended Experiment 1 was conducted using two-way cross (line 7×11) female Taiwan native breeder chicks, and two-way cross (line 9×12) male Taiwan native breeder chicks were employed for experiment 2 (Livestock Research Institute, Council of Agriculture, Hsinhua, Tainan 712, Taiwan). VITAMIN E ON IMMUNE RESPONSE OF CHICKENS 885 Table 1. Composition of basal diets and weeks fed in experiment 1 and 2 Growing period (Exp. 1 and 2) Laying period (Exp. 1) Mature period (Exp. 2) Ingredients 0 to 6 wk 6 to 12 wk Over 12 wk Over 17 wk Over 23 wk Ground yellow corn (%) 64.6 65.1 70.9 67.0 68.0 Soybean meal (43.5%) (%) 25.0 25.0 16.0 12.9 9.0 Fish meal (64%) (%) 3.0 0.0 0.0 4.0 0.0 Wheat bran (%) 4.5 6.8 10.0 6.4 20.0 Dicalcium phosphate (%) 1.2 1.3 1.3 1.1 1.4 Calcium carbonate (%) 1.2 1.3 1.3 8.1 1.1 Salt (%) 0.3 0.3 0.3 0.3 0.3 Vitamin premix1 (%) 0.1 0.1 0.1 0.1 0.1 Mineral premix2 (%) 0.1 0.1 0.1 0.1 0.1 Total (%) 100.0 100.0 100.0 100.0 100.0 Calculated analysis Protein (%) 18.6 17.1 14.1 14.5 12.4 Metabolizable energy (kcal/kg) 2,858 2,827 2,862 2,719 2,739 Calcium (%) 0.92 0.88 0.85 3.53 0.79 Available phosphorus (%) 0.42 0.37 0.35 0.40 0.37 Total sulfur amino acids (%) 0.66 0.59 0.52 0.55 0.47 Linoleic acid (%) 1.60 1.65 1.79 1.64 1.87 Selenium (mg/kg) 0.12 0.10 0.12 0.14 0.04 Vitamin E (mg/kg) 15.5 15.8 17.3 16.1 17.8 1 Provided per kilogram of diet: vitamin A (retinyl acetate), 1.9 mg; cholecalciferol, 27.5 µg; thiamine, 1.1 mg; riboflavin, 4.4 mg; pyridoxine 2.2 mg; vitamin B12 6.6 µg; menadione, 1.1 mg; biotin 0.11 mg; folic acid, 0.55 mg; niacin 44 mg; pantothenic acid, 12 mg. 2 Provided per kilogram of diet: iron, 64 mg; copper, 12 mg; manganese, 64 mg; cobalt, 0.2 mg; zinc, 40 mg; iodine, 0.68 mg. Table 1. Composition of basal diets and weeks fed in experiment 1 and 2 Provided per kilogram of diet: vitamin A (retinyl acetate), 1.9 mg; cholecalciferol, 27.5 µg; thiamine, 1.1 mg; riboflavin, 4 vitamin B12 6.6 µg; menadione, 1.1 mg; biotin 0.11 mg; folic acid, 0.55 mg; niacin 44 mg; pantothenic acid, 12 mg. 2 Provided per kilogram of diet: iron, 64 mg; copper, 12 mg; manganese, 64 mg; cobalt, 0.2 mg; zinc, 40 mg; iodine, 0.68 mg. provided in meal form. Experimental animals To minimize vitamin E interactions with other nutrients, selenium, synthetic sulfur amino acids or oils were not added to the basal diets (Lin et al., 1989; Levander 1992), because the base ingredient composition was considered sufficient to meet the minimum dietary requirements of native chickens. Birds from each of the five treatment groups were fed the basal diet supplemented with five graded levels of vitamin E (all-rac-α-tocopherol acetate) (Roche, F. Hoffmann-La Roche Ltd, 4002 Basel, Switzerland), respectively as described in Table 2. Three hundred day-old female chicks in Experiment 1, and 90 day-old male chicks in Experiment 2 were reared in concrete floor pens padded with rice hull litter in an open- sided growing house till 16 weeks and 22 weeks of age, respectively. At 16 weeks and 22 weeks of age, birds from the flock were randomly allotted to five treatment groups and moved into the open-sided cage breeder house. Each bird was housed in an individual cage measuring 36×25×39 cm for females and 47×32×61 cm for males. Each treatment group, containing 60 pullets in Experiment 1, and 18 cockerels in Experiment 2, was equally separated into three experimental units (replicates). Units were randomly distributed to minimize the cage effect. One feeder trough was used for a unit (replicate) of 20 birds for females and 6 birds for males. Feed and water were supplied ad libitum. All experimental procedures were approved by the Laboratory Animal Management Committee of the Livestock Research Institute. provided in meal form. To minimize vitamin E interactions with other nutrients, selenium, synthetic sulfur amino acids or oils were not added to the basal diets (Lin et al., 1989; Levander 1992), because the base ingredient composition was considered sufficient to meet the minimum dietary requirements of native chickens. Birds from each of the five treatment groups were fed the basal diet supplemented with five graded levels of vitamin E (all-rac-α-tocopherol acetate) (Roche, F. Hoffmann-La Roche Ltd, 4002 Basel, Switzerland), respectively as described in Table 2. Where, post PHA-P inj. = thickness of left wattle 24 h after injection of 0.1 ml PHA-P; pre PHA-P inj. = thickness of left wattle before injection; Antibody titer to NDV was measured using haemagglutination inhibition (HI) technique. Twenty five µl of serum containing antibody was serially diluted into 96- well ground bottom plate with phosphate buffer saline (PBS). The same volume of virus antigen was added to react and bind with the antibody. Addition of 2% red blood cell (RBC) solution in each well will show the ability of ND virus left to agglutinate with RBC. If there was enough antibody bound to virus during the incubation period, haemagglutination would be inhibited completely. The titers were expressed as log2 of the reciprocal of the last serum dilution showing haemagglutation inhibition. post PBS inj. = thickness of right wattle 24 h after injection of 0.1 ml PBS; pre PBS inj. = thickness of right wattle before injection. Diets and experimental design Based on the guidelines suggested by NRC (1994) and the Extension Booklet for Taiwan Native Chicken (1995), basal corn-soybean meal diets (calculated vitamin E contents have been shown in Table 1) were formulated to meet the nutrient requirements for Taiwan native breeder chickens during different periods, except that vitamin E was omitted from the vitamin premix (Table 1). The premixes were stored in airtight container at -20°C until being mixed with the diets. All diets were prepared biweekly and Twelve birds in each treatment group were randomly chosen and injected intraperitoneally with 0.5 ml/bird of 5% SRBC diluted in phosphate-buffered saline (PBS) at 34 weeks of age in Experiment 1 and 26 and 30 weeks of age in Experiment 2 (Table 2). The birds injected with SRBC at 886 LIN AND CHANG Table 2. Experimental design and vaccination and challenge schedule1 Parameter Experiment 1 Experiment 2 Vitamin E added ------------------------------ mg/kg of diets -------------------------------------- Growing period none none Laying (mature) period 0, 40, 80, 120, 160 0, 20, 40, 80, 160 Vaccination2 ------------------------------------ age ------------------------------------------ MD 1 day 1 day ND (B1, live) 4 days 4 days IBD (Lukert, live) 1 week, 3 weeks 1 week, 3 weeks ND (Lasota, live) 2 weeks 2 weeks ND (Lasota, killed) 4 weeks, 28 weeks 4 weeks, 28 weeks ND+IB+IBD (killed) 14, 29 weeks 14, 29 weeks Antigen ------------------------------------ age ------------------------------------------ SRBC 34 weeks 26, 30 weeks PHA 36 weeks none 1 MD = Marek’s disease; ND = Newcastle disease; IBD = infectious bursal disease; IB = infectious bronchitis; SRBC = sheep red blood cell; PHA = phytohemagglutinin 2 ND: Kimber; IB: Massachusetts M41; IBD: Lukert. different ages in Experiment 2 were not reused to avoid secondary response. After 7 days, blood samples were collected and sera from each sample were stored at -20°C for subsequent measurement of antibody titer to SRBC and the vaccines. et al., 1984), according to manufacturer’s instructions. Twenty µl of 100-fold buffer diluted serum from each chicken were used and read at 650 nm in a microplate reader. Cell-mediated immune response Cell-mediated response was measured by reaction to phytohaemagglutinin-P (PHA-P) at 36 weeks of age in Experiment 1. Twelve birds from each treatment group were randomly chosen and intradermally injected with 0.1 ml PHA-P (Sigma chemical Co., St. Louis, MO 63178) at left wattle. Besides, the other wattle (right) was also injected with the same volume of PBS as control. The accumulation of lymphocytes which stand for cell-mediated immune response will reflect in the swelling of injected skin. The PHA-P skin test of chicken was considered to be a thymus-dependent response using swelling of wattle caused by PHA-P injection (Goto et al., 1978). Wattle thickness was measured using a pressure-sensitive meter. Cell- mediated immune response (wattle index) was calculated as the difference between the PHA-P and PBS (control) injected sites. Wattle response to PHA-P, in millimeters, was calculated as follows: Measurement of humoral immune response PHA-P value = (post PHA-P inj.-pre PHA-P inj.) -(post PBS inj.-pre PBS inj.), PHA-P value = (post PHA-P inj.-pre PHA-P inj.) -(post PBS inj.-pre PBS inj.), Antibody response to SRBC : Sheep red blood cells were used as T-dependent antigen to quantify the antibody response. The antibody assay procedures described by Munns and Lamont (1991) were followed. The agglutination titer was expressed as the log2 of the highest titer giving 50% agglutination. Where, Weight gain and packed cell volume (PCV) Weight gain and PCV were used as indicators to evaluate nutritional status in chickens. Body weight and weight gain for each female and male bird were recorded every 2 to 3 weeks beginning at the age of 17 and 23 weeks, respectively. Hematocrits were measured at 31 weeks of age in Experiment 1 and 27 and 31 weeks of age in Experiment 2. Blood samples were collected into haparinized Antibody titer to IBDV and IBV were determined by ELISA kits (IDEXX Inc., Westbrook, ME 04092) (Snyder VITAMIN E ON IMMUNE RESPONSE OF CHICKENS 887 Table 3. Effect of supplemental vitamin E on body weight (BW) and body weight gain (BWG) of Taiwan native breeder chickens1 Added vitamin E2 (mg/kg) Variables Wks post- adding Vit. E 0 20 40 80 120 160 SEM p -------------------------------- Experiment 1 (female) -------------------------- BW (g) Wk 17 0 1,382 1,399 1,389 1,386 1,371 19.1 0.89 Wk 26 9 1,534 - 1,557 1,568 1,579 1,530 25.7 0.62 Wk 35 18 1,572 - 1,588 1,636 1,668 1,624 30.6 0.19 BWG, g Wk 17 to 26 0 to 9 153 - 158 180 193 160 17.1 0.42 Wk 26 to 35 9 to 18 37.6bc - 31.1c 67.6abc 89.0ab 97.3a 14.5 <0.01 -------------------------------- Experiment 2 (male) -------------------------- BW Wk 23 0 2,053 2,089 2,043 2,027 - 2,032 50.6 0.92 Wk 27 4 2,244 2,232 2,312 2,259 - 2,295 55.0 0.82 Wk 31 8 2,330 2,312 2,384 2,348 - 2,379 58.5 0.89 BWG (g) Wk 23 to 27 0 to 4 191 143 269 232 264 32.6 0.04 Wk 27 to 31 4 to 8 86.1 80.1 72.8 89.1 83.6 23.3 0.99 a, b Values in a row without the same superscript are significantly different (p<0.05). 1 Composition of basal diets and experiment design in experiment 1 and 2 are described as Table 1 and 2. min E on body weight (BW) and body weight gain (BWG) of Taiwan native breeder chickens1 2 Table 4. Effects of supplemental vitamin E on antibody titers in response to sheep red blood cell (SRBC) and packed cell volume (PCV) of Taiwan Native breeder cockerels1 (Experiment 2) (n =12) Diets (added vitamin E, mg/kg) Variables Age (wks) Wks post - adding vit. Weight gain and packed cell volume (PCV) E 0 20 40 80 160 Age x 3 SRBC2 (log2) 27 4 3.59 4.68 4.08 3.82 4.10 4.05 31 8 4.42 5.17 4.33 4.17 3.67 4.35 Diets x 3 4.02b 4.93a 4.20ab 4.00b 3.86b PCV (%) 27 4 40.3 38.8 38.9 40.6 38.8 39.5b 31 8 41.3 41.4 40.8 42.8 40.9 41.5a Diets x 3 40.8 40.1 39.9 41.7 39.9 Main sources of variation Diets Age Diets×age ANOVA variables SELSM4 p value SELSM p value SELSM p value SRBC 0.21 0.005 0.13 0.115 0.30 0.345 PCV 0.72 0.338 0.46 0.003 1.02 0.96 a, b Diets x and age x with no common superscript differ significantly (p<0.05). 1 Composition of basal diets, experiment design, and SRBC challenge schedule are described as Table 1 and 2. 2 Hemagglutinin titers. 3 The statistical model used in the analyses of variance included added vitamin E levels in the different diets, and weeks of age as main effects. 4 SELSM = Standard error of least squares means. supplemental vitamin E on antibody titers in response to sheep red blood cell (SRBC) and packed cell volume (PCV) reeder cockerels1 (Experiment 2) (n =12) Table 4. Effects of supplemental vitamin E on antibody titers in response to sheep red blood cell (SRBC) and packed of Taiwan Native breeder cockerels1 (Experiment 2) (n =12) tamin E on antibody titers in response to sheep red blood cell (SRBC) and packed cell volume (PCV) 1 (Experiment 2) (n 12) Hemagglutinin titers. 3 The statistical model used in the analyses of variance included added vitamin E levels in the different diets, and weeks of age as main effects. 4 SELSM = Standard error of least squares means. Statistical analysis Pullets fed diets supplemented with different levels (0 to 160 mg/kg) of vitamin E during laying period (Experiment 1) exhibited no significant difference in average body weight at 17, 26 and 35 weeks of age, or in weight gain from 17 to 26 weeks of age (Table 3). However, pullets fed 160 mg/kg supplemental vitamin E had significantly higher (p<0.01) body weigh gain during 26 to 35 weeks of age than those fed 0 and 40 mg/kg vitamin E. Cockerels fed different levels (0 to 160 mg/kg) of supplemental vitamin E during mature period (Experiment 2) showed no significant Random samples for determination of antibody titers etc. were from individual birds (experimental unit) (n = 12) of different treatments. Data were analyzed using the General Linear Model (GLM) procedure of SAS (SAS Institute, 1996) for the effects of treatment, age and their interactions. Statements of statistical significance were based on p<0.05. Least-squares means of the five treatments were compared by Tukey’s honestly significant difference (HSD) test. 888 LIN AND CHANG Table 5. Effects of supplemental vitamin E on antibody titers in response to Newcastle disease (ND), infectious bursal disease (IBD), and infectious bronchitis (IB) vaccine of Taiwan Native breeder cockerels1 (Experiment 2) (n = 12) ---------------------- Diets (added vitamin E, mg/kg) ---------------------------- Antigens Age (wks) Wks post-adding vit. E 0 20 40 80 160 p ND2 (log2) 31 8 11.8±0.9 11.8±0.4 11.9±0.3 11.8±0.5 11.9±0.3 0.73 IBD3 (×103) 31 8 14.7±2.5ab 15.4±1.1a 12.5±2.0bc 12.1±1.6c 11.0±3.3c <0.01 IB3 (×103) 31 8 7.44±3.28 7.80±2.92 6.18±2.26 6.94±2.14 5.31±2.18 0.16 a, b, c Values in a row without the same superscript are significantly different (p<0.05). 1 Composition of basal diets, experiment design and vaccination schedule are described as Table 1 and 2. 2 Hemagglutinin inhibition titers. 3 ELISA titers. a, b, c Values in a row without the same superscript are significantly different (p<0.05). 1 Composition of basal diets, experiment design and vaccination schedule are described as Table 1 and 2. 2 Hemagglutinin inhibition titers. 3 corn-soybean meal diets without supplemental vitamin E should have also received minimum requirement for growth maintenance. Similar report was found by Siegel et al. (2001) who observed greater BW gains from laying pullets fed on a 300 mg vitamin E/kg than those fed on 10 mg/kg, whereas BW gain was not significantly affected by the vitamin E treatments in broilers (Bartov and Frigg, 1992). Immune response Supplemental vitamin E was added beginning at 17 weeks of age for breeder pullets. There was no significant difference among the treatments for antibody titer in responses to SRBC, NDV at 35 weeks of age, or for skin response to PHA-P and for PCV percentage at 36 and 31 weeks of age, respectively (data not shown). Humoral immune response was evaluated by antibody response to SRBC, NDV, IBDV and IBV. Cell-mediated immune response was evaluated by skin response to PHA-P (mitogen-driven proliferation). Compared with growth performance, immune response affected by supplemental vitamin E for laying pullets appeared to be less sensitive. In this study, female chickens fed corn-soy bean diets without supplemental E did not appear significantly lower in humoral and cell-mediated immune responses than those fed supplemental vitamin E. Richter et al. (1986) also reported no relation between the humoral immune response and vitamin E supplementation (0 and 20 mg/kg) for laying pullets. Cockerels fed the diets supplemented with 20 mg/kg vitamin E had the highest antibody titers in response to SRBC at 27 and 31 weeks of age (p<0.05) (Table 4). Significantly lower titers were found in cockerels fed with 0 and 160 mg/kg supplemental vitamin E at 27 and 31 weeks of age, respectively. Pooled data indicated that cockerels fed the diets supplemented with 20 mg/kg vitamin E had significant higher anti-SRBC titers than those fed with 0, 80 and 160 mg/kg vitamin E (p<0.01). Diets supplemented with vitamin E had no significant difference on cockerels’ PCV. Cockerels’ PCV at 31 weeks of age was higher than that at 26 weeks of age. From the results of cockerels, it was shown that moderate supplementation of vitamin E (20 mg/kg) could enhance immune response to SRBC. Previous research indicated that deficiency of vitamin E caused a number of reduced immune responses and deficiency of both vitamin E and selenium in chicks impaired bursal growth and reduced the number of lymphocytes in the bursa and the thymus gland (Marsh et al., 1986). From this point of view, vitamin E seemed to be beneficial for the ontogeny of immune response in terms of humoral immunity. Compared with cockerels fed supplemental vitamin E in the mature period, long-term (27 to 31 weeks) omission of supplemental vitamin E appeared to depress antibody titers in response to SRBC but not in response to ND, IBD or IB vaccine. Immune response We assume that long term omission of supplemental vitamin E might reach the margin of vitamin deficiency for specific immune responses. For antibody titers in response to NDV, IBDV and IBV at 31 weeks of age, the results indicated that cockerels’ diets supplemented with 20 mg/kg vitamin E had an IBD-titer comparable with the control birds and birds supplemented with more than 20 mg/kg had lower (p<0.01) antibody levels (Table 5). There was no significant difference in anti- ND and IB titers. Statistical analysis These results led to the assumption that the vitamin E existing in the ingredients of corn-soybean meal diets was sufficient to meet the minimum requirement for growth performance of chickens, exclusive of laying pullets. difference in body weight and body weight gain through 31 weeks of age. However, cockerels fed 40 to 160 mg/kg of supplemental vitamin E tended to have higher (p = 0.04) body weight gain than those fed 0 to 20 mg/kg of supplemental vitamin E during week 23 to 27. DISCUSSION Results of this study showed that high levels (120 and 160 mg/kg) of supplemental vitamin E significantly increased body weight gain only when the pullets reached their peak egg-production period (26 to 35 weeks of age), suggesting that breeder pullets fed corn-soybean meal diets without supplemental vitamin E had received minimum nutrient requirement for maintenance of growth before peak egg-production period. In this study, cockerels fed with Previous works were not consistent on relation between supplemental vitamin E and immune response. Some VITAMIN E ON IMMUNE RESPONSE OF CHICKENS 889 research indicated that high levels of vitamin E (greater than 10 times the required level) were immunostimulatory in chicks (Latshaw, 1991). However, Qureshi et al. (1993) and Marsh et al. (1981) found no effect by supplementing 100 or 250 mg vitamin E/kg diet. The present study in cockerels showed that high levels of supplemental vitamin E (80 to 160 mg/kg) had no or adverse effects on antibody titers in response to SRBC or IBD vaccine. Our results are consistent with those reported by Leshchinsky and Klasing (2001) who reported that moderate levels of supplemental vitamin E (25 to 50 mg/kg) modulate the immune system more than high levels (100 to 200 mg/kg). antioxidant system are able to regenerate each other (Beyer 1994; Packer et al., 1997). However, under various conditions, vitamin E has been found to have diverse roles; that is neutral, anti-, or pro-oxidant activity. For example, Thomas and Stocker (2000) reported that vitamin E at high concentrations can serve as a prooxidant. In addition, Yang et al. (1976) reported that both excess and deficiency of vitamin E in rats significantly depress the activity of glutathione peroxidase and thus modulates the intracellular glutathione pool. Thus, we speculated that excess dose of vitamin E could modify the overall balance of antioxidants in a cell, resulting in different pools of antioxidant compound that carry different immunoregulatory properties. Significant differences in immune response to SRBC and IBDV were observed but not in NDV or IBV. It might be due to different types of those antigens. Antibody levels of IBDV and NDV were determined following vaccination with a live vaccine and booster with inactivated vaccines. It is known that SRBC and IBDV are antigens which require the presence of T helper cells (CD4) in order to induce humoral immune response (T-cell dependent), whereas NDV does not (T-cell independent). DISCUSSION Research has shown that more T helper cells (CD4) were presented with increased dietary vitamin E (0 to 87 mg/kg) and thus improved responsiveness to immunologic stimuli (Erf et al., 1998). Further study is needed to clarify whether the different results of immune response to SRBC and IBDV but not to NDV and IBV by vitamin E supplementation correlate with the views described above. Supplemental vitamin E had no effect on PCV of pullets or cockerels. Similar results were obtained in broiler chickens (Jakobsen et al., 1995; Tras et al., 2000). In addition, this study has shown that older cockerel’s PCV (41.5) was higher than younger PCV (39.5) and cockerel’s PCV (40.8 to 42.8) was higher than hen’s PCV (29.1 to 30.3). Sex and age effects on PCV in this study may partly account for previous reports that broilers selected for high body weight, rapid protein accretion, or superior feed efficiency have higher PCV (Lubritz and McPherson 1994; Silversides et al., 1997) and PCV increased with age (Price et al., 1998). Yahav (1997) reported that a difference of temperature of 10°C or more may affect PCV. In this study, the difference of mean temperature in the determined weeks (week 27 and 31) was only 1°C, thus excluding the temperature effect. There were no diet×age interaction on SRBC or PCV. However more data are needed to elucidate the age effect. Although higher antibody response to vaccination of chickens may result in lower mortality caused by infectious diseases (Yunis et al., 2000) and higher clinical protection against virulent challenge (Maas et al., 1999), the beneficial effect of supplemental vitamin E in terms of disease resistance needs to be further investigated. In conclusion, the results of the present study indicated that vitamin E existing in the ingredients of corn-soybean meal diets was sufficient to meet the minimum requirement for growth performance of breeder chickens, exclusive of peak-laying pullets. Supplemental vitamin E has no effect on immune response of laying pullets. For cockerels, moderate supplementation of vitamin E was shown to enhance immune responses to SRBC, whereas for antibody titers in response to IBD depression was observed when supplementation level was over 80 mg/kg. Although a vitamin E deficiency would not be expected to occur in practice from the results of this study, caution must be taken as excessive vitamin E may depress specific immune responses. DISCUSSION For laying hens, vitamin E is one of the nutrients that can be increased in the egg by increasing the dietary level of this vitamin (Cherian and Sim, 1997; Grobas et al., 2001). Meydani and Hayek (1992) reported that low vitamin E levels led to unstable immune cell membranes, which led to enhanced production of immunosuppressors (eg, prostaglandins). In addition, laying hens showed higher serum concentration of prostaglandins than nonlaying hens or cocks (Takahashi et al., 1999). In the study of mice, Meydani et al. (1986) reported that the enhancement of immune response of aged mice by vitamin E appeared to be mediated by decreased prostaglandin synthesis. It remains an unsettled question whether egg-laying itself or depletion of supplemental vitamin E for laying influences the immune response. REFERENCES The potent impact of nutritional factors on immune response. In Nutrition and Immunity. pp. 1-7. Academic Press, New York, NY. Goto, N., H. Kodama, K. Okada and Y. Fujimoto. 1978. Suppression of phytohemagglutinin skin response in thymectomized chickens. Poult. Sci. 57:246-250. Meydani, S. N. and M. Hayek. 1992. Vitamin E and the immune response. In International Conference on Nutrition, Immuniity, and Illness in the Elderly (Ed. R. K. Chandra), ARTS Biomedical Publ, St. Johns, Newfoundland, pp. 105-128. Grobas, S., J. Mendez, B. C. Lopez, B. C. De and G. G. Mateos. 2001. Effect of vitamin E and A supplementation on egg yolk alpha-tocopherol concentration. Poult. Sci. 81:376-381. Meydani, S. N., S. N. Han and D. Wu. 2005. Vitamin E and immune response in the aged: molecular mechanisms and clinical implications. Immunol. Rev. 205:269-284. Jakobsen, K., R. M. Engberg, J. O. Andersen, S. K. Jensen, C. Lauridsen, P. Sorensen, P. Henckel, G. Bertelsen, L. H. Skibsted and C. Jensen. 1995. Supplementation of broiler diets with all-rac-alpha- or a mixture of natural source RRR-alpha- ,gamma-,delta-tocopheryl acetate. 1. effect on vitamin E status of broilers in vivo and at slaughter. Poult. Sci. 74:1984-1994. Munns, P. L. and S. J. Lamont. 1991. Research note: effects of age and immunization interval on the anamnestic response to T- cell-dependent and T-cell-independent antigens in chickens. Poult. Sci. 70:2371-2374. National Research Council. 1994. Nutrient Requirement of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Latshaw, J. D. 1991. Nutrition--mechanisms of immunosuppression. Vet. Immunol. Immunopathol. 30:111-120. Packer, L., H. J. Tritschler and K. Wessel. 1997. Neuroprotection by the metabolic antioxidant alpha-lipoic acid. Free Radic. Biol. Med. 22:359-378. Leshchinsky, T. V. and K. C. Klasing. 2001. Relationship between the level of dietary vitamin E and the immune response of broiler chickens. Poult. Sci. 80:1590-1599. Price, S. E., E. A. Dunnington and P. B. Siegel. 1998. Hematocrit values in weight-selected and relaxed lines of White Rock chickens. Poult. Sci. 77:1478-1480. Levander, O. A. 1992. Selenium and sulfur in antioxidant protective systems: relationships with vitamin E and malaria. Proc. Soc. Exp. Biol. Med. 200:255-259. Qureshi, M. A., P. R. Ferket and J. D. Garlich. 1993. Effect of dietary supplementation of vitamin E on the immune function of turkey poults. Poult. Sci. 72(Suppl. 1):56 (Abstr.). Lin, C. F., A. J. Asghar, I. D. Gray, J. Buckley, A. M. Booren, R. L. Crackel and C. J. Flegal. 1989. REFERENCES Lohakare, J. D., J. Y. Choi, J. K. Kim, J. S. Yong, Y. H. Shim, T.-W. Hahn and B. J. Chae. 2005. Effects of dietary combinations of vitamin A, E and methionine on growth performance, meat quality and immunity in commercial broilers. Asian-Aust. J. Anim. Sci. 18:516-523. Bartov, I. and M. Frigg. 1992. Effect of high concentrations of dietary vitamin E during various age periods on performance, plasma vitamin E and meat stability of broiler chicks at 7 weeks of age. Br. Poult. Sci. 33:393-402. Beyer, R. E. 1994. The role of ascorbate in antioxidant protection of biomembranes: interaction with vitamin E and coenzyme Q. J. Bioenerg. Biomembr. 26:349-358. Lubritz, D. L. and B. N. McPherson. 1994. Effect of genotype and cold stress on incidence of ascites in cockerels. J. Appl. Poult. Res. 3:171-178. Cherian, G. and J. S. Sim. 1997. Egg yolk polyunsaturated fatty acids and vitamin E content alters the tocopherol status of hatched chicks. Poult. Sci. 76:1753-1759. Maas, R. A., H. L. Oei, S. Venema-Kemper, G. Koch and J. Bongers. 1999. Dose-response effects of inactivated Newcastle disease vaccines: influence of serologic assay, time after vaccination, and type of chickens. Avian Dis. 43:670-677. Di Mascio, P., M. E. Murphy and H. Sies. 1991. Antioxidant defense systems: the role of carotenoids, tocopherols, and thiols. Am. J. Clin. Nutr. 53(Suppl):194S-200S. Marsh, J. A., G. F. Jr. Combs, M. E. Whitacre and R. R. Dietert. 1986. Effect of selenium and vitamin E dietary deficiencies on chick lymphoid organ development. Proc. Soc. Exp. Biol. Med. 182:425-436. Erf, G. F., W. G. Bottje, T. K. Bersi, M. D. Headrick and C. A. Fritts. 1998. Effects of dietary vitamin E on the immune system in broilers: altered proportions of CD4 T cells in the thymus and spleen. Poult. Sci. 77:529-537. Marsh, J. A., R. R. Dietert and G. F. Jr. Combs. 1981. Influence of dietary selenium and vitamin E on the humoral immune response of the chick. Proc. Soc. Exp. Biol. Med. 166:228-236. Extension Booklet of Taiwan Native Chicken. 1995. Nutrient Requirement of Native Chicken. Taiwan Livestock Research Institute Press, Tainan 712, Taiwan. Meydani, S. N., M. Meydani, C. P. Verdon, A. A. Shapiro, J. B. Blumberg and K. C. Hayes. 1986. Vitamin E supplementation suppresses prostaglandin E1(2) synthesis and enhances the immune response of aged mice. Mech. Ageing Dev. 34:191- 201. Gershwin, M., R. Beach and L. Hurley. 1985. ACKNOWLEDGMENTS The authors thank Dr. Yu-Shin Cheng and Dr. Yu-Chia Huang of the Livestock Research Institute, Council of Agriculture for technical assistance. Appreciation is also expressed to professor Yan-Nian Jiang of National Taiwan University for their useful comments in this study. It is well-known that vitamin E works as a part of cellular antioxidant systems in close cooperation with other cellular antioxidants, particularly with the ascorbate and glutathione systems (Di Mascio et al., 1991). Parts of this 890 LIN AND CHANG the reproduction performance of Taiwan Native Chicken cockerels. Br. Poult. Sci. 46:366-373. REFERENCES Effects of oxidised dietary oil and antioxidant supplementation on broiler growth and meat stability. Br. Poult. Sci. 30:855-864. Richter, G. E., A. Hennig and G. Steinbach. 1986. Vitamin E requirements of laying hens. Arch. Anim. Nutr. 36:1133-1143. Lin, Y. F., S. J. Chang and A. L. Hsu. 2004. Effects of supplemental vitamin E during the laying period on the reproductive performance of Taiwan native chickens. Br. Poult. Sci. 45:807-814. Richter, G., I. Rodel, E. Wunderlich and E. Marckwardt. 1985. Evaluation of laying-hen feed with varied vitamin E and antioxidant supplementation. Arch. Anim. Nutr. 35:707-714. AS Institute. 1996. Version 6.11. SAS Institute Inc., Cary, N Lin, Y. F., H. L. Tsai, Y. C. Lee and S. J. Chang. 2005a. Maternal vitamin E supplementation affects the antioxidant capability and oxidative status of hatching chicks. J. Nutr. 135:2457-2461. Siegel, P. B., S. E. Price, B. Meldrum, M. Picard and P. A. Geraert. 2001. Performance of pureline broiler breeders fed two levels of vitamin E. Poult. Sci. 80:1258-1262. Lin, Y. F., S. J. Chang, J. R. Yang, Y. P. Lee and A. L. Hsu. 2005b. Effects of supplemental vitamin E during the mature period on Silversides, F. G., M. R. Lefrancois and P. Villeneuve. 1997. The VITAMIN E ON IMMUNE RESPONSE OF CHICKENS VITAMIN E ON IMMUNE RESPONSE OF CHICKENS 891 effect of strain of broiler on physiological parameters associated with the ascites syndrome. Poult. Sci. 76:663-667. effect of strain of broiler on physiological parameters associated with the ascites syndrome. Poult. Sci. 76:663-667. Tras, B., F. Inal, A. L. Bas, V. Altunok, M. Elmas and E. Yazar. 2000. Effects of continuous supplementations of ascorbic acid, aspirin, vitamin E and selenium on some haematological parameters and serum superoxide dismutase level in broiler chickens. Br. Poult. Sci. 41:664-666. Snyder, D. B., W. W. Marquardt, E. T. Mallinson, P. K. Savage and D. C. Allen. 1984. Rapid serological profiling by enzyme- linked immunosorbent assay. III. Simultaneous measurements of antibody titers to infectious bronchitis, infectious bursal disease, and Newcastle disease viruses in a single serum dilution. Avian Dis. 28:12-24. Yahav, S., A. Straschnow, I. Plavnik and S. Hurwitz. 1997. Blood system response of chickens to changes in environmental temperature. Poult. Sci. 76:627-633. Yang, N. Y. J., I. B. MacDonald and I. J. Desai. 1976. Vitamin E supplementation and glutathione peroxidase activity. Proc. Soc. Exp. Biol. Med. 151:770-774. Takahashi, T., M. Kawashima, T. Yasuoka, T. Kuwayama and K. Tanaka. 1999. Prostaglandin F concentration in serum of hens and cocks. Poult. Sci. 78:906-908. Yunis, R., A. Ben-David, E. D. Heller and A. Cahaner. 2000. Immunocompetence and viability under commercial conditions of broiler groups differing in growth rate and in antibody response to Escherichia coil vaccine. Poult. Sci. 79:810-816. Thomas, S. R. and R. Stocker. 2000. Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis. Free Radic. Biol. Med. 28:1795-1805.
https://openalex.org/W2889731832	https://www.nature.com/articles/s41598-018-32578-w.pdf	English	null	Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy	Scientific reports	2,018	cc-by	13,850	Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy Received: 23 April 2018 Accepted: 23 August 2018 Published online: 21 September 2018 Received: 23 April 2018 Accepted: 23 August 2018 Published online: 21 September 2018 Guido Frosina 1, Jean Louis Ravetti2, Renzo Corvò3,4, Mauro Fella5, Maria Luisa Garrè6, Fabrizio Levrero7, Diana Marcello1, Daniela Marubbi8,9, Giovanni Morana6, Michele Mussap5, Carlo Emanuele Neumaier10, Aldo Profumo11, Alessandro Raso6, Francesca Rosa10, Stefano Vagge3, Donatella Vecchio1, Antonio Verrico6, Gianluigi Zona12,13 & Antonio Daga 8 It has been reported that the ATM kinase inhibitor KU60019 preferentially radiosensitizes orthotopic high grade gliomas (HGG) driven by established U87 and U1242 cell lines bearing specific TP53 mutations. We wished to determine whether those results could be extended to tumors driven by primary glioma initiating cells (GIC) that closely mimic clinical tumors. Orthotopic HGG were developed in immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by intracranial injection of primary GIC isolated from the adult glioblastoma COMI (acronym of patient’s name) and the pediatric anaplastic astrocytoma 239/12. Similar to the clinical tumors of origin, the orthotopic tumors COMI and 239/12 displayed different growth properties with a voluminous expansive lesion that exerted considerable mass effect on the adjacent structures and an infiltrating, gliomatosis-like growth pattern with limited compressive attitude, respectively. Significant elongations of median animal survival bearing the adult COMI tumor was observed after one KU60019 convection enhanced delivery followed by total 7.5 Gy of ionizing radiation delivered in fifteen 0.5 Gy fractions, as compared to animals treated with vehicle + ionizing radiation (105 vs 89 days; ratio: 0.847; 95% CI of ratio 0.4969 to 1.198; P:0.0417). Similarly, a trend to increased median survival was observed with the radiosensitized pediatric tumor 239/12 (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891). Our results indicate that radiosensitization by KU60019 is effective towards different orthotopic gliomas that faithfully mimic the clinical tumors and that multiple GIC-based animal models may be essential to develop novel therapeutic protocols for HGG transferable to the clinics. High grade gliomas (HGG-WHO grades III and IV) are the most frequent gliomas accounting for almost 50% of cases in Europe1,2. Despite advances in surgery and radiotherapy (RT), the prognosis for HGG remains poor, with median patient’s survival of 12–14 months for grade IV [glioblastoma(GB)] and 24–60 months for grade III [anaplastic astrocytoma (AA)]3,4. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy (J) Dose calibration vi: example of dose distribution quantification after scanning of radiochromic film. (K) Dose calibration vii: the irradiated region under the head as indicated by the darkened region of the bottom radiochromic film. (L) Dose calibration viii: the 70°-rotated animal allowing direct irradiation of the left, tumor-bearing brain hemisphere. (M) Dose calibration ix: as in K but with high-sensitivity radiochromic film allowing visualization of the guide screw position (indicated with arrow). (N) Dose calibration x: magnification of L with site of guide screw indicated with arrow. (O) Dose calibration xi: example of RS 2000 parameters for irradiation with 0.5 Gy to the head and virtually 0.0 Gy to the body. Figure 1. Experimental set up for multiple radiosensitizations in vivo. (A) Device i. In order to reduce toxicity during multiple radiosensitization cycles, CED was carried out under inhalation anesthesia with isoflurane as described under Materials and methods and13. Top left and top central insets are detail snapshots showing the location of CED guide screw on the mouse skull with distances from the left ear and eye and the simultaneous inhalation anesthesia of three mice, respectively. (B) Device ii: the RS 2000 Biological Irradiator (Rad Source Technologies, Alpharetta, GA, USA) equipped with the inhalation anesthesia device. (C) Device iii: the irradiation chamber with inhalation anesthesia tubing. (D) Device iv: the X-ray collimator with positioning laser beams. (E) Dose calibration i: the mouse polystyrene support with RadCal X-ray calibrating probe. (F) Dose calibration ii: the RadCal dosimeter display. (G) Dose calibration iii: radiochromic films placed under and over the mouse body and positioning the X-rays beam to the brain by the collimator laser. (H) Dose calibration iv: the upper radiochromic film before irradiation. (I) Dose calibration v- the upper radiochromic film after irradiation. The irradiated region of top head is indicated by the darkened region of the radiochromic film. (J) Dose calibration vi: example of dose distribution quantification after scanning of radiochromic film. (K) Dose calibration vii: the irradiated region under the head as indicated by the darkened region of the bottom radiochromic film. (L) Dose calibration viii: the 70°-rotated animal allowing direct irradiation of the left, tumor-bearing brain hemisphere. (M) Dose calibration ix: as in K but with high-sensitivity radiochromic film allowing visualization of the guide screw position (indicated with arrow). (N) Dose calibration x: magnification of L with site of guide screw indicated with arrow. Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy The ineffectiveness of currently available chemotherapeutic agents and RT depends on intrinsic tumor properties including elevated infiltration capacity and resistance to therapies5. In 1Mutagenesis & Cancer Prevention, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 2Pathological Anatomy and Histology, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 3Radiation Oncology, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 4Department of Health Sciences (DISSAL) University of Genoa, 16132, Genova, Italy. 5Laboratory Medicine, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 6IRCCS Istituto Giannina Gaslini, 16147, Genova, Italy. 7Medical Physics, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 8Regenerative Medicine, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 9Department of Experimental Medicine (DIMES), University of Genova, 16132, Genova, Italy. 10Diagnostic Imaging and Interventional Oncology, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 11Biopolymers and Proteomics, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 12Neurosurgery and Neurotraumatology, IRCCS Ospedale Policlinico San Martino, 16132, Genova, Italy. 13Department of Neurology, Ophthalmology, Genetics and Paediatrics (DINOGMI), University of Genova, 16132, Genova, Italy. Correspondence and requests for materials should be addressed to G.F. (email: guido.frosina@hsanmartino.it) Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 1 www.nature.com/scientificreports/ Figure 1. Experimental set up for multiple radiosensitizations in vivo. (A) Device i. In order to reduce toxicity during multiple radiosensitization cycles, CED was carried out under inhalation anesthesia with isoflurane as described under Materials and methods and13. Top left and top central insets are detail snapshots showing the location of CED guide screw on the mouse skull with distances from the left ear and eye and the simultaneous inhalation anesthesia of three mice, respectively. (B) Device ii: the RS 2000 Biological Irradiator (Rad Source Technologies, Alpharetta, GA, USA) equipped with the inhalation anesthesia device. (C) Device iii: the irradiation chamber with inhalation anesthesia tubing. (D) Device iv: the X-ray collimator with positioning laser beams. (E) Dose calibration i: the mouse polystyrene support with RadCal X-ray calibrating probe. (F) Dose calibration ii: the RadCal dosimeter display. (G) Dose calibration iii: radiochromic films placed under and over the mouse body and positioning the X-rays beam to the brain by the collimator laser. (H) Dose calibration iv: the upper radiochromic film before irradiation. (I) Dose calibration v- the upper radiochromic film after irradiation. The irradiated region of top head is indicated by the darkened region of the radiochromic film. Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy (O) Dose calibration xi: example of RS 2000 parameters for irradiation with 0.5 Gy to the head and virtually 0.0 Gy to the body. particular, resistance of HGG to RT may in part be linked to temporary quiescence of subpopulations of cells driving tumor development and progression [glioma initiating cells (GIC)], in turn caused by constitutive acti- vation of the response to DNA damage (DDR)6,7. Ataxia Telangiectasia Mutated (ATM) kinase is a major sensor of DNA damage and activator of DDR8. In vitro studies in our and other laboratories have shown that inhibition of ATM protein by the specific inhibitor KU60019 can flush GIC out of their quiescence and sensitize them to ionizing radiation (IR)9–17. In vivo, inhibition of ATM kinase by KU60019 has been reported as an effective radi- osensitization strategy of orthotopic gliomas driven by the established U87 and U1242 cell lines bearing specific TP53 mutations17. We show here that primary GIC-driven orthotopic tumors faithfully mimic the growth prop- erties of the clinical tumors and that the in vivo radiosensitization and survival advantages achievable by the ATM inhibitor KU60019 can be partially extended to these models. Materials and Methods As a chromatographic marker, 10 nM KU60019 was run under the same conditions. Experimentation, Ospedale Policlinico San Martino, Genova, Italy (record n. P.R.216REG2015). Similarly, the 239/12 pediatric patient’s informed consent for study participation was obtained by parents and the study was approved by the Regional Committee for Ethical Experimentation with protocol n. HERBY B025041/ITCC019/ HIGH GRADE GLIOMA – 001 Version Ita 01231358v01 of 25/10/2011). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. In particular, all animal experiments were performed in compliance with guidelines approved by the Italian Ministry of Health and the committee for animal well-being in cancer research (OPBA) at Ospedale Policlinico S.Martino - Genova, Italy (Italian Ministry of Health authorization n. 1309/2015-PR). The ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines were followed throughout this report1. Chemicals. The ATM inhibitor 2-[(2R, 6S)-2, 6-dimethylmorpholin-4-yl]-N-[5-(6-morpholin-4-yl-4-oxo- 4H-pyran-2-yl)-9H-thioxanthen-2-yl]-acetamide (KU60019) was purchased from Selleck Chemicals (Houston, TX, USA, product code: S1570). Experimental design. Mice were provided by the Breeding Unit of the Animal Facility at IRCCS Ospedale Policlinico S.Martino - Genova, Italy and by Envigo (San Pietro al Natisone, Udine, Italy). Immunodeficient non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice were housed in dedicated premises of the animal facility under maximum barrier conditions, one animal per cage to facilitate health status inspection. Barrier conditions included: use of microisolator (filter bonneted) cages; sterilized or disinfected food, water, bed- ding, cages and anything that come in contact with the mice; only personnel involved in care of the mice had access to the mouse rooms and caretakers wore personal, protective equipment at all times; cages were changed under a laminar flow hood weekly. Immunocompetent mice were housed under ordinary pathogen-free conditions, one animal/cage. Husbandry conditions included a 12 hours light/dark cycle and ad libitum access to standard laboratory chow and water. The experimental unit was the single animal that was uniquely identified by a number marked on the tail and coded by ear punches. This mouse ID number was in the format XX.XX where the first two digits indicate the experiment and the second two digits indicate the animal. In radiosensitization experiments, twenty mice were randomly assigned to the experimental (ten animals) and control (ten animals) groups: unless otherwise indicated, mice in the experimental group were i.c. infused with 12.5 µl of 250 µM KU60019 in 2.5% eth- anol/0.9% NaCl. Animals in the control groups were i.c. Materials and Methods Approvals. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committees and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. In particular, the adult COMI patient’s informed consent for study participation was obtained and the study was approved by the Regional Committee for Ethical Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 2 tificreports/ Figure 2. Inability of KU60019 to cross the BBB after chronic administration. Healthy C57/Black mice were inoculated i.p. daily during two consecutive weeks (total of 10 injections) with 30 µl/g body weight of (A) vehicle (2.5% ethanol in 0.9% NaCl) or (B) 250 µM KU60019. Mice were then euthanized by CO2 asphyxiation, the brain removed and whole brain extracts prepared as described in Materials and Methods and13. KU60019 content was determined by HPLC/MS as described in Materials and Methods and13. KU60019 amounts higher than limit of quantitation (LoQ - 5 nM) were never found in brain tissues. C. As a chromatographic marker, 10 nM KU60019 was run under the same conditions. www.nature.com/scientificreports/ Figure 2. Inability of KU60019 to cross the BBB after chronic administration. Healthy C57/Black mice were inoculated i.p. daily during two consecutive weeks (total of 10 injections) with 30 µl/g body weight of (A) vehicle (2.5% ethanol in 0.9% NaCl) or (B) 250 µM KU60019. Mice were then euthanized by CO2 asphyxiation, the brain removed and whole brain extracts prepared as described in Materials and Methods and13. KU60019 content was determined by HPLC/MS as described in Materials and Methods and13. KU60019 amounts higher than limit of quantitation (LoQ - 5 nM) were never found in brain tissues. C. As a chromatographic marker, 10 nM KU60019 was run under the same conditions. Figure 2. Inability of KU60019 to cross the BBB after chronic administration. Healthy C57/Black mice were inoculated i.p. daily during two consecutive weeks (total of 10 injections) with 30 µl/g body weight of (A) vehicle (2.5% ethanol in 0.9% NaCl) or (B) 250 µM KU60019. Mice were then euthanized by CO2 asphyxiation, the brain removed and whole brain extracts prepared as described in Materials and Methods and13. KU60019 content was determined by HPLC/MS as described in Materials and Methods and13. KU60019 amounts higher than limit of quantitation (LoQ - 5 nM) were never found in brain tissues. C. Materials and Methods Immunostaining at d52 revealed that most of the orthotopic tumor expressed the stem cell marker nestin, indicating its stem cell-driven character (H). Mouse ID numbers 25.12 and 27.3 are indicated for the sake of reference with the days (d) of tumor development. Figure 3. The GIC-driven COMI tumor. (A,B) Patient’s tumor i: coronal and axial T1-weighted post-contrast MRI showing a left parieto-occipital intra-axial tumor with irregular contrast enhancement and necrotic core. (C,D) Patient’s tumor ii: T2-weighted images showing significant peritumoral edema and infiltrative behavior. (E) GIC i: the GIC component of the tumor was isolated immediately after surgery. Under matrigel-coating and serum-free conditions, the cells grew and layered onto a monolayer, maintaining intact self-renewal capacity. (F) GIC ii: the COMI GIC could be effectively radiosensitized in vitro by exposure to 1 µM KU60019 30 min prior to irradiation with three 2.5 Gy IR fractions [reproduced from12, with permission]. (G) GIC iii: removal of growth factors and addition of 10% FCS to the proliferation medium after approximately two weeks induced GIC differentiation with acquisition of astrocytic morphology and altered refractory index. (H–K) Orthotopic tumor. 3 × 105 COMI serum-free grown GIC were stereotactically injected in the left corpus striatum of immunodeficient NOD SCID mice. Staining with hematoxylin/eosin of brain tissue sections revealed 52 (I,J) and 108 (K) days later a growth pattern of the orthotopic tumor reflecting the characteristics of the clinical tumor: a voluminous expansive lesion (I,K) with an irregular infiltrating wall (J) exerting mass effect on the adjacent structures (K). Immunostaining at d52 revealed that most of the orthotopic tumor expressed the stem cell marker nestin, indicating its stem cell-driven character (H). Mouse ID numbers 25.12 and 27.3 are indicated for the sake of reference with the days (d) of tumor development. 30 µl/g body weight of vehicle (2.5% ethanol in 0.9% NaCl) or 250 µM KU60019. 16 hours after the last adminis- tration, blood was collected by retro-orbital bleeding, the mice were euthanized by CO2 asphyxiation and their brains explanted. Blood and brain tissues were immediately extracted for KU60019 content determination as described13. Briefly, blood and brain samples were suspended in water and homogenized for 30 sec using an Ystral X1020 homogenizer (Ystral GMBH, Dottingen, Germany). An equal volume of pure methanol was then added, the samples were homogenized for further 60 sec, centrifuged and their supernatants were stored at −80 °C. Materials and Methods infused with an equal volume of vehicle. Considering an average mouse brain volume of 415 µl18, the brain ethanol concentration after CED was 0.075% that does not cause any serious health effect (limited euphoric status at most)12. All other procedures (e.g. radiotherapeutic treatments – Fig. 1) were identical and concomitant for the two groups. The order in which the animals in the experimental and control groups were treated, as well as the animal sex, were alternate in subsequent experiments. Animals were monitored daily in blind by experienced researchers assigning to each animal a health score (H1 moribund status – H10 full health) based on neurological symptoms including posture changes, arched back, tail weakness, diminished activity and skin turgor. Euthanasia was performed by CO2 asphyxiation at the moribund status H1 that was the primary experimental outcome in this study. Pharmacokinetics of KU60019 after intraperitoneal (i.p.) delivery. Tumor-free immunocompetent C57/Black mice were inoculated i.p. daily on weekdays for two consecutive weeks (total of 10 i.p. injections) with Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 3 www.nature.com/scientificreports/ Figure 3. The GIC-driven COMI tumor. (A,B) Patient’s tumor i: coronal and axial T1-weighted post-contrast MRI showing a left parieto-occipital intra-axial tumor with irregular contrast enhancement and necrotic core. (C,D) Patient’s tumor ii: T2-weighted images showing significant peritumoral edema and infiltrative behavior. (E) GIC i: the GIC component of the tumor was isolated immediately after surgery. Under matrigel-coating and serum-free conditions, the cells grew and layered onto a monolayer, maintaining intact self-renewal capacity. (F) GIC ii: the COMI GIC could be effectively radiosensitized in vitro by exposure to 1 µM KU60019 30 min prior to irradiation with three 2.5 Gy IR fractions [reproduced from12, with permission]. (G) GIC iii: removal of growth factors and addition of 10% FCS to the proliferation medium after approximately two weeks induced GIC differentiation with acquisition of astrocytic morphology and altered refractory index. (H–K) Orthotopic tumor. 3 × 105 COMI serum-free grown GIC were stereotactically injected in the left corpus striatum of immunodeficient NOD SCID mice. Staining with hematoxylin/eosin of brain tissue sections revealed 52 (I,J) and 108 (K) days later a growth pattern of the orthotopic tumor reflecting the characteristics of the clinical tumor: a voluminous expansive lesion (I,K) with an irregular infiltrating wall (J) exerting mass effect on the adjacent structures (K). Materials and Methods 50 days later, mice were i.c.-administered with vehicle (2.5% ethanol in 0.9% NaCl) and then irradiated with one (total: 2.5 Gy), three (total: 7.5 Gy), four (total: 10.0 Gy) or five (total: 12.5 Gy) 2.5 Gy fractions to the head. Increased survival was observed up to the 10.0 Gy cumulative IR dose. Additional 2.5 Gy (cumulating to a total of 12. 5 Gy) were rapidly lethal with all mice dying in 24–48 hours (solid line). Differences between survival curves with P < 0.05, 0.01, 0.001 are indicated with one, two and three asterisks, respectively. (B) For comparison, the heads of tumor-free wild type C57Black mice (left) were irradiated every weekday with 2.5 Gy over five weeks (total dose delivered: 62.5 Gy). Blood samples were then drawn for hematology and the brains explanted for histopathology. No significant variation of (C) animal survival; (D) hematocrit; (E) hemoglobin; (F) normal brain histology was observed in irradiated (IR) versus unirradiated (C) mice. Figure 4. RT of GIC-driven orthotopic tumors. (A) 4–5 weeks old NOD-SCID mice were stereotactically injected in the left corpus striatum with 2–5 × 105 COMI GIC. 50 days later, mice were i.c.-administered with vehicle (2.5% ethanol in 0.9% NaCl) and then irradiated with one (total: 2.5 Gy), three (total: 7.5 Gy), four (total: 10.0 Gy) or five (total: 12.5 Gy) 2.5 Gy fractions to the head. Increased survival was observed up to the 10.0 Gy cumulative IR dose. Additional 2.5 Gy (cumulating to a total of 12. 5 Gy) were rapidly lethal with all mice dying in 24–48 hours (solid line). Differences between survival curves with P < 0.05, 0.01, 0.001 are indicated with one, two and three asterisks, respectively. (B) For comparison, the heads of tumor-free wild type C57Black mice (left) were irradiated every weekday with 2.5 Gy over five weeks (total dose delivered: 62.5 Gy). Blood samples were then drawn for hematology and the brains explanted for histopathology. No significant variation of (C) animal survival; (D) hematocrit; (E) hemoglobin; (F) normal brain histology was observed in irradiated (IR) versus unirradiated (C) mice. The adult GIC line COMI. The primary GIC line COMI derived from an adult GB has been previously described10,12,13,19 (Fig. 3E–G). Its authentication was performed by determining the proliferation rate, the expres- sion of DDR, stem, PI3K/Akt pathway genes as well as the IDH1, TP53, H3F3A, PDGFRA, CDKN2A and EGFR status as previously described10,12,13,19. Materials and Methods KU60019 content was determined by High Performance Liquid Chromatography (HPLC)/mass spectrometry (MS) using an Agilent 1200 series chromatographic system (Agilent Technologies, Palo Alto, CA, USA). 10 µl of filtrated sample were injected onto a Symmetry 300 C18 column (Waters Corp., Milford, MA, USA). After sepa- ration, the eluent flow was directly sent to an Agilent 6210 TOF mass spectrometer equipped with an electrospray (ESI) ion source operating in positive polarity (Agilent Technologies). High-resolution spectra were recorded in the range m/z 100–1000. The Mass Hunter acquisition software ver.A.02.02 (Agilent Technologies, Palo Alto, CA, USA) was autocalibrating, continuously recording the results of internal reference masses along with the raw data, thereby allowing accurate mass correction. The full-scan data recorded were processed by using Mass Hunter Qualitative Analysis ver.B.02.00 (Agilent Technologies, Palo Alto, CA, USA) and the relative amounts of KU60019 were measured by evaluating the peak areas of the extracted ion currents (EIC m/z 548.22 [M+H]+). Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 4 www.nature.com/scientificreports/ p Figure 4. RT of GIC-driven orthotopic tumors. (A) 4–5 weeks old NOD-SCID mice were stereotactically injected in the left corpus striatum with 2–5 × 105 COMI GIC. 50 days later, mice were i.c.-administered with vehicle (2.5% ethanol in 0.9% NaCl) and then irradiated with one (total: 2.5 Gy), three (total: 7.5 Gy), four (total: 10.0 Gy) or five (total: 12.5 Gy) 2.5 Gy fractions to the head. Increased survival was observed up to the 10.0 Gy cumulative IR dose. Additional 2.5 Gy (cumulating to a total of 12. 5 Gy) were rapidly lethal with all mice dying in 24–48 hours (solid line). Differences between survival curves with P < 0.05, 0.01, 0.001 are indicated with one, two and three asterisks, respectively. (B) For comparison, the heads of tumor-free wild type C57Black mice (left) were irradiated every weekday with 2.5 Gy over five weeks (total dose delivered: 62.5 Gy). Blood samples were then drawn for hematology and the brains explanted for histopathology. No significant variation of (C) animal survival; (D) hematocrit; (E) hemoglobin; (F) normal brain histology was observed in irradiated (IR) versus unirradiated (C) mice. Figure 4. RT of GIC-driven orthotopic tumors. (A) 4–5 weeks old NOD-SCID mice were stereotactically Figure 4. RT of GIC-driven orthotopic tumors. (A) 4–5 weeks old NOD-SCID mice were stereotactically injected in the left corpus striatum with 2–5 × 105 COMI GIC. www.nature.com/scientificreports/ The region grows as a frame starting from a seed identified by the operator, including pixels that are spatially connected and have their signal level above a threshold. The procedure ends when the latter conditions are no more satisfied. i For histological analysis, animals were euthanized by CO2 asphyxiation and brains were cryopreserved. Coronal sections obtained at a cryostat microtome were fixed and stained with hematoxylin/eosin (H/E) or with an anti-human nestin mouse monoclonal primary antibody (1 µg/ml - Abcam, Cambridge, UK) followed by a FITC-conjugated goat anti-mouse secondary IgG and DAPI counterstaining. Hematological analyses and ATM inhibition. For hematological analyses (Fig. 4D,E), 0.5–0.7 ml of blood was obtained from C57 Black mice by retro-orbital withdrawal in lithium-heparinized vessels without separating gel. Samples were then analyzed by a Sysmex sp-1000i automated slide preparer-stainer (Sysmex Corporation, Kobe, Japan) equipped with CellaVision® DM-96 morphological cell analyzer (CellaVision AB Lund, Sweden).hl ) The capacity of the KU60019 preparations to inhibit ATM kinase in vivo was determined by (a) immunofluo- rescence (IF – Fig. 5A,B): mice bearing orthotopic adult COMI GB developed for 52 days were inoculated by CED at the tumor site with 12.5 µl of 250 µM KU60019 or vehicle (12.5 µl of 2.5% ethanol in 0.9% NaCl) followed by irradiation to the head with 2.5 Gy at d56, 57 and 58. The radiosensitization cycle was then repeated with CED at d59 followed by 2.5 Gy at d63 and d64 (total dose delivered: 12.5 Gy). At d65, moribund animals were euthanized by CO2 asphyxiation, the brains removed and slices obtained by cryostat sectioning. The orthotopic tumor tissue was fixed, permeabilized, blocked with normal goat serum and incubated with rabbit monoclonal antibody to ATM-phospho S1981 (1:500 - Abcam, Cambridge, UK) followed by fluorochrome-conjugated antibody (goat anti-rabbit Alexa Fluor-488, Molecular Probes, Oregon, USA). After counterstaining with DAPI (Sigma-Aldrich, Milano, Italy), nuclei were scored at 400X magnification and considered positive when containing 3 or more ATM-phospho S1981 foci. The percentage of ATM-phospho S1981-positive cells was calculated after counting in blind at least 740 cells in 45 randomly selected fields. Images were always taken under equal conditions of brightness and contrast. (b) enhancement of KI67 immunostaining in orthotopic tumors specimens, as described (Fig. www.nature.com/scientificreports/ exposed. 12.5 µl of KU60019 solution in 2.5% ethanol/0.9%NaCl or vehicle were directly infused into the brain via a cannula inserted into the guide-screw using a BeeHive electrically-motorized pump (BeeHive™, Bioanalytical Systems, West Lafayette, Indiana) set at rate of 0.5 µl/minute. This procedure did not cause any evident inter- ference with the cognitive behavior of the mice nor increased susceptibility to bacterial infections, suggesting absence of effects on the immune system. f y RT of orthotopic GB was performed by an RS 2000 Biological Irradiator (Rad Source Technologies, Alpharetta, GA, USA - Fig. 1B) whose “in vivo” delivered dose was verified by a RadCal Accu-Gold system (Monrovia, CA, USA) equipped with a 10 × 6–0.6 High Dose Rate Chamber (Fig. 1E,F). The dose was confirmed by two radi- ochromic films (Gafchromic® EBT3, Ashland Inc., Covington, KY, USA) placed over and under the mouse body (Fig. 1G–O). The prescription dose was 0.5 or 2.5 Gy. In some experiments, RT was also performed on the head of mice by a HDR (high dose rate) brachytherapy delivery system (microSelectron Digital, Nucletron Elekta, Stockholm, Sweden) as previously described13. Radiation toxicity studies were performed on both immune/ repair-deficient NOD-SCID mice bearing adult orthotopic tumors and immune/repair-competent tumor-free wild type C57/Black mice (Fig. 4). In order to reduce toxicity of anesthesia during repeated drug adminis- trations and RT, an isoflurane inhalation anesthesia apparatus was used including: an Isoflurane Vaporizer (Rothacher-Medical, CH-3000, Berne Switzerland); a 2B LFY-I-5A Medical Oxygen Concentrator (2Biol, Besozzo, Italy); an anesthesia induction chamber (E-Z Anesthesia, E-Z Systems Corporation, Palmer, PA) and a dispersed isoflurane captation device (Fluovac Harvard Apparatus with Veterinary Fluosorber) (Fig. 1B–D). pl p ( pp y ) ( g ) MRI of the orthotopic tumors was performed under ketamine-xylazine i.p. anesthesia by a clinical 3 T whole body scanner (Signa EXCITE®HDxT, GE, Milwaukee, USA) with mice positioned in a prototype coil (linear birdcage transmit/receive coil, Flick Engineering Solutions BV-General Electric) as described12. Tumor volumes were considered as secondary experimental outcomes. They were determined by Growing Region Segmentation Software (GRES) for quantitative magnetic resonance imaging as described20. Briefly, the software determines the tumor area (segmentation) in any single slice and the tumor volume is calculated multiplying the areas for the slice thickness (1 mm). Materials and Methods COMI GICs have wild type TP53 sequence but they express the TP53 RNA at low levels12. COMI GICs initiate orthotopic glioma development with >95% efficiency when injected i.c. into immunodeficient NOD-SCID mice12,13,19 (Fig. 3H–K). To this aim, 4–5 weeks old NOD/SCID mice were anes- thetized with i.m. ketamine and xylazine. Thereafter, the animals were positioned into a stereotaxic frame (David Kopf instruments) and a hole was made, using a 21-gauge needle, 2.5 mm lateral and 1 mm anterior from the intersection of the coronal and sagittal sutures (Bregma). 2–5 × 105 GIC were injected at a depth of 3 mm in cor- respondence of the left corpus striatum. A guide screw with a central hole fitting a 26 gauge needle (PlasticsOne, Inc, Roanoke, VA) was then inserted into the skull hole to facilitate subsequent drug infusion via convection enhanced delivery [(CED) – drug delivery performed through a catheter connected to an electricity-operated pump] and the skin closed using metal staples (Martin GMBH, Tuttingen, Germany). At least two weeks were left elapsing before CED in order to achieve appropriate guide screw stabilization into the skull bone. In order to avoid significant subpopulation selection during prolonged cell culture, GIC samples frozen after no more than 30 days of culture were used for orthotopic tumor development. The pediatric GIC line 239/12. The pediatric GIC line 239/12 was obtained from surgical resection of a left temporo-parietal anaplastic astrocytoma (AA-WHO grade III) in a 14-year-old male. Pediatric GIC were cultured and characterized under the same conditions used for adult COMI GIC. 239/12 GICs expressed glial fibrillary acidic protein (GFAP), vimentin and nestin by immunohistochemistry. Mutation analysis by direct-sequencing and multiplex ligation-dependent probe amplification demonstrated in 239/12 the presence of a frameshift mutation in the TP53 tumor suppressor gene causing early termination and loss of function of the protein (c.819_820insT; p.V274Cfs*. Reference sequence NM000546 – Supplementary Fig. 1). CED, RT, MRI, histology. Intracranial (i.c.) KU60019 administrations to the tumor site were carried out in the surgery room by convection enhanced delivery (CED) as previously described12,13 (Fig. 1A). Briefly, at the indicated day after tumor implant, a mid-sagittal skin incision was made on the head and the guide screw Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5C,D)13: briefly, mice bearing orthotopic COMI GB developed for 52 days were inoculated by CED at the tumor site with 20 µl of 250 µM KU60019 or vehicle (20 µl of 2.5% ethanol in 0.9% NaCl) followed by irradiation with 0.5 Gy at d56, 57 and 58. The radiosensitization cycle was then repeated with CED at d59 followed by 1.5 Gy at d63 and with CED at d66 followed by 1.5 Gy at d70, 71 and 72 (total dose delivered: 7.5 Gy). At the indicated times [(d); Fig. 5C], animals were euthanized by CO2 asphyxiation, the brains removed and fixed in 10% for- maldehyde. Sections of the brain were stained with hematoxylin/eosin (H/E) and the orthotopic tumor tissue subjected to immunohistochemistry with anti-KI67 antibody (5 µg/L - Ventana-Roche, Tucson, AZ, USA). The percentage of KI67-positive cells was evaluated at 200X magnification counting in blind at least 1350 cells in 30 randomly selected fields. Statistical analysis. Ten mice per treatment group were used. This number was calculated a priori by the Gpower software (http://www.gpower.hhu.de/) based on an alpha error probability value of 0.05 for a 10% dif- ference of median survivals in the experimental and control groups and taking into account occasional losses of animals unrelated to tumor progression (e.g. during CED - Figs 4A, 6A,E and 7Q). Percentages of ATM-phospho S1981- and KI67-postive cells were compared by one-way ANOVA with Tukey’s Multiple Comparison test after counting in blind at least 740 cells in 45 randomly selected fields and 1350 cells in 30 randomly selected fields, respectively (Fig. 5B,D). Tumor volumes as determined by the GRES method (Fig. 6D) were compared by two-tailed unpaired t-test. The GraphPad Prism 5.01 statistical software was used. 6 Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w www.nature.com/scientificreports/ Figure 5. In vivo ATM inhibition by KU60019. (A) Immunodeficient mice bearing orthotopic COMI GB developed for 52 days were inoculated by CED at the tumor site with 12.5 µl of 250 µM KU60019 or vehicle (2.5% ethanol in 0.9% NaCl) followed by irradiation with 2.5 Gy at d56, 57 and 58. The radiosensitization cycle was then repeated with CED at d59 followed by 2.5 Gy at d63 and d64 (total dose delivered: 12.5 Gy). At d65, moribund animals were euthanized by CO2 asphyxiation, the brains removed and slices obtained by cryostat sectioning. www.nature.com/scientificreports/ The orthotopic tumor tissue was processed for IF analysis with rabbit monoclonal antibody to ATM-phospho S1981 as described under Materials and Methods. Reduced pATM foci in [KU60019 + IR (2.5 Gy × 5)] as compared to [Vehicle + IR (2.5 Gy x 5)] -treated tumors is shown. Mouse ID numbers 25.12, 25.5 and 25.2 are indicated for the sake of reference with the days (d) of tumor development. (B) The frequency distributions of the percentages of ATM-phospho S1981-positive cells in 45 randomly selected fields are shown. The three asterisks indicate P < 0.0001 for the differences of the frequency distributions between the indicated groups as determined by ANOVA (25.5 vs 25.12 mean difference +57% of p(S1981)ATM+ cells, 95% CI of difference +50 to +64%; 25.2 vs 25.5 mean difference −26% of p(S1981)ATM+ cells, 95% CI of difference −19 to −33%; 25.12 vs 25.2 mean difference −31% of p(S1981) ATM+ cells, 95% CI of difference −24 to −38%). (C) Persistent increase of KI67 staining in KU60019-treated mice 42 days after CED. At d52 of tumor development, COMI tumor-bearing mice were i.c.- injected by CED with 20 µl of 250 µM KU60019 or vehicle (2.5% ethanol in 0.9% NaCl) followed by irradiation with 0.5 Gy at d56, 57 and 58. The radiosensitization cycle was then repeated with CED at d59 followed by 1.5 Gy at d63 and with CED at d66 followed by 1.5 Gy at d70, 71 and 72 (total dose delivered: 7.5 Gy) Mouse ID numbers 12.1, 27.13 and 27.3 are indicated for the sake of reference with the days (d) of tumor development. (D) The frequency distributions of the percentages of KI67-positive cells in 30 randomly selected fields are shown. The three asterisks indicate P < 0.0001 for the differences of the frequency distributions between the indicated groups as determined by ANOVA (27.13 vs 12.1 mean difference −25% of KI67+ cells, 95% CI of difference −18 to −32%; 27.3 vs 27.13 mean difference +12% of KI67+ cells, 95% CI of difference +5 to +19%; 12.1 vs 27.3 mean difference +13% of KI67+ cells, 95% CI of difference +6 to +19%). Figure 5. In vivo ATM inhibition by KU60019. www.nature.com/scientificreports/ (A) Immunodeficient mice bearing orthotopic COMI GB developed for 52 days were inoculated by CED at the tumor site with 12.5 µl of 250 µM KU60019 or vehicle (2.5% ethanol in 0.9% NaCl) followed by irradiation with 2.5 Gy at d56, 57 and 58. The radiosensitization cycle was then repeated with CED at d59 followed by 2.5 Gy at d63 and d64 (total dose delivered: 12.5 Gy). At d65, moribund animals were euthanized by CO2 asphyxiation, the brains removed and slices obtained by cryostat sectioning. The orthotopic tumor tissue was processed for IF analysis with rabbit monoclonal antibody to ATM-phospho S1981 as described under Materials and Methods. Reduced pATM foci in [KU60019 + IR (2.5 Gy × 5)] as compared to [Vehicle + IR (2.5 Gy x 5)] -treated tumors is shown. Mouse ID numbers 25.12, 25.5 and 25.2 are indicated for the sake of reference with the days (d) of tumor development. (B) The frequency distributions of the percentages of ATM-phospho S1981-positive cells in 45 randomly selected fields are shown. The three asterisks indicate P < 0.0001 for the differences of the frequency distributions between the indicated groups as determined by ANOVA (25.5 vs 25.12 mean difference +57% of p(S1981)ATM+ cells, 95% CI of difference +50 to +64%; 25.2 vs 25.5 mean difference −26% of p(S1981)ATM+ cells, 95% CI of difference −19 to −33%; 25.12 vs 25.2 mean difference −31% of p(S1981) ATM+ cells, 95% CI of difference −24 to −38%). (C) Persistent increase of KI67 staining in KU60019-treated mice 42 days after CED. At d52 of tumor development, COMI tumor-bearing mice were i.c.- injected by CED with 20 µl of 250 µM KU60019 or vehicle (2.5% ethanol in 0.9% NaCl) followed by irradiation with 0.5 Gy at d56, 57 and 58. The radiosensitization cycle was then repeated with CED at d59 followed by 1.5 Gy at d63 and with CED at d66 followed by 1.5 Gy at d70, 71 and 72 (total dose delivered: 7.5 Gy) Mouse ID numbers 12.1, 27.13 and 27.3 are indicated for the sake of reference with the days (d) of tumor development. (D) The frequency distributions of the percentages of KI67-positive cells in 30 randomly selected fields are shown. www.nature.com/scientificreports/ The three asterisks indicate P < 0.0001 for the differences of the frequency distributions between the indicated groups as determined by ANOVA (27.13 vs 12.1 mean difference −25% of KI67+ cells, 95% CI of difference −18 to −32%; 27.3 vs 27.13 mean difference +12% of KI67+ cells, 95% CI of difference +5 to +19%; 12.1 vs 27.3 mean difference +13% of KI67+ cells, 95% CI of difference +6 to +19%). Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w Results KU60019 does not cross the blood brain barrier (BBB). Previous in vitro work in our laboratory on cultured GIC has shown that an increasing number of radiosensitizing cycles each consisting of exposure of cells to 1 µM KU60019 followed 30 min later by irradiation with 2.5 Gy could substantially delay (Figs 3F and 7J) and even eliminate the recovery of radioresistant cells, as compared to irradiated GIC exposed to vehicle only12. In order to translate this radiosensitizing effect to the animal setting, we had also investigated the pharmacoki- netics of KU60019 in tumor-free mice finding that, unlike blood, liver and kidneys, no drug could be detected in the brain parenchyma after one i.p. administration, indicating that KU60019 could not cross the BBB13. However, since traces of drug close to the limit of quantitation could be detected in the brain parenchyma by HPLC/MS and by virtue of the established low/null toxicity of KU6001912, we wished to investigate whether the chronic i.p administration may produce a KU60019 accumulation in the brain, reaching a concentration suffi- cient to radiosensitize the orthotopic tumors (Fig. 2). Ten i.p injections of 250 µM KU60019 at 30 µl/g body weight were carried out in tumor-free mice every weekday during two consecutive weeks (Fig. 2B). In agreement with previous data, no significant signs of toxicity were observed12,13. 16 hours after the last i.p. injection the animals Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 7 www.nature.com/scientificreports/ Figure 6. Increased survival of NOD-SCID mice bearing orthotopic adult COMI GB treated by KU60019 plus RT. At d41 of tumor development, animals were i.c.-injected by CED with 12.5 µl of 250 µM KU60019 (blue) or vehicle (ethanol in 0.9% NaCl - black) followed by irradiation with 2.5 Gy to the head at d45, 46 and 48 (total dose delivered: 7.5 Gy). (A) Kaplan-Meier survival curves. Median survival of KU60019-treated animals was significantly longer than that of vehicle-treated controls (101 vs 91 days; ratio: 0.901; 95% CI of ratio: 0.5384 to 1.264; P: 0.0409 indicated by one asterisk). (B) H/E staining of tumors at euthanasia (d91 and 104 in vehicle and KU60019-injected mice, respectively). Mouse ID numbers 22.1 and 22.15 are indicated for the sake of reference. Left: 2X snap photographs. Right: 100X magnifications showing tumor infiltrating cells. (C) 3 T MRI of orthotopic tumors carried out at d62. Top: tumors treated with vehicle+ (2.5 Gy × 3). Results Bottom: tumors treated with KU60019+ (2.5 Gy × 3). Mouse ID numbers are indicated for the sake of reference. (D) Determination of tumor volumes by region segmentation software (GRES). Top: example of segmented tumor. The mouse ID number (22.14) is indicated for the sake of reference. Bottom: mean ± SEM volumes of vehicle + (2.5 Gy × 3) (black) and KU60019+ (2.5 Gy × 3) (blue) – treated tumors. The tumor volumes in KU60019+ (2.5 Gy × 3) – treated mice were significantly reduced as compared to vehicle + (2.5 Gy × 3) -treated mice. Mean ± SEM of vehicle+ (2.5 Gy × 3): 140 ± 17 mm3; mean ± SEM of KU60019+ (2.5 Gy × 3): 97 ± 11 mm3; P: 0.047 after two-tailed unpaired t-test. (E) Further beneficial effect on animal survival after RT hyperfractionation. At d16 of tumor development, animals were i.c.-injected by CED in the surgery room with 12.5 µl of 250 µM KU60019 (blue) or vehicle (ethanol in 0.9% NaCl - black) followed by irradiation with 15 fractions of 0.5 Gy IR delivered to the head of mice 2–4 days apart (total dose delivered: 7.5 Gy). Kaplan-Meier survival curves are shown. Median survival of KU60019-treated animals was further increased (105 vs 89 days; ratio: 0.847; 95% CI of ratio: Figure 6. Increased survival of NOD-SCID mice bearing orthotopic adult COMI GB treated by KU60019 plus RT. At d41 of tumor development, animals were i.c.-injected by CED with 12.5 µl of 250 µM KU60019 (blue) or vehicle (ethanol in 0.9% NaCl - black) followed by irradiation with 2.5 Gy to the head at d45, 46 and 48 (total dose delivered: 7.5 Gy). (A) Kaplan-Meier survival curves. Median survival of KU60019-treated animals was significantly longer than that of vehicle-treated controls (101 vs 91 days; ratio: 0.901; 95% CI of ratio: 0.5384 to 1.264; P: 0.0409 indicated by one asterisk). (B) H/E staining of tumors at euthanasia (d91 and 104 in vehicle and KU60019-injected mice, respectively). Mouse ID numbers 22.1 and 22.15 are indicated for the sake of reference. Left: 2X snap photographs. Right: 100X magnifications showing tumor infiltrating cells. (C) 3 T MRI of orthotopic tumors carried out at d62. Top: tumors treated with vehicle+ (2.5 Gy × 3). Bottom: tumors treated with KU60019+ (2.5 Gy × 3). Mouse ID numbers are indicated for the sake of reference. Results Post-contrast T1-weighted images (G,H) do not show blood brain barrier disruption or necrotic areas. (I) GIC i: the GIC component of the tumor was isolated immediately after surgery. Under matrigel-coating and serum-free conditions, the cells grew and layered onto a monolayer, maintaining intact self-renewal capacity. (J) GIC ii: the 239/12 GIC could be effectively radiosensitized in vitro by exposure to 1 µM KU60019 30 min prior to irradiation with three 2.5 Gy IR fractions [blue versus black circles; reproduced from13, with permission]. The in vitro growth pattern of unirradiated cells is indicated by black squares. (K–O) Orthotopic tumor. 2 × 105 239/12 serum-free grown GIC were stereotactically injected into the left corpus striatum of NOD SCID mice. Immunostaining with an antibody directed against human nestin revealed at both d29 (K) and d138-150 (M,P) an infiltrating, gliomatosis-like, growth pattern of the orthotopic tumor exerting limited mass effect and reflecting the characteristics of the clinical tumor (A–H). Staining with hematoxylin/ eosin of brain tissue could reveal the presence of this highly infiltrating tumor only at late times of tumor development and elevated magnification (d138; N,O). Mouse ID numbers 32.8, 32.19 and 32.14 are indicated for the sake of reference with the days (d) of tumor development. (Q) Beneficial effect on animal survival after RT hyperfractionation in the presence of KU60019. At d25 of tumor development, animals were i.c.-injected by CED in the surgery room with 12.5 µl of 250 µM KU60019 (blue) or vehicle (ethanol in 0.9% NaCl - black) followed by irradiation with 15 fractions of 0.5 Gy IR delivered to the head of mice 2–4 days apart from d29 until d94 (total dose delivered: 7.5 Gy). Kaplan-Meier survival curves are shown. Median survival of KU60019- treated animals was increased (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891) with respect to controls. Figure 7. The GIC-driven pediatric 239/12 tumor and its radiosensitization by KU60019. (A–D) Patient’s tumor at admission: axial and coronal brain MRI FLAIR images demonstrate a diffusely infiltrating lesion (arrows, A,B) involving the cortical-subcortical region of the left temporo-parietal junction without contrast enhancement (C,D, post-contrast T1-weighted images). (E–H) Patient’s tumor at progression: axial and coronal FLAIR images shows subtle diffuse infiltration of the brain parenchyma adjacent to the surgical bed and extending to the ipsilateral insular and temporal region with concomitant bi-thalamic involvement (arrows, E,F). Results (D) Determination of tumor volumes by region segmentation software (GRES). Top: example of segmented tumor. The mouse ID number (22.14) is indicated for the sake of reference. Bottom: mean ± SEM volumes of vehicle + (2.5 Gy × 3) (black) and KU60019+ (2.5 Gy × 3) (blue) – treated tumors. The tumor volumes in KU60019+ (2.5 Gy × 3) – treated mice were significantly reduced as compared to vehicle + (2.5 Gy × 3) -treated mice. Mean ± SEM of vehicle+ (2.5 Gy × 3): 140 ± 17 mm3; mean ± SEM of KU60019+ (2.5 Gy × 3): 97 ± 11 mm3; P: 0.047 after two-tailed unpaired t-test. (E) Further beneficial effect on animal survival after RT hyperfractionation. At d16 of tumor development, animals were i.c.-injected by CED in the surgery room with 12.5 µl of 250 µM KU60019 (blue) or vehicle (ethanol in 0.9% NaCl - black) followed by irradiation with 15 fractions of 0.5 Gy IR delivered to the head of mice 2–4 days apart (total dose delivered: 7.5 Gy). Kaplan-Meier survival curves are shown. Median survival of KU60019-treated animals was further increased (105 vs 89 days; ratio: 0.847; 95% CI of ratio: 0.4969 to 1.198; P: 0.0417) with respect to controls as compared to standard 2.5 Gy fractions as shown in A. were euthanized, the brains explanted and the content of KU60019 in brain extracts measured by HPLC/MS as described under Materials and Methods and in13. No significant amounts of KU60019 were detected in the brain of animals even under those conditions of chronic administration (Fig. 2B), confirming the impermeability of the BBB to KU60019. 8 Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w www.nature.com/scientificreports/ re.com/scientificreports/ The GIC-driven adult COMI tumor. Biddlestone-Thorpe and coworkers have reported that ATM kinase inhibition by KU60019 preferentially sensitizes TP53 mutant orthotopic gliomas to IR17 In that study 250µM Figure 7. The GIC-driven pediatric 239/12 tumor and its radiosensitization by KU60019. (A–D) Patient’s tumor at admission: axial and coronal brain MRI FLAIR images demonstrate a diffusely infiltrating lesion (arrows, A,B) involving the cortical-subcortical region of the left temporo-parietal junction without contrast enhancement (C,D, post-contrast T1-weighted images). (E–H) Patient’s tumor at progression: axial and coronal FLAIR images shows subtle diffuse infiltration of the brain parenchyma adjacent to the surgical bed and extending to the ipsilateral insular and temporal region with concomitant bi-thalamic involvement (arrows, E,F). Results Post-contrast T1-weighted images (G,H) do not show blood brain barrier disruption or necrotic areas. (I) GIC i: the GIC component of the tumor was isolated immediately after surgery. Under matrigel-coating and serum-free conditions, the cells grew and layered onto a monolayer, maintaining intact self-renewal capacity. (J) GIC ii: the 239/12 GIC could be effectively radiosensitized in vitro by exposure to 1 µM KU60019 30 min prior to irradiation with three 2.5 Gy IR fractions [blue versus black circles; reproduced from13, with permission]. The in vitro growth pattern of unirradiated cells is indicated by black squares. (K–O) Orthotopic tumor. 2 × 105 239/12 serum-free grown GIC were stereotactically injected into the left corpus striatum of NOD SCID mice. Immunostaining with an antibody directed against human nestin revealed at both d29 (K) and d138-150 (M,P) an infiltrating, gliomatosis-like, growth pattern of the orthotopic tumor exerting limited mass effect and reflecting the characteristics of the clinical tumor (A–H). Staining with hematoxylin/ eosin of brain tissue could reveal the presence of this highly infiltrating tumor only at late times of tumor development and elevated magnification (d138; N,O). Mouse ID numbers 32.8, 32.19 and 32.14 are indicated for the sake of reference with the days (d) of tumor development. (Q) Beneficial effect on animal survival after RT hyperfractionation in the presence of KU60019. At d25 of tumor development, animals were i.c.-injected by CED in the surgery room with 12.5 µl of 250 µM KU60019 (blue) or vehicle (ethanol in 0.9% NaCl - black) followed by irradiation with 15 fractions of 0.5 Gy IR delivered to the head of mice 2–4 days apart from d29 until d94 (total dose delivered: 7.5 Gy). Kaplan-Meier survival curves are shown. Median survival of KU60019- treated animals was increased (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891) with respect to controls. The GIC-driven adult COMI tumor. Biddlestone-Thorpe and coworkers have reported that ATM kinase inhibition by KU60019 preferentially sensitizes TP53 mutant orthotopic gliomas to IR17. In that study, 250 µM KU60019 was i.c.- delivered by osmotic pumps or CED to orthotopic tumors driven by the established U1242 and U87 cell lines carrying the R175H and D281G TP53 mutations, respectively. Here we wished to investigate Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 9 www.nature.com/scientificreports/ Figure 8. Proposed mechanism for in vivo radiosensitization by ATMi in GIC-driven orthotopic tumors. Results This lesion, compatible in the first instance with a HGG lesion, exerted considerable mass effect on the adjacent structures (Fig. 3B,D). The patient was subjected to surgery to remove the lesion and histological examination of the tumor led to diagnosis of GB (WHO grade IV). Despite postoperative chemo- and radiotherapy the patient eventually died 413 days after diagnosis. Immediately after surgery, tissue surplus to diagnostic requirements was processed for isolation of GIC, according to10. Under matrigel-coating and serum-free conditions, the cells grew and layered into a monolayer (Fig. 3E), maintaining intact self-renewal capacity. In the absence of matrigel, cells predominantly grew forming suspended neuros- pheres. The COMI GIC could be effectively dislodged from their quiescence (population doubling time decreased from 60 to 49 hours) and radiosensitized, after exposure to 1 µM KU60019 30 min prior to irradiation with three 2.5 Gy IR fractions (Fig. 3F)12. Removal of growth factors and addition of 10% FCS to the proliferation medium, after approximately two weeks induced GIC differentiation with acquisition of astrocytic morphology, altered refractory index (Fig. 3G)12, increased expression of a number of differentiation markers including GFAP and beta III tubulin and refractoriness to radiosensitization by KU6001910,19. 3 × 105 COMI serum-free grown GIC were then stereotactically injected into the left corpus striatum of NOD SCID mice. Staining with hematoxylin/ eosin of brain tissue sections revealed 52 (Fig. 3I,J) and 108 (Fig. 3K) days later a growth pattern of the ortho- topic tumor reflecting the characteristics of the clinical tumor: a voluminous expansive lesion (Fig. 3I,K) with whether KU60019 displayed similar radiosensitization properties towards GIC-driven orthotopic tumors that might more faithfully mimic the growth patterns of clinical tumors. The selected adult patient was a 48-year-old man who, in full well-being, complained about the onset of two episodes of violent headache in the left parieto-occipital region which resolved spontaneously. In the following days his relatives noticed a progressive space-time disorientation so that, after neurological examination, the patient was subjected to brain MRI which showed the presence of a voluminous expansive lesion in the left temporo-parieto-occipital site, associated with perilesional edema, with a central necrotic-colliquative component and an irregular wall that showed remarkable and inhomogeneous intake of contrast medium (Fig. 3A–D). This lesion, compatible in the first instance with a HGG lesion, exerted considerable mass effect on the adjacent structures (Fig. 3B,D). Results The patient was subjected to surgery to remove the lesion and histological examination of the tumor led to diagnosis of GB (WHO grade IV). Despite postoperative chemo- and radiotherapy the patient eventually died 413 days after diagnosis. Immediately after surgery, tissue surplus to diagnostic requirements was processed for isolation of GIC, according to10. Under matrigel-coating and serum-free conditions, the cells grew and layered into a monolayer (Fig. 3E), maintaining intact self-renewal capacity. In the absence of matrigel, cells predominantly grew forming suspended neuros- pheres. The COMI GIC could be effectively dislodged from their quiescence (population doubling time decreased from 60 to 49 hours) and radiosensitized, after exposure to 1 µM KU60019 30 min prior to irradiation with three 2.5 Gy IR fractions (Fig. 3F)12. Removal of growth factors and addition of 10% FCS to the proliferation medium, after approximately two weeks induced GIC differentiation with acquisition of astrocytic morphology, altered refractory index (Fig. 3G)12, increased expression of a number of differentiation markers including GFAP and beta III tubulin and refractoriness to radiosensitization by KU6001910,19. 3 × 105 COMI serum-free grown GIC were then stereotactically injected into the left corpus striatum of NOD SCID mice. Staining with hematoxylin/ eosin of brain tissue sections revealed 52 (Fig. 3I,J) and 108 (Fig. 3K) days later a growth pattern of the ortho- topic tumor reflecting the characteristics of the clinical tumor: a voluminous expansive lesion (Fig. 3I,K) with whether KU60019 displayed similar radiosensitization properties towards GIC-driven orthotopic tumors that might more faithfully mimic the growth patterns of clinical tumors. The selected adult patient was a 48-year-old man who, in full well-being, complained about the onset of two episodes of violent headache in the left parieto-occipital region which resolved spontaneously. In the following days his relatives noticed a progressive space-time disorientation so that, after neurological examination, the patient was subjected to brain MRI which showed the presence of a voluminous expansive lesion in the left temporo-parieto-occipital site, associated with perilesional edema, with a central necrotic-colliquative component and an irregular wall that showed remarkable and inhomogeneous intake of contrast medium (Fig. 3A–D). This lesion, compatible in the first instance with a HGG lesion, exerted considerable mass effect on the adjacent structures (Fig. 3B,D). The patient was subjected to surgery to remove the lesion and histological examination of the tumor led to diagnosis of GB (WHO grade IV). Results (A) RT initially exerts its limited cytocidal effect on the radioresistant, quiescent GIC component of glioma (indicated in red). Once RT is over, the GICs resume proliferation driving tumor progression. (B) ATMi such as KU60019 may radiosensitize the GICs and initially augment their killing by dislodging them from quiescence but, once RT is over, the persistent ATMi-mediated stimulus to proliferate causes the GIC recovery and expansion, thus mitigating the initial radiosensitization benefit. (C) Hyperfractionation of RT has little or no beneficial effect in limiting tumor progression as compared to standard RT fractionation in (A). (D) In the presence of ATMi, RT hyperfractionation may allow to take advantage of the long-term proliferation-boosting, radiosensitizing effect of the drug towards the GIC component of the tumor and significantly delay tumor progression. Figure 8. Proposed mechanism for in vivo radiosensitization by ATMi in GIC-driven orthotopic tumors. (A) RT initially exerts its limited cytocidal effect on the radioresistant, quiescent GIC component of glioma (indicated in red). Once RT is over, the GICs resume proliferation driving tumor progression. (B) ATMi such as KU60019 may radiosensitize the GICs and initially augment their killing by dislodging them from quiescence but, once RT is over, the persistent ATMi-mediated stimulus to proliferate causes the GIC recovery and expansion, thus mitigating the initial radiosensitization benefit. (C) Hyperfractionation of RT has little or no beneficial effect in limiting tumor progression as compared to standard RT fractionation in (A). (D) In the presence of ATMi, RT hyperfractionation may allow to take advantage of the long-term proliferation-boosting, radiosensitizing effect of the drug towards the GIC component of the tumor and significantly delay tumor progression. 10 Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w whether KU60019 displayed similar radiosensitization properties towards GIC-driven orthotopic tumors that might more faithfully mimic the growth patterns of clinical tumors. The selected adult patient was a 48-year-old man who, in full well-being, complained about the onset of two episodes of violent headache in the left parieto-occipital region which resolved spontaneously. In the following days his relatives noticed a progressive space-time disorientation so that, after neurological examination, the patient was subjected to brain MRI which showed the presence of a voluminous expansive lesion in the left temporo-parieto-occipital site, associated with perilesional edema, with a central necrotic-colliquative component and an irregular wall that showed remarkable and inhomogeneous intake of contrast medium (Fig. 3A–D). KU60019 radiosensitizes GIC-driven adult orthotopic tumors and improves animal survivalhhi KU60019 radiosensitizes GIC-driven adult orthotopic tumors and improves animal survival. The (12,5 µl of 250 µM) dose employed by Biddlestone-Thorpe and coworkers was confirmed in our studies to be effective in inhibiting the ATM protein with consequent release of ATM-mediated checkpoints and induc- tion of the KI67 proliferative marker in the GIC-driven orthotopic tumors10,12,13. Figure 5 shows the inhibition of ATM protein by KU60019 in the irradiated TP53-deficient COMI adult orthotopic tumors. In Fig. 5A,B COMI tumors were treated with two radiosensitizing cycles, the first consisting of one CED with 12.5 µl of 250 µM KU60019 followed by three 2.5 Gy IR fractions and the second of one CED with the same KU60019 dose fol- lowed by two 2.5 Gy IR fractions. Significant activation of ATM protein, as indicated by phosphorylation at S1981 detected through IF foci with a specific antibody, was observed in vehicle + IR - treated mice (Fig. 5A central panel, Fig. 5B black symbols) as compared to untreated mice (Fig. 5A left panel, Fig. 5B green symbols) (25.5 vs 25.12 mean difference: +57% of p(S1981)ATM+ cells; 95%; CI of difference +50 to +64%). The p(S1981)ATM foci detected 24 hours after the fifth 2.5 Gy IR fraction were significantly reduced in the KU60019-treated tumors (Fig. 5A right panel, Fig. 5B blue symbols) (25.2 vs 25.5 mean difference: −26% of p(S1981)ATM+ cells; 95% CI of difference −19 to −33%). These results confirm and extend previous evidences of effective ATM inhibition by the employed doses of KU60019 as determined by TP53-phosphorylation assays, radiosensitization of the same GIC lines used for orthotopic tumor development and the induction of the proliferative marker KI67 in vivo, consequent to release of the G1/S, intra-S and G2/M checkpoints10,12,13. Figure 5C,D show that the elevated pro- liferative activity in the untreated tumor (Fig. 5C left panel, mouse 12.1, Fig. 5D green symbols) was reduced after RT (Fig. 5C central panel, mouse 27.13, Fig. 5D black symbols) (27.13 vs 12.1 mean difference: −25% of KI67+ cells, 95% CI of difference −18 to −32%). KU60019 exposure induced proliferation boosting (Fig. 5C right panel, mouse 27.3, Fig. 5D blue symbols) (27.3 vs 27.13 mean difference: +12% of KI67+ cells; 95% CI of difference +5 to +19%). Importantly, the KU60019-mediated stimulus to GIC-mitosis in vivo was prolonged in time (Fig. Results Despite postoperative chemo- and radiotherapy the patient eventually died 413 days after diagnosis. Immediately after surgery, tissue surplus to diagnostic requirements was processed for isolation of GIC, according to10. Under matrigel-coating and serum-free conditions, the cells grew and layered into a monolayer (Fig. 3E), maintaining intact self-renewal capacity. In the absence of matrigel, cells predominantly grew forming suspended neuros- pheres. The COMI GIC could be effectively dislodged from their quiescence (population doubling time decreased from 60 to 49 hours) and radiosensitized, after exposure to 1 µM KU60019 30 min prior to irradiation with three 2.5 Gy IR fractions (Fig. 3F)12. Removal of growth factors and addition of 10% FCS to the proliferation medium, after approximately two weeks induced GIC differentiation with acquisition of astrocytic morphology, altered refractory index (Fig. 3G)12, increased expression of a number of differentiation markers including GFAP and beta III tubulin and refractoriness to radiosensitization by KU6001910,19. 3 × 105 COMI serum-free grown GIC were then stereotactically injected into the left corpus striatum of NOD SCID mice. Staining with hematoxylin/ eosin of brain tissue sections revealed 52 (Fig. 3I,J) and 108 (Fig. 3K) days later a growth pattern of the ortho- topic tumor reflecting the characteristics of the clinical tumor: a voluminous expansive lesion (Fig. 3I,K) with Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 10 www.nature.com/scientificreports/ an irregular infiltrating wall (Fig. 3J) that exerted considerable mass effect on the adjacent structures (Fig. 3K). Immunostaining at d52 revealed that most of the orthotopic tumor expressed the stem cell marker nestin, indi- cating its stem cell-driven character (Fig. 3H). CED and RT of GIC-driven orthotopic tumors. Due to the impermeability of the BBB to KU60019 (Fig. 2) and based on previous data, in order to effectively radiosensitize the brain tumor, KU60019 must be delivered i.c. and multiple radiosensitization cycles have to be carried out12,13,17. To this aim and in accordance with the principle of 3Rs (replacement, refinement, reduction) in animal research, two major technical improvements were adopted. First, since animals would not survive to frequent systemic ketamine-xylazine anesthesia, a less toxic isoflurane (inhalation anesthetic) delivery system was employed (Fig. 1A,B). Second, one on-site dedicated animal irradiator equipped with a collimator directing the radiation beam to the brain only was calibrated and employed (Fig. 1B–O).h p y ( g ) The maximum deliverable radiation dose to the mice bearing the COMI tumor was preliminarily determined (Fig. 4A). Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w Results In radiosensitive NOD-SCID mice bearing orthotopic adult COMI tumors, a dose – therapy relationship was observed in the range 0–10 Gy, delivered by 2.5 Gy fractions to the head every 24 hours (2.5 vs 10 Gy, 91 vs 136 days of median survival, ratio: 0.669, 95% CI of ratio: 0.351 to 0.986, P: 0.0040 indicated by two asterisks; 7.5 vs 10 Gy, 100.5 vs 136 days of median survival, ratio: 0.739, 95% CI of ratio: 0.328 to 1.150, P: 0.0310 indicated by one asterisk; Fig. 4A, right). After addition of one fifth 2.5 Gy fraction [bringing the total cumulative (∑=) dose to 12.5 Gy] all animals rapidly died (in 24–48 hours), indicating that a cumulative radiation dose higher than 10 Gy to the head is invariably lethal to the DNA repair–deficient NOD-SCID mice (10 vs 12.5 Gy, 136 vs 65 days of median survival, ratio: 2.092, 95% CI of ratio: 1.756 to 2.428, P: 0.0005 indicated by three asterisks; 7.5 vs 12.5 Gy, 100.5 vs 65 days of median survival, ratio: 1.546, 95% CI of ratio: 1.155 to 1.937, P < 0.0001 indicated by three asterisks; 2.5 vs 12.5 Gy, 91 vs 65 days of median survival, ratio: 1.400, 95% CI of ratio: 1.095 to 1.705, P:0.0034 indicated by two asterisks; Fig. 4A, right). The multiple defects in both innate and adaptive immunity in NOD-SCID mice are necessary in order to achieve reproducible engraftment of GIC-driven orthotopic tumors in virtually 100% of mice21. For comparison and in the view of the possible clinical translation of these studies, we wished to deepen our understanding of radiation toxicity to the head of wild type DNA repair competent C57/Black mice (Fig. 4B). No significant variation of survival (Fig. 4C), hematocrit (HCT-Fig. 4D), hemoglobin (HGB-Fig. 4E) or brain his- tology (Fig. 4F) were observed after twenty-five 2.5 Gy fractions to the head each other 24 hours apart (cumulative radiation dose: 62.5 Gy) (Fig. 4B), thus showing the feasibility of an elevated number of radiosensitization cycles in DNA repair competent organisms such as GB patients usually are. Discussion In agreement with previous studies13,17, KU60019 could not cross the BBB even after chronic i.p administrations (Fig. 2). Hence, in order to exploit its remarkable and specific radiosensitizing capacity towards GIC, KU60019 was delivered i.c. to the tumor site. This was accomplished by CED12,13, an efficient and clinically used i.c. delivery technique22, allowing for steady, finely-tunable locoregional drug infusion13,17. CED was effective allowing signif- icant survival improvements of mice bearing the TP53-deficient COMI tumor treated with one single radiosen- sitization cycle consisting of one KU60019 administration followed by three 2.5 Gy IR fractions (Fig. 6). Albeit those results were encouraging, being obtained in orthotopic tumors that faithfully mimic the growth pattern of the clinical tumors, the survival improvements were lower than those observed in previous studies performed on orthotopic tumors driven by the established U87 and U1242 glioma cell lines bearing specific TP53 mutations. Several factors may have contributed to this difference. IR fractions were slightly lower (2.5 Gy) than those used by Biddlestone-Thorpe and coworkers (3 Gy) with orthotopic tumor-bearing athymic nude mice, due to the radi- osensitivity of NOD-SCID mice, whose deeper immunodeficiency level is strictly required for reproducible and complete (>95%) engraftment of GIC-driven tumors21. Further, each cell line is different in terms of doubling time and aggressiveness and tumor volume/mass represent critical factors when different types of orthotopic tumors are compared. Lastly,.the starting of the radiosensitization was delayed (d16-d42 of tumor development) as compared to U87-U1242-driven tumors (d6-d7 of tumor development)17, due to the significantly slower growth rate of orthotopic GIC-driven tumors. While untreated U87-U1242 tumors usually kill xenografted mice in less than 50 days, GIC-driven tumors employ more than 90 days[17 Frosina et al., unpublished], reflecting the slower proliferation rate of GIC7,16,19. We believe unlikely that the above minor protocol variations may have caused the significant decrease of KU60019-mediated tumor radiosensitization observed here in comparison to the experiments reported by Biddlestone-Thorpe and coworkers17. The validation of ATM inhibition in the tumor by p(S1981)ATM and KI67 immunostaining under our experimental conditions (Fig. 5), supports this opinion and indicates the following three reasons as more likely explaining the discrepancy with results reported by Biddlestone-Thorpe and coworkers17: first, the nature and extent of the TP53 deficiency in the orthotopic tumors used in the two studies have probably contributed to the differences. KU60019 radiosensitizes pediatric GIC-driven orthotopic tumors. 239/12 was a 14 b ith i k bl li i l hi t h t d t th E U it f Gi i KU60019 radiosensitizes pediatric GIC-driven orthotopic tumors. 239/12 was a 14 years-old boy with a previous unremarkable clinical history who presented at the Emergency Unit of Giannina Gaslini Children’s Hospital because of generalized seizures unresponsive to first line therapy. After CT and MR imaging suggestive of a HGG, biopsy of the lesion led to diagnosis of anaplastic astrocytoma (WHO grade III), positive for GFAP, negative for TP53 overexpression, exhibiting a KI67 proliferation index of 25%. A subtotal resection was thereafter performed. Patient was referred for focal RT and was treated using intensity modulated radiotherapy (IMRT) to a total dose of 60 Gy in 2 Gy daily fractions 5 days per week. Simultaneously, temozolomide chemo- therapy was started. The patient died because of progressive disease at d551 after diagnosis.hi pyh p p gt g Figure 7A–H show the MRI of the 239/12 pediatric patient’s tumor. The growth pattern is that of an infil- trating, gliomatosis-like tumor that, differently from the adult COMI tumor, does not exert a significant mass effect at early stages of development. Similar to COMI, the tumor growth pattern was faithfully repro- duced in the GIC-driven orthotopic tumor model. Pediatric 239/12 GIC cultured under adherent conditions in matrigel-coated flasks are shown in Fig. 7I. These cells can be radiosensitized in vitro by exposure to 1 µM KU60019 30 minutes prior to irradiation (Fig. 7J – blue versus black circles)13. The in vitro growth pattern of unir- radiated cells is indicated by black squares. After orthotopic injection of 2 × 105 239/12 GIC in NOD SCID mice, the tumor engrafted with elevated (>95%) frequency but its progression was very slow and one month later (d29) no tumor was evident after hematoxylin/eosin staining (Fig. 7L). At this stage, immunostaining with an antibody for human nestin allowed to detect scattered, isolated tumor cells in the normal brain parenchyma (Fig. 7K). At d138-d150, a highly infiltrating tumor with little mass effect on adjacent structures could be observed after staining for nestin (Fig. 7M,P). At this late stage of development, tumor cells could be detected at elevated mag- nification after H/E staining as well (Fig. 7N,O). KU60019 radiosensitizes GIC-driven adult orthotopic tumors and improves animal survivalhhi 5C,D), since the significantly increased proliferative activity of GIC, as indicated by KI67 tumor staining, was detected 42 days (d108 of tumor development) after the last drug CED (d66 of tumor development) (Fig. 5C,D).it y ( p )t g ( p ) ( g ) Significant elongation of median survival was observed in COMI-bearing animals after one single CED of KU60019 (performed at d41 of tumor development) followed four-seven days later (d45,46,48) by three 2.5 Gy RT fractions as compared to vehicle + IR - treated mice (101 vs 91 days of median survival; ratio: 0.901; 95% CI of ratio: 0.5384 to 1.264; P: 0.0409 indicated by one asterisk; Fig. 6A). Histological analyses performed at eutha- nasia, showed in both groups large tumor masses whose development was delayed in the KU60019- treated ani- mals (Fig. 6B). 3T MRI imaging performed at d62 and determination of tumor volumes by GRES, consistently showed significant reductions of tumor volumes in radiosensitized mice [mean ± SEM of vehicle + (2.5 Gy × 3): 140 ± 17 mm3; mean ± SEM of KU60019 + (2.5 Gy × 3): 97 ± 11 mm3; P: 0.047 after two-tailed unpaired t-test indicated by one asterisk (Fig. 6C,D)]20. The reduced tumor volumes as assessed by histological analysis and MRI were considered as a secondary experimental outcome in this study. Increasing the radiation dose to total 10 Gy (∑ = 10 Gy) by addition of a fourth 2.5 Gy fraction to mice intracranially infused with KU60019 using osmotic pumps, could not further improve longevity. On the contrary, the beneficial effect observed in Fig. 6A was lost (Supplementary Fig. 2). To better exploit the prolonged proliferation-boosting, radiosensitizing effect Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 11 www.nature.com/scientificreports/ of KU60019, an hyperfractionated RT schedule was adopted (Fig. 6E). Tumor bearing mice were i.c.-injected at d16 of tumor development by CED with 12.5 µl of 250 µM KU60019 (blue) or vehicle (2.5% ethanol in 0.9% NaCl - black) followed by irradiation with 15 fractions of 0.5 Gy IR delivered to the head of mice 2–4 days apart from d20 to d76 of tumor development (total dose delivered: 7.5 Gy). Median survival of KU60019-treated animals was further increased with respect to controls as compared to standard fractionation with 2.5 Gy doses in Fig. KU60019 radiosensitizes pediatric GIC-driven orthotopic tumors. 239/12 was a 14 b ith i k bl li i l hi t h t d t th E U it f Gi i Exposure of mice bearing 239/12 orthotopic tumor to 12.5 µl of 250 µM KU60019 via CED performed at d25 of tumor development followed by 15 IR fractions of 0.5 Gy to the head delivered from d29 to d94, resulted in a trend to increased median survival as compared to mice treated by vehicle + IR (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891; Fig. 7Q). KU60019 radiosensitizes GIC-driven adult orthotopic tumors and improves animal survivalhhi 6A (105 vs 89 days; ratio: 0.847; 95% CI of ratio 0.4969 to 1.198; P = 0.0417 indicated by one asterisk; Fig. 6E) showing that it is possible to take advantage of the long term proliferation-boosting, radiosensitizing effect of KU60019 over the GIC component of the tumor by adoption of an hyperfractionated RT schedule. Discussion Although the COMI tumor has a low expression level of the TP53 gene as assessed by quantitative polymerase chain reaction (qPCR)12, its TP53 gene sequence is wild type thus making possible that the residual TP53 activity is sufficient to fade the in vivo radiosensitization response to KU60019. The highest radiosensitization efficacy of ATM inhibition by KU60019 was observed by Biddlestone-Thorpe and coworkers towards U87 tumors harboring the TP53 D281G mutation. Since the D281G is a gain of function TP53 mutation causing disruption of a spindle checkpoint and promot- ing genetic instability23, it might be possible that disruption of DDR checkpoints due to ATM inhibition and of spindle checkpoints linked to the D281G mutation synergized and cooperated to inhibit the tumor growth in the Biddlestone-Thorpe study. In clinical perspective, albeit we confirm that loss of TP53 function (as well as gain of Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 12 www.nature.com/scientificreports/ PI3K function) do enhance the response to KU60019 radiosensitization12, we have shown by analysis of multiple patients, that even tumors with unfavorable properties (elevated TP53 and reduced PI3K wild type expressions) can be radiosensitized by KU60019, although less promptly than GIC-driven tumours bearing the “responder” profile, thus making reasonable the treatment of an elevated percentage of HGG patients with ATMi13.f profile, thus making reasonable the treatment of an elevated percentage of HGG patients with ATMi . Second, the pharmacodynamics of KU60019 may be related as well to the different responses of the two types of orthotopic tumor used in17 and here: the GIC proliferation-stimulating effect exerted by KU60019 that reaches a peak 96 hours (four days) after drug infusion to the orthotopic tumor13, may in fact sustain the growth of the GIC-driven tumors well beyond that time, as indicated by the persistent, significant increase of KI67-positive cells 42 days after the last drug infusion (d108-d66; Fig. 5C,D). The KU60019-mediated DDR inhibition may thus not be completely reversible and the prolonged stimulus to proliferate, persisting after the end of RT, may cancel most radiosensitizing benefit initially achieved on the slow-growing GIC-driven tumors (Fig. 8) (but not on the fast-growing/killing U87/U1242 tumors used in17). In this regard, it is not surprising that the survival benefit given by the hyperfractionated radiosensitization schedule was mitigated and did not achieve statistical significance in pediatric 239/12-bearing mice (Fig. 7Q). References 1. Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. & Altman, D. G. Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research. Plos Biol. 8(6) (2010). p g ( ) ( ) 2. Crocetti, E. et al. Epidemiology of glial and non-glial brain tumours in Europe. Eur J Cancer. 48(10), 1532–1542 (2012). 3. Ahmed, R., Oborski, M. J., Hwang, M., Lieberman, F. S. & Mountz, J. M. Malignant gliomas: current perspectives in dia treatment, and early response assessment using advanced quantitative imaging methods. Cancer Manag Res. 6, 149–170 (20 y p g q g g g 4. Gately, L., McLachlan, S. A., Dowling, A. & Philip, J. Life beyond a diagnosis of glioblastoma: a systematic review of the literature J Cancer Surviv. 11(4), 447–452 (2017). 5. Osuka, S. & Van Meir, E. G. Overcoming therapeutic resistance in glioblastoma: the way forward. J Clin Invest. 127(2), 415–426 (2017). 6. Bao, S. et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 444(7120), 756–760 (2006). ( ) 7. Chen, W., Dong, J., Haiech, J., Kilhoffer, M. C. & Zeniou, M. Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy. Stem Cells Int. 2016, 1740936 (2016). 7. Chen, W., Dong, J., Haiech, J., Kilhoffer, M. C. & Zeniou, M. Cancer Stem Cell Quiesce Cancer Therapy. Stem Cells Int. 2016, 1740936 (2016). h py 8. Paull, T. T. Mechanisms of ATM Activation. Annu Rev Biochem. 84, 711–738 (2015). h 8. Paull, T. T. Mechanisms of ATM Activation. Annu Rev Bioc 9. Golding, S. E. et al. Improved ATM kinase inhibitor KU-60019 radiosensitizes glioma cells, compromises insulin, AKT and ERK prosurvival signaling, and inhibits migration and invasion. Mol Cancer Ther. 8(10), 2894–2902 (2009).i 9. Golding, S. E. et al. Improved ATM kinase inhibitor KU-60019 radiosensitizes gliom prosurvival signaling, and inhibits migration and invasion. Mol Cancer Ther. 8(10), 2 h 10. Raso, A. et al. Characterization of glioma stem cells through multiple stem cell markers and their specific sensitization to double- strand break-inducing agents by pharmacological inhibition of ataxia telangiectasia mutated protein. Brain Pathol. 22(5), 677–688 (2012). 1. Golding, S. E. et al. Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control. Cell Cycle. 11(6), 1167–1173 (2012).fi g y 12. Vecchio, D. et al. Discussion Because of its histopathologic features and gliomatosis-like growth mode, the 239/12 tumor did not exert considerable mass effect early after implant/diagnosis (Fig. 7A–H; K–P) and caused the death of the affected organisms more slowly than the adult COMI tumor [median survivals of vehicle + (0.5 Gy × 15) – treated mice: 167 and 89 days, respectively (88% longer in 239/12 than in COMI); post-diagnosis survival of chemo/radiotherapy-treated patients: 551 and 413 days, respectively (33% longer in 239/12 than in COMI)]. Thus, in 239/12 bearing mice, the tumor had longer time to recover aggressiveness after the last, 15th radiosensitized fraction [92 days = 186 (median survival in radiosensitized 239/12 mice with 15 frac- tions) - 94 (day of 15th RT fraction to 239/12 mice) – Fig. 7Q] as compared to COMI mice [29 days = 105 (median survival in radiosensitized COMI mice with 15 fractions) −76 (day of 15th fraction to COMI mice) Fig. 6E]. In the clinical perspective, the growth properties of each tumor will have to be carefully considered in order to plan the most effective radiosensitization protocol. In this setting, the persistent pharmacodynamics effect of KU60019 on GIC-driven tumors (Fig. 5C,D) may represent a drug administration advantage: one single intraop- erative KU60019 delivery will exert its radiosensitizing effects over a significant number of post-surgery adjuvant radiotherapeutic fractions24. If required, subsequent i.c. administrations might follow by different techniques including CED, an increasingly employed administration procedure in the clinics22, nanoparticle vectors25, per- meabilization enhanced delivery and others26.h y Third, primary GIC driven-tumors such as COMI and 239/12 maintain, similar to the clinical tumors, elevated infiltrative behavior and intratumoral heterogeneity (Figs 3H–J and 7K–P) conferring higher refractoriness to the radiosensitization procedure, as compared to U87 and U1242 tumors which are poorly infiltrating and more contained, thus providing an accessible, physical target for KU60019. Further, the DNA profile of currently used U87 is different from that of the original cells thus indicating a possible mix-up with another human GB cell line of unknown origin27. The use of multiple GIC-driven orthotopic tumors that faithfully mimic the patient’s tumor (Figs 3 and 7) may be essential to develop effective therapeutic protocols for HGG transferable to the clinics. Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w References B , g y g g g ( ), ( ) 27. Allen, M., Bjerke, M., Edlund, H., Nelander, S. & Westermark, B. Origin of the U87MG glioma cell line: Good news and bad news. Sci Transl Med. 8(354), 354re3 (2016). Acknowledgements g We thank Carlo Palmisani, Radiation Oncology, IRCCS Ospedale Policlinico San Martino, for skillful technical work on lead animal protection masks; Michele Cilli, Laura Emionite, Emanuela Ognio, Animal Facility, IRCCS Ospedale Policlinico San Martino for support to animal experimentation; Elisabetta Rosellini, Mutagenesis & Cancer Prevention, IRCCS Ospedale Policlinico San Martino for administrative work. G.F. and A.D. are supported by Compagnia San Paolo, Turin, Italy, Grant number: 2015.0643. A.R. and M.L.G. are supported by Associazione Italiana Ricerca Tumori Cerebrali del Bambino (ARTUCEBA), Genova, Italy. Grant number: 917-17. We thank Carlo Palmisani, Radiation Oncology, IRCCS Ospedale Policlinico San Martino, for skillful technical work on lead animal protection masks; Michele Cilli, Laura Emionite, Emanuela Ognio, Animal Facility, IRCCS Ospedale Policlinico San Martino for support to animal experimentation; Elisabetta Rosellini, Mutagenesis & Cancer Prevention, IRCCS Ospedale Policlinico San Martino for administrative work. G.F. and A.D. are supported by Compagnia San Paolo, Turin, Italy, Grant number: 2015.0643. A.R. and M.L.G. are supported by Associazione Italiana Ricerca Tumori Cerebrali del Bambino (ARTUCEBA), Genova, Italy. Grant number: 917-17. Author Contributions Conceptualization: Guido Frosina. Data curation: Guido Frosina, Jean Louis Ravetti, Mauro Fella, Fabrizio Levrero, Diana Marcello, Daniela Marubbi, Giovanni Morana, Aldo Profumo, Alessandro Raso, Francesca Rosa, Antonio Verrico, Gianluigi Zona, Antonio Daga. Formal analysis: Guido Frosina, Jean Louis Ravetti, Fabrizio Levrero, Giovanni Morana, Aldo Profumo, Alessandro Raso, Francesca Rosa, Antonio Verrico, Gianluigi Zona and Antonio Daga. Funding acquisition: Guido Frosina, Maria Luisa Garrè, Alessandro Raso, Antonio Daga. Investigation: Guido Frosina, Jean Louis Ravetti, Mauro Fella, Diana Marcello, Daniela Marubbi, Giovanni Morana, Aldo Profumo, Alessandro Raso, Francesca Rosa, Stefano Vagge, Donatella Vecchio, Antonio Verrico, Gianluigi Zona, Antonio Daga. Methodology: Guido Frosina, Jean Louis Ravetti, Mauro Fella, Fabrizio Levrero, Diana Marcello, Daniela Marubbi, Aldo Profumo, Alessandro Raso, Francesca Rosa, Stefano Vagge, Antonio Daga. Project administration: Guido Frosina. Resources: Guido Frosina, Maria Luisa Garrè, Alessandro Raso, Antonio Daga. Software: Guido Frosina, Fabrizio Levrero, Aldo Profumo, Alessandro Raso, Francesca Rosa, Antonio Daga. Supervision: Guido Frosina, Renzo Corvò, Maria Luisa Garrè, Michele Mussap, Carlo Emanuele Neumaier, Gianluigi Zona. Validation: Guido Frosina, Jean Louis Ravetti, Mauro Fella, Fabrizio Levrero, Aldo Profumo, Alessandro Raso, Stefano Vagge, Gianluigi Zona, Antonio Daga. Writing ± original draft: Guido Frosina, Renzo Corvò, Fabrizio Levrero, Giovanni Morana, Aldo Profumo, Alessandro Raso, Gianluigi Zona, Antonio Daga. References Predictability, efficacy and safety of radiosensitization of glioblastoma-initiating cells by the ATM inhibitor KU- 60019. Int J Cancer 135(2), 479–491 (2014).fi 13. Vecchio, D. et al. Pharmacokinetics, pharmacodynamics and efficacy on pediatric tumors of the glioma radiosensitizer KU60019. Int J Cancer 136(6), 1445–1457 (2015). 4. Carruthers, R. et al. Abrogation of radioresistance in glioblastoma stem-like cells by inhibition of ATM kinase. Mol Oncol. 9(1) 192–203 (2015). 15. Ahmed, S. U. et al. Selective Inhibition of Parallel DNA Damage Response Pathways Optimizes Radiosensitization of Glioblastoma Stem-like Cells. Cancer Res. 75(20), 4416–4428 (2015). ( ) ( ) 16. Frosina, G. DNA repair and resistance of gliomas to chemotherapy and radiotherapy. Mol Cancer Res. 7(7), 989–999 (2009). ddl Th l h b f ll l d l 17. Biddlestone-Thorpe, L. et al. ATM Kinase Inhibition Preferentially Sensitizes p53-Mutant Glioma to Ionizing Radiation. Clin C Res. 19(12), 3189–3200 (2013). 18. Kovacevic, N. et al. A three-dimensional MRI atlas of the mouse brain with estimates of the average and variability. Cereb Cortex. 15(5), 639–645 (2005). ( ) ( ) 9. Ropolo, M. et al. Comparative analysis of DNA repair in stem and nonstem glioma cell cultures. Mol Cancer Res. 7(3), 383–392 (2009). Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 13 www.nature.com/scientificreports/ 20. Parodi, R. C. et al. Growing Region Segmentation Software (GRES) for quantitative magnetic resonance imaging of multiple sclerosis: intra- and inter-observer agreement variability: a comparison with manual contouring method. Eur Radiol. 12(4), 866–871 (2002). ( ) 21. Shultz, L. D. et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol. 15 180–191 (1995). ( ) 22. Jahangiri, A. et al. Convection-enhanced delivery in glioblastoma: a review of preclinical and clinical studies. J Neurosurg. 126(1), 191–200 (2017). ( ) 23. Gualberto, A., Aldape, K., Kozakiewicz, K. & Tlsty, T. D. An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control. Proc Natl Acad Sci USA 95(9), 5166–5171 (1998). p p p 24. Weller, M. et al. EANO guideline for the diagnosis and treatment of anaplastic gliomas and glioblastoma. Lancet Oncol. 1 e395–403 (2014). ( ) 25. Frosina, G. Nanoparticle-mediated drug delivery to high-grade gliomas. Nanomedicine 12(4), 1083–1093 (2016 Advances in drug delivery to high grade gliomas. Brain Pathol. 26( drug delivery to high grade gliomas. Brain Pathol. 26(6), 689–700 ( 26. Frosina, G. Advances in drug delivery to high grade gliomas. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-32578-w.h 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. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific Reports \| (2018) 8:14191 \| https://doi.org/10.1038/s41598-018-32578-w 14
https://openalex.org/W2911741418	https://radar.brookes.ac.uk/radar/file/492c4ef7-46ea-4261-8a6f-97e7af797699/1/Exercising control at the urban scale - 2019 - Reeve.pdf	English	null	Exercising Control at the Urban Scale: Towards a Theory of Spatial Organisation and Surveillance	Springer eBooks	2,019	public-domain	16,152	Exercising control at the urban scale: towards a theory of spatial organisation and surveillance Exercising control at the urban scale: towards a theory of spatial organisation and surveillance Exercising control at the urban scale: towards a theory of spatial organisation and surveillance Introduction The quotation at the head of this chapter taken from a text in the fields of sociology and criminology on the nature of surveillance in the contemporary city, reminds us that the control of urban space has a long history, and is even perhaps, a necessary condition of a phenomenon in which power is always exercised and exercised unequally. The purpose of this chapter is to explore the forms that surveillance as a dimension of control takes in the contemporary and emerging urban settings. In order to do this, we start with an analysis of what has been termed social-spatialisation (Shields, 1992): the particular regulatory, aesthetic and material strategies by which control and the subversion of control are exercised; and how the meaning and directedness of such control - through various forms of surveillance – is predicated on notions of space as instrumental and, particularly in the case of city centres, commodified as a space for individuated and life-style consumption. The chapter is also concerned with articulating how surveillance operates both from the outside, and as an apparently natural response to contemporary narratives of safety, conformity and moral propriety: but also from the inside with how individuals. reflexively self-monitor in response to imposed clues and signals from increasingly aestheticized public environments such as theme parks, through shopping malls to the managed High Street. Finally, the chapter discusses how emerging technologies embodied in the mobile phone both extend the possibilities of a controlling and externalized surveillance, and at the same time offer the opportunity for the individual to appropriate space and its narrative meaning in a paradoxical privileging of the very private in the context of the public realm. The chapter covers a range of themes, starting with the question of how notions of the public and the private are central to understanding what surveillance is. Building on this, it sets out a theory social spatialisation, and provides a model of the relationship between the three dimensions of public space –material or formal, regulatory or managerial, and aesthetic or felt. This section places the concept of surveillance/ the gaze, in its broader theoretical setting related to notions of propriety, safety, image and identity, and in terms of the political economy of the city. Abstract The purpose of this chapter is to explore how urban spaces are implicated in the control and surveillance of users in a culture saturated by the notion of the self as a consuming body or entity. Using the work of Foucault on disciplinary cultures, Lefebvre in relation to the production of space, and other seminal theorists such as Jean Baudrillard, Zygmunt Bauman, Rob Shields and Micahel Walzer, a model for analysing the three dimensions of social spatialisation is proposed and illustrated by reference to contemporary public spaces, and specifically spaces of mundane leisure such as shopping malls and high streets. The chapter examines space in terms of its moral, aesthetic and cognitive dimensions – both as produced and as consumed – and how control is exercised over the individual’s sense of identity as well as over the social aspects of place through material, managerial and aesthetic strategies. The chapter also discusses the dialogue that exists between constructed technologies of surveillance – CCTV, architectural elements such as windows and their placing in relation to the street, and internalised expectations and the self- censorship of identity and behaviour of consumers induced in a culture of highly aestheticized and depoliticised consumption. The chapter also thinks through the implications of contemporary approaches to the design of commodity dominated and privatized public space in relation to notions of the private and the public, and the idea of ‘third’ spaces, as they have been called. Finally, the chapter deals with how the public realm as a controlling space has been theorised in terms of opposition to such controlling tendencies – from the flaneur, through the self-constructed narratives of De Certeau’s walker, to the digitally ‘enhanced’ individual today, appropriating space via technology and their own projects in tinder etc., and other potentially transgressive media. 1 \| P a g e 1 \| P a g e The city is, and always has been, constituted as a contest over space – over its production, representation and regulation; over who is authorised to be in it, and who is kept out; over what constitutes an unpolluted space and what constitutes a transgression of space. Coleman, R (2007) (p240) Introduction The chapter then briefly examines how control is exercised in different public settings, from the passive and incidental to the increasingly remote and digital and briefly how different narratives have been constructed to rationalise and justify these. In particular, the focus is on how space is 2 \| P a g e 2 \| P a g e socially organised through range of tangible and intangible devices of control as a form of social spatialisation. The third section focuses on a the idea of the entrepreneurial city (Harvey, 1990; Coleman, 2007), and the use of surveillance technologies along with other management and aesthetic strategies intended to create a new type of public realm – of the depoliticised and atomised consumer as against the civic and the collective – focused in the notion of the panopticon of leisure and consumption. The third section focuses on a the idea of the entrepreneurial city (Harvey, 1990; Coleman, 2007), and the use of surveillance technologies along with other management and aesthetic strategies intended to create a new type of public realm – of the depoliticised and atomised consumer as against the civic and the collective – focused in the notion of the panopticon of leisure and consumption. The final section discusses notions of appropriation and transgression of public space, and the subversion of surveillance by turning the technology on itself - effectively the digitisation of the 19th century flaneur as celebrated by Walter Benjamin in the Arcades Project. This section speculates on the relationship between notions of the individual as gazed upon by the other – both as individual and present, but also as remote and ‘managerial’, or present by their absence; and of the individual as observer or voyeur. The public and the private That is, within any city it is represented by those spaces, such as streets, squares, plazas, market places, theatres, malls, and railway stations, which are social to the extent that they can be occupied or used by more than the solitary individual, and which exist outside of the spaces of work or domestic reproduction. As Henri Lefebvre (1991), amongst others (Mumford, 1987; Morris, 1979) has suggested, public space is an inevitable dimension of the urban. So, for example, his discussion of space within Roman society, provides a clear definition from the perspective of a spatial dichotomy between the private and the public, (public space) is the space of social relationships (as against) private areas (spaces for contemplation and retreat). (Lefebvre, 1991, 153) This is the sense in which public space is often treated by physical designers, as if it were a homogenised product where distinctions can only be made between different kinds of public spaces, according to their pragmatic and cultural functions, de-situated from their ethical and political context: streets as routes, markets as places of commercial exchange, theatres as spaces for performance, or theme parks as places of escape. The design literature approaches the problem of public space as a product precisely from the view that its cultural and functional performance can be measured and designed for (see Whyte, 1980; Cooper Marcus and Francis, 1998; Carmona et al. 2010). 4 \| P a g e However, Lefebvre also argues that the meaning of the public as against the private realm has shifted, historically, with changes in the mode of production, and within the present mode of production, in which the definition of what is public and private and what belongs under each realm depends on who owns what and how it fits within a system of production and social reproduction, used to maintain hegemonic interests. This hegemony can be seen to be exercised through various means of control – including the deployment of technologies of surveillance – whether by the state, or by state sanctioned, but essentially non-civic interests such as those of capital, in this context. Ownership precedes rights of use and is overlaid by class and gender specific expectations of power. In Victorian society (Daunton, 1983; Jackson, 1990), for example, the private sphere was the home, the space of the family and of social and sexual reproduction, with its own rigid power structure. The public and the private 3 \| P a g e Before examining how surveillance as one dimension of control is exercised in the built environment, it is useful to think about two apparently complementary concepts – the public and the private – in so far as the relate to urban space, its design, and management. The reason for this is that the idea of surveillance, at first sight, only has meaning in the context of the notion of the public realm or the public domain – that is, of place (spatial or non-spatial) as being open to more than the individual. As the seminal literature on surveillance (Lyon, 1994, Coleman, 2007) indicates, the electronic observation of real space in real time is only one dimension of a much larger phenomenon which is concerned with the collection and analysis, for whatever ends, of digital information about individuals as they engage with other individuals. What is important here is that technologies of surveillance which collect and interpret digital data and ‘footprints’, whilst they refer to specific geographically locatable acts, exist in a sense outside of time and place, in a virtual public space. Moreover, digital data is essentially private in the sense that it is accessed and used by individuals, arguably, in the private virtual ‘space’ of the electronic device - phones, computers, tablets etc. which provide its interface. The surveillance of the spatial public realm, on the other hand, exists in specific geographical locations and, for the most part, in real time. In this sense, it is helpful to distinguish between public space, as a site of surveillance, and private space - essentially the domestic space of the home – where even today most people would regard surveillance by ‘outsiders’ as unjustifiably intrusive or voyeuristic. Whilst the technology for remotely monitoring behaviour in domestic spaces, or spaces of privacy outside the home, undoubtedly exists (and there is anecdotal evidence of the use of cameras on mobile phones and other devises to observe users 3 \| P a g e without their consent ref); this discussion is concerned with public physical space as properly distinct from the space of the private functioning of the individual. The meaning of public space At its simplest, physical public space is space which is given over to public use. The public and the private A society dominated by the male 'breadwinner', with social roles and property rights organised along 4 \| P a g e the 'natural' divisions of gender. The public space, the unassigned space of the street at least, was the responsibility of the state as owner in terms of control and organisation. Rights of use and powers to proscribe public behaviour were based on the claims of the state as owner of the space and these were given particular form by the class in control of capital, law making and ownership of the space. Its dominant (class) interests were the preservation of social order, the maintenance of standards of conduct in which the proletariat, for example, were regarded as a permanent threat in the form of the crowd or mob (Golby & Purdue, 1984). Jackson (1990) has claimed that this separation between private and public was lived in the imagination of the dominant group in society to the extent that the public domain became a demonised territory, and represented a potential threat to the sanctity of the home, The street was symbolically opposed to the home, a profane versus a sacred world....A clear association was assumed between the private virtues of family life in the home and the public dangers of the street. (Jackson, 1990, 100) The street was symbolically opposed to the home, a profane versus a sacred world....A clear association was assumed between the private virtues of family life in the home and the public dangers of the street. (Jackson, 1990, 100) As we shall see, the sense of the street as a threat to the moral sanctity of the home may have decreased – not least because the home is no longer a space so completely closed off from the world of the street as it was in the 19th Century – but this has been replaced by a different sense of the threat represented by the street as a place of economic immorality or anarchy which needs to be disciplined (See Coleman, 2007) , in part by the use of technologies of surveillance. 5 \| P a g e Daunton (1983) also argued that the street as a public place was a space with no particular purpose or prescription except, on the face of it, as a route or passageway. The public and the private In contemporary culture, from the later part of the last century, it has been argued that the distinction between the private and public is being transformed, if it has not already been entirely lost (Harvey, 1990; Bauman, 1994, 1993; Jackson, 1990 ; Shields, 1992; Giddens, 1990, 1992). The home remains the private space for accommodating the interests of the nuclear family and of social reproduction, as well as a space for the retreat of the individual from the collective project and gaze. In contemporary culture, from the later part of the last century, it has been argued that the distinction between the private and public is being transformed, if it has not already been entirely lost (Harvey, 1990; Bauman, 1994, 1993; Jackson, 1990 ; Shields, 1992; Giddens, 1990, 1992). The home remains the private space for accommodating the interests of the nuclear family and of social reproduction, as well as a space for the retreat of the individual from the collective project and gaze. But the public sphere, as the space of public duty and responsibility, of collective and community identification, and as a territory for the expression of the community interest, has been argued to be under threat (see, for example, Heller and Feher, 1988; Bauman, 1992, 1994). The question is whether this threat is because of the privatisation of space or the privatisation of interests, or both. But the public sphere, as the space of public duty and responsibility, of collective and community identification, and as a territory for the expression of the community interest, has been argued to be under threat (see, for example, Heller and Feher, 1988; Bauman, 1992, 1994). The question is whether this threat is because of the privatisation of space or the privatisation of interests, or both. The physical nature, the apparent social space of the city of the 2010s is not, arguably, much different to what it was in the nineteenth century. It may still be physically categorised under the headings already alluded to: the domestic space of the family1, or of social reproduction, the public domain of the street as a pathway, the nodes or dedicated spaces within the urban fabric, such as squares and plazas which are capable of being appropriated for collective ends, and the 'third places', of entertainment, worship or refreshment. The public and the private It therefore came to represent a threat because it was indiscriminately available to any class of individual for anything. This is a key point in our argument: the street in Victorian England stands as a type of public space towards which the state had, at best, an ambivalent attitude: protecting rights of use, but limiting or controlling uses, according to a particular social and moral conception of public order and efficiency. It is no accident that the Victorian period saw a proliferation of bye-laws regulating the use of public spaces, including the street and the new public parks. Little has changed, arguably, since the Victorian period in terms of the presumption that whilst the state has some rights to control, regulate and legislate over behaviours out of the public gaze (ie the home); it has an absolute right to observe behaviours in public space. Indeed, over the last twenty years in the UK there has a been a further proliferation of laws to control and demonise forms of behaviour in public spaces in the UK – to which we will return. The key point here is that the distinction between the 5 \| P a g e public and the private is central to assumptions about what spaces can and ought to be surveilled, and to how these are then justified or legitimated by agencies who have the resources the desire to exercise such surveillance in its various guises. It is also significant that with respect to what have been termed 'third places', as they have been called (Oldenburg, 1989), surveillance technologies have become commonplace. Third places are public spaces assigned to specific semi-public functions, in which codes of conduct, customs and practices, were historically prescribed by 'in-house' rules as well as social norms particular to those spaces. Such spaces would include theatres and concert halls, pubs and buses, and even hospitals and schools. These 'third places' are actually quite complex in the sense that they may also be seen as spaces for private, or at least personal projects. But what is interesting today is that few people would object to the presence of CCTV cameras in such settings, as though being observed anywhere except the home has become normal and even expected – particularly in the UK. 1The family, of course, is different today to that of the nineteenth century. in the sense of having contracted to two parents and 2.5 children, or the (contradiction of?) the one parent family. But the point still holds, that the family, whatever its actual make up in terms of numbers or extension, can be conceptualised in terms of the social unit of parent(s) and children. Social spatialisation Having briefly sketched in the nature of the complexity of the two concepts of the public and the private, we can now begin to think about the place that surveillance as a concept has within the broad field of social-spatialisation – which is, the theory of the relationship between or interdependence of space – here urban space – and the social. The term has been used to relate social practices with spatial organisation (Shields, 1991, 1992; Jackson, 1990). It understands the meaning of all social space as socially constructed, both from the social situation of a particular place (its historical associations, and the control over its uses and values ascribed to it by competing interests), and objective material relations (in particular, access to the means by which spaces are manufactured and managerially organised). This construction is an active and dynamic process. There are discrete spatial practices within design, management and promotion which can be analyzed in terms of their relationship to the issue of spatial control. In essence, the term social spatialisation has been used to designate the ongoing social construction of the spatial at the level of the social imaginary (collective mythologies, presuppositions) as well as interventions in the landscape (for instance the built environment). (Shields, 1991, 31). In terms of understanding the place of surveillance in the physical public realm, on the basis of this concept, we can begin to grasp that the use of different devices or spatial strategies of control – including the technological such as CCTV, as well as the architectural such as the placing of windows to provide eyes on the street – are only one part of a larger whole. This larger construction of the urban is made up of a number of components, both tangible and intangible, which create or impose identity on place. These components include designed elements- such as street furniture, and building facades; as well as discursive elements –narratives of place either deliberately constructed through place promotion campaigns to attract, say, tourists or visitors; or manufactured in a more ad hoc fashion in press articles, usually focusing on the negative aspects of identity such as crime or anti-social behaviour. The public and the private There is also a relatively new type of public space, the mall, the internalised common space for personal consumption in which surveillance takes on a particularly intensive policing, managerial and even aesthetic function . 1The family, of course, is different today to that of the nineteenth century. in the sense of having contracted to two parents and 2.5 children, or the (contradiction of?) the one parent family. But the point still holds, that the family, whatever its actual make up in terms of numbers or extension, can be conceptualised in terms of the social unit of parent(s) and children. 6 \| P a g e Social spatialisation Strategies and instruments of control and surveillance represent one of the means by which a normative sense of place is created or sustained , in conjunction with other both physical (design) and non-physical (advertising and place promotion, management and policing) tactics of place making. Surveillance as Spatial Practice. Surveillance as Spatial Practice. 7 \| P a g e Social spatialisation is not limited to explaining the existence of social meanings and how these are reflexively modified over time. It can also be used as a way in to understanding the material and technical practices used in the manufacture of real spaces, alongside the tendency of such practices to be complicit in the creation and enforcing of a particular hegemonic identity – bound up in notions of what is acceptable and appropriate in relation to behaviour and individual performance, or identity. The term 'spatial practices' derives originally from Lefebvre's (1991) philosophical analysis of the production of space. According to this view, space in both its material form (as built environments and existing landscapes), as well as in how it is represented in maps and discourse and in what it is imagined to be for (utopian and other visions of the future), comes into being as a social fact through distinct technical, intellectual, artistic and other practices of individuals, institutions and other agencies. Every social space can be understood in terms of the particular spatial practices which have brought it into being, and which maintain its meanings and availability for particular ends. Lefebvre’s contention is that space is a product of particular interests – generally class interests- and its design, along with its management in the broadest sense, and its appropriation in use are conditioned by these particular hegemonic interests. Again, surveillance can be regarded under this view as an aspect of this exercise of hegemonic interest, and one which is, as we shall see, foregrounded or hidden in particular narratives of the city: for instance in the promotion of the idea of city centres as safe from crime or social disorder, or terror. Developing the concept of spatial practice and how surveillance as a practice in itself can be understood today, it is helpful to analyse it in terms of what the sociologist, Zygmunt Bauman (1994) implied as distinct ‘fields of experience’ and how these are addressed in the manufacture and control of public space. social space is...far from simple and needs...unpacking. Surveillance as Spatial Practice. In particular it ought to be seen as a complex interaction of three interwoven yet distinct processes - those of cognitive, aesthetic and moral 'spacings - and their respective products...If the cognitive space is constructed intellectually, by acquisition and distribution of knowledge, aesthetic space is plotted affectively, by the attention guided by curiosity and the search for experiential intensity, while moral space is 'constructed' through an uneven distribution of felt/assumed responsibility. (Bauman, 1994, 145-6) 8 \| P a g e What Bauman is suggesting is that social or public space is experienced in terms of three things: its 8 \| P a g e What Bauman is suggesting is that social or public space is experienced in terms of three things: its 8 \| P a g e cognitive possibilities (what we believe or think it is for, functionally) its moral possibilities (how we ought to act in it, particularly in relation to others), and its affective possibilities (how we experience it as a site of pleasure, or discomfort). What I want to argue is that these three modes of experience are provided for in the design and management of public space, consciously or not, in order to reinforce or instantiate particular hegemonic interest. In neo-liberal settings, these interests are generally commercial, and focus on the individual as consumer whose behaviours must conform to the interests of the neo-liberal economic system, be predictable, but also feel as though they are voluntary and undirected – all within a context that feels both safe and comfortable/fun. Different public space settings demonstrate this hegemonic tendency or reality in different degrees – ie some public spaces are more controlling and surveilled than others – from the domestic or residential street at one end of the spectrum, to the mall or theme park at the other. It goes without saying, that the more physically or discursively controlled access to a particular part of a city is, the less it needs to be surveilled and controlled through CCTV and other such methods. Surveillance as Spatial Practice. Indeed, highly aestheticized places of leisure (of play or shopping) exhibit the co-ordination of design and management strategies at perhaps their greatest intensity, and for the reasons suggested again by Bauman in his trenchant analysis: If reality is oozy, ubiquitous, straggly, spattered all over the place, play is securely protected behind its spatial and temporal walls...play has its place...as well marked, by stage frame, fence, guarded entrance. (Bauman, 1994, 173) Here cognitive distinctions and moral responsibilities can be suspended. However, this may only happen in a situation in which cognitive spacing has already occurred so that the aesthetic experience can take place free from the threat of the intrusion of the unwanted other, 'Only in the well administered and policed space can the aesthetic enjoyment of the city take off' (Bauman, 1994, 169). Likewise, moral claims are anathema to aesthetic experience, and the aesthetic experience can only be enjoyed in a space free from the pressing presence of, say, 'real' suffering or poverty. The best aesthetic spaces in this sense are the liminal, bounded, separated off (temporally or spatially) and which have their own artificial game rules or codes which inform users how to play in an 'as if world '. 9 \| P a g e Bauman cites the Arcades and streets of Baron Hausmann's Paris in the second part of the 19th Century, as a type of the space of play for the late nineteenth century French bourgeoisie, the game 9 \| P a g e played to its fullest by the flaneur (see below) However, the street as the broader territory of the flaneur, is now too dangerous, too cognitively indiscriminate, to allow the play it once guaranteed. That play - the safety of the aesthetic experience - has retreated to the well surveilled and guarded spaces, such as the mall. But that play has in turn been transformed by the very nature of the mall as an over-specifying2 space. The other characteristic of the aesthetic which is of interest is its contemporary ambiguity. Spaces such as malls appear, on the one hand, to be places of the liberation of experience in which outer reality is held at bay or apparently suspended, and on the other are surveilled and produced spaces with 'real' intentions. 2 This term refers to the tendency of spaces such as the mall to be designed, managed and controlled in such a way as to powerfully imply certain requirements in the way that individuals ought to regard themselves: as players acting out a script which they have not written. way as to powerfully imply certain requirements in the way that individuals ought to regard themselves: a acting out a script which they have not written. 2 This term refers to the tendency of spaces such as the mall to be designed, managed and controlled way as to powerfully imply certain requirements in the way that individuals ought to regard themselves: a Surveillance as Spatial Practice. At once an apparent space of freedom they can be read as spaces in which freedom is curtailed by being directed towards particular and instrumental goals which are not voluntarily scripted or invented by the spectator in any authentic sense. . The three fields of experience designated by Bauman, the cognitive, moral and aesthetic, can be turned around for the purposes of understanding surveillance as a dimension of public space, by examining how these are addressed in the design and operation of such space. That is, it is possible to categorise techniques of management, design, promotion and so on under the three headings: form, image and rules. Image involves both aesthetic and cognitive spacing and has to do with the 'look' of a place as well as its discursive status and its manufactured affective associations. In practice, image is produced through strategies such as advertising and place promotion, as well as through retrofitting or redressing public space to make it more aesthetically attractive, or the appearance of being well maintained. Likewise, rules have to do with moral and cognitive spacing, controlling through both explicit and informal codes how space may and may not be used, and how individuals may relate to each other in them - what is permissible. These would include both criminal and civil law – (such as Anti-Social Behaviour Orders in the UK) expressed through the presence of CCTV and signage warning against such things as drinking in public. Form, on the other hand, has to do with the material organisation and disposition of parts according to a cognitive strategy, but with moral and aesthetic outcomes. This is illustrated in gated communities, where public space is privatised through physical exclusivity; or shopping malls which either have doors, or a particular aesthetic to signal that the user is moving from the civic space of the street to the commercial space of the shopping centre. Particular techniques of social control, such as the use of closed circuit television, can be interpreted as having a complex function: enforcing rules, 2 This term refers to the tendency of spaces such as the mall to be designed, managed and controlled in such a way as to powerfully imply certain requirements in the way that individuals ought to regard themselves: as players acting out a script which they have not written. Surveillance as Spatial Practice. 10 \| P a g e contributing to an image and having formal characteristics, as well as moral, cognitive and aesthetic implications. Fig 1. illustrates how these categories are interrelated contributing to an image and having formal characteristics, as well as moral, cognitive and aesthetic implications. Fig 1. illustrates how these categories are interrelated INSERT FIG 1 – Diagramme of spatial practices and public space Each of these strategies (rules, image, form) in practice, affects and is affected, by each of the others; the dominant quality of any public place will depend directly on the degree to which and by whom these strategies are deliberately articulated together to add up to a particular environment. The ontology of a place which is the product of a unified intention in its image, form and rule strategies is arguably quite different to that where a number of intentions exercised at different times, have had an influence over each of these qualities. 'Single Minded' and 'Open Minded' Social Space. 'Single Minded' and 'Open Minded' Social Space. We have already touched on the idea that some types of public spaces are more controlling – and more co-ordinated in terms of their formal, aesthetic and managerial characteristics, than others. It is worth expanding on this a little, since it has a direct bearing on the question of both where and in what ways forms of surveillance operate. In the 1980s Michael Walzer (1986) proposed a distinction between what he called 'single minded' and 'open minded' space: In the 1980s Michael Walzer (1986) proposed a distinction between what he called 'single minded' and 'open minded' space: Zoned business and residential areas are single minded, as is the modern dormitory suburb. The central city (as it once was) and also the 'quarter', the neighbourhood with its own shops and small factories constitute 'open minded' space. (Walzer, 1986, 471) Essentially 'open minded' space is, in theory, social space that is available and accessible for more than one purpose and to more than one interest. It is also space which is capable of being experienced for more than one end by its users. It is pluralistic both in its appeal and function. It is space which is reflexive or responsive over time; capable of being imaginatively and functionally appropriated for different ends. Walzer clearly associates it with the space of the 'traditional city'. 'Single minded space', on the other hand, is space dedicated to particular ends, and intended to be used, in its contemporary context, for the satisfaction of private goals. He implies at least that it is coming to replace 'open minded' space, 11 \| P a g e Designed by planners or entrepreneurs who have only one thing in mind, and used by similarly single-minded citizens.... Designed by planners or entrepreneurs who have only one thing in mind, and used by similarly single-minded citizens.... The reiteration of single mindedness at one public site after another (is) something societies should avoid. (Walzer, 1986, 470) The reiteration of single mindedness at one public site after another (is) something societies should avoid. (Walzer, 1986, 470) He claims that we not only use these spaces differently because of the single function that they house, but we also feel differently in them. We recognise an intention towards us that the space contains in its design, management and in the type of individual it attempts exclusively to attract. 'Single Minded' and 'Open Minded' Social Space. He also suggests that this 'single mindedness' is a general cultural characteristic of modern day life, in which time is increasingly spent in spaces either physically separate and private, such as the car, or instrumentally separate and given over to personal ends (see Table 1 for a summary of spaces dichotomised as 'open minded' and 'single minded' by Walzer). 12 \| P a g e 12 \| P a g e Single minded space Open minded space zoned business district quarter/neighbourhood dormitory suburb the street the highway forum government centre square medical centre courtyard green belt city park/playground housing project urban block urban mall cafe/pub fast food restaurant urban hotel motel long distance train airplane theatre cinema urban fair ground exhibition centre row of shops department store university campus Table 1: Open Minded and Single Minded Spaces (After Walzer, 1986) Single minded space Open minded space zoned business district quarter/neighbourhood dormitory suburb the street the highway forum government centre square medical centre courtyard green belt city park/playground housing project urban block urban mall cafe/pub fast food restaurant urban hotel motel long distance train airplane theatre cinema urban fair ground exhibition centre row of shops department store university campus Table 1: Open Minded and Single Minded Spaces (After Walzer, 1986) Open minded space Table 1: Open Minded and Single Minded Spaces (After Walzer, 1986) Helpful as Walzer’s distinction is, we need to make an important caveat thrown up by thinking about surveillance as an aspect of the contemporary public realm. The first is that, as the work of Rustin (1986 ) and Vidler (1978) imply, 'open minded' space can be historically located as the chaotic space of the petite bourgeoisie, the space of the nineteenth century public street or square. That is, it is in part a romantic ideal which no longer effectively pertains, precisely because of the technological possibilities of remote and ubiquitous surveillance, alongside the neo-liberal turn represented by both the commodification of even mixed use streets combined with the rise of the solipsistic and aesthetic project of the individual. Helpful as Walzer’s distinction is, we need to make an important caveat thrown up by thinking about surveillance as an aspect of the contemporary public realm. 'Single Minded' and 'Open Minded' Social Space. The first is that, as the work of Rustin (1986 ) and Vidler (1978) imply, 'open minded' space can be historically located as the chaotic space of the petite bourgeoisie, the space of the nineteenth century public street or square. That is, it is in part a romantic ideal which no longer effectively pertains, precisely because of the technological possibilities of remote and ubiquitous surveillance, alongside the neo-liberal turn represented by both the commodification of even mixed use streets combined with the rise of the solipsistic and aesthetic project of the individual. Despite these qualifications, one measure of the success of an urban space must be how pluralistic it is, how constraining, and in whose interests it is managed and designed. 13 \| P a g e Panopticon. Surveillance as an aspect of the urban, as we have seen, is a highly complex phenomenon, in that it can be exercised in different ways – formally and informally, and for different purposes, from state control to individual safety and even pleasure. In order to unpack the nature of this complexity and to better understand how surveillance operates as a dimension of public space, this section briefly highlights the key narratives which have either critiqued or been used to justify surveillance within public space over the last few decades, and attempts to link these with a broader political perspective in relation to notions of individual agency as against hegemonic control. The following analysis suggests first that surveillance is a strategy that has to be understood in relation to other methods, tactics or tropes of control (discursive, managerial and formal) and is co-opted to reinforce the power of these other aspects of place; second, that the function of surveillance as a ‘protective’ mode of the urban can be understood in relation to the dominant interests within a place – from the domestic, suburban street, to the High Street, and the shopping mall. A helpful starting point for this analysis is the work of Gold and Revill (1983), who have attempted to list the common features of ‘safer cities’ as they called them (Table ). It is worth highlighting that the features that they identified range from the physical, such as walls, the regulatory, such as signage, the managerial, such as street ambassadors, the technological, such as CCTV, and the ‘natural’ (what they refer to as the ‘animated’), such as the presence of people. Different narratives of the city, as we shall see, justify or explain and even celebrate the use of these various components of public space depending on their underlying ideological perspective, and their alignment or otherwise with the dominant narrative of the city – for instance currently that of neo-liberalism, anti-terror, and entrepreneurialism. Panopticon. 14 \| P a g e 14 \| P a g e Table 2 Urban design approaches to safer city centres From Gold and Revill (1983, 193): Safer city approach Common features Fortress • Walls • Barriers • Gates • Physical segregation • Privatisation of territory • exclusion Panoptic • control of public space • privatisation of space • explicit police presence • presence of security guards • CCTV systems • covert surveillance systems • erosion of civil liberties • exclusion • the ‘police state’ Regulatory • management of public space • explicit rules and regulations • temporal regulations • spatial regulations • CCTV • the ‘policed state’ Table 2 Urban design approaches to safer city centres From Gold and Revill (1983, 193): Table 2 Urban design approaches to safer city centres 15 \| P a g e • ambassadors/ city centre reps. Animated • people presence • people generators • activities • welcoming ambience • accessibility • inclusion • 24-hour/ evening economy strategies Having set out an at least provisional set of spatial strategies through which control is exercised in public space, we can now turn to different perspectives and narratives of public space, and of how such surveillance should operate and to what ends. Essentially there are two largely opposed positions in the debates about how cities should be survellied – on the one hand, the liberal view that takes the urban as a place of community and neighbourliness; on the other, the instrumental view that public space is part of a neo-liberal political economy where it’s economic security and performance is paramount. I want to argue that these opposing positions are apparent in those strategies of surveillance argued for and deployed in practice. For the sake of simplicity, and drawing on Gold and Revill’s analysis, I have termed these the animated city, and the panotpticon city. The animated city The underlying presumption in the view that streets and other public spaces are genuinely public if they are animated – ie well used, by a variety of individuals for multi-valent ends – is that the public realm is the civic realm, a democratic place that is pluralistic and open minded. This is an essentially romantic ideal that has its origins in the Greek Agora and the Roman Forum. One text, above all, that has been deeply influential in promoting the idea that public space is safe and even humane if it allows for ‘eyes on the street’ – ie natural surveillance – is The Death and Life of Great American Cities, by Jane Jacobs, first published in . Jane Jacobs was a journalist and 16 \| P a g e 16 \| P a urban activist, whose views on the quality of urban spaces, and the need for design to enable and encourage diversity of use and users, arose from her personal experience of living in a mixed neighbourhood in Boston in the 1950s and early 60s. Her writing powerfully conveys her sense of the humanity of city streets characterised by mixed use – combining residential apartments above independent shops, bars and cafes, in high density neighbourhoods. Her seminal book is highly anecdotal, as here where she describes the response of her neighbours to an attempted abduction of a young child: As I watched from the second-floor window, making up my mind how to intervene if it seemed advisable, I saw it was not going to be necessary. From the butcher beneath the tenement had emerged the woman who, with her husband runs the shop; se was standing within earshot of the man, her arms folded, a look of determination on her face. Joe Cornaccchia, who with his sons-in –law keeps the delicatessen, emerged about the same moment and stood solidly to the other side. Several heads poked out of the tenement windows above, one was withdrawn quickly and its owner reappeared a moment later in the doorway. Two men from the bar next to he butcher shop came to the doorway and waited. On my side of the street, I saw the locksmith, the fruit man and the laundry proprietor had all come out of their shops and that he scene was being surveyed from a number of windows besides our own. Jane Jacobs (1984, 48) Jane Jacobs (1984, 48) Jane Jacobs (1984, 48) From this and other personal experiences of living in this particular neighbourhood, she generalised a set of design principles which still dominate the thinking and practice of many urban designers to this day, although often observed more in the breach. The following extract captures part of her thinking: There must be eyes upon the street, eyes belonging to those we might call the natural proprietors of the street. The buildings on a street equipped to handle strangers and to ensure the safety of both residents and strangers must be oriented to the street. They cannot turn their backs or blank sides on it and leave it blind… Jane Jacobs (1984, 45) Jane Jacobs (1984, 45) 17 \| P a g e What she successfully advocated was a response to a narrative of the city as a sociable, self- policing and largely self-managing setting where civilised neighbourliness would be enabled through a combination of particular elements – doors and windows onto public space, a mixture of different land uses (including housing) to ensure variety and, as far as possible, 24 hour vitality and activity, and connected and permeable route systems to ensure both choice in movement, and a pedestrian friendly layout. What she successfully advocated was a response to a narrative of the city as a sociable, self- policing and largely self-managing setting where civilised neighbourliness would be enabled through a combination of particular elements – doors and windows onto public space, a mixture of different land uses (including housing) to ensure variety and, as far as possible, 24 hour vitality and activity, and connected and permeable route systems to ensure both choice in movement, and a pedestrian friendly layout. The notion of a naturally surveilled place was taken up by Oscar Newman in his book Design Guidelines for Creating Defensible Space (Newman 1973) in relation to residential development. Again, the underlying presumption is that spaces are safe if they are naturally surveilled by users – in this case residents – where strangers can easily be identified and where there is a sufficient sense of community for those residents to feel they own their ‘territory’. Jane Jacobs (1984, 48) This principle of self- surveillance, and natural policing can be seen to be expressed in quite detailed design terms in the context of domestic environments: for instance, housing design which provides a set-back in the forms of a defined front garden; and the use of windows at ground floor which allow for an unobstructed view of the street (see fig 2) Fig. 2 Residential street and surveillance from the house Fig. 2 Residential street and surveillance from the house 18 \| P a g e In addition, in the UK – following precedents in the United States – many residential streets employ Neighbourhood Watch schemes, involving local residents, police, local authorities and other agencies with the intention of making such environments safer through active monitoring of activity and users, and providing mechanisms for reporting unusual or potentially criminal acts. There is, of course, a double edge to surveillance strategies such as Neighbourhood Watch (Fig 3), that whilst intended to create a sense of neighbourliness and community, also reinforce a sense of the otherness of strangers, and invoke notions of the acceptable and unacceptable which are highly normative. Fig 3 Surveilling the domestic street – Neighbourhood Watch Schemes Fig 3 Surveilling the domestic street – Neighbourhood Watch Schemes Much of the design guidance literature (for example Responsive Environments, Bentley (1985), and the Urban Design Compendium, English Partnerships (2000)), that has informed the approach taken by urban designers over the last fifty years, has adopted both the implicit rules of good public space making in the work of Jacobs, as well as the underlying liberal notion of the city and its streets as civic and social space which implicitly treats users as ends in themselves as against either instrumentalising or ‘othering’ them. On the face of it, it makes no presumptions about who belongs or has rights to the city and who does not. 19 \| P a g e Other narratives of the city – often to do with place promotion and marketing – ironically draw on images of the animated street and public space, but are underpinned by conceptions of place as exclusive, and as ‘performative’ (see Coleman, 2007), where users are judged in terms of their contribution to the public realm as safe, economically productive, and conforming to a limited notion of the aesthetically acceptable and morally appropriate, as we shall see. The panoptic city The so called panoptic city, might be seen as the opposite of the animated city and it is also worth sketching in the background to this commonly used trope employed in debates about the surveilled city (which Gold and Revill’s analysis mention); and also how it links to the broader notion of the disciplinary society (see also Reeve (1996, 1998), originating in Foucault’s seminal text Discipline and Punish (1973). On the face of it Foucault’s book is simply an historical account of the disciplining and punishment practices of European societies. It examines the legal institution and cultural practices of punishment, incarceration, torture and surveillance. The fundamental idea of a society as a whole, managed through the exercise of physical constraint, of power of the state or other institutions over the body, extends as a metaphor in Foucault's work, however, beyond the actual spaces of imprisonment. In a similar vein to Guy Debord's notion of spectacle (1973), punishment and discipline are seen as ideological and material techniques, used to maintain the hegemonic interests of particular dominant classes or groups, at different historical moments. Discipline, Foucault argues, suffuses modern, rational society to such a degree that it has become an internalised value: we discipline ourselves whilst being reminded, through the physical institution of prisons, that discipline is necessary for the healthy functioning of society. What crimes are considered worthy or necessary to punish is an indication of the interests of the dominant groups in any particular society. Attitudes inscribed in punishment, proscribed for particular crimes against property or crimes against the person or state, will vary depending on how these relate to the threat they represent to the dominant interests within a society. 20 \| P a g e But the exercise of power through discipline and punishment has an important spatial dimension. As Shields has said, 'spatial control is an essential constituent of modern technologies of discipline and power' (Shields, 1991, 39). For Foucault, the disciplining mode of nineteenth century capitalism, for example, in part took spatial forms: in certain building typologies associated with certain social practices - the prison, the clinic, the asylum. Markus (1993) has demonstrated that 20 \| P a g e 20 \| P the internal layout of many nineteenth century and earlier institutional buildings can be interpreted as 'texts' of social control exercised through spatial dispositions. The governor was in a tower at the centre of a cylinder of cells....invisible to the prisoners and to the turnkeys who patrolled an intermediate annular passage, whilst the prisoners, lit from behind, were continuously visible. (Markus, 1993, 123) As Rabinow and Dreyfus (1982) have noted, commenting on Foucault's work, The panoptic city One of Foucault's particular interests was, as suggested above, the prison, and particularly the Panopticon designed by Jeremy Bentham in the late Eighteenth century. The peculiar characteristic of this spatial arrangement was the organisation of cells off of a central observation tower from which all prisoners could be observed whilst the design of the building ensured that prisoners were not able to see each other. Again, Markus (after Foucault, 1973) has subjected this type to a close spatial scrutiny, The governor was in a tower at the centre of a cylinder of cells....invisible to As Rabinow and Dreyfus (1982) have noted, commenting on Foucault's work, Discipline proceeds by the organisation of individuals in space, and it therefore requires a specific enclosure of space. In the hospital, the school, or the military field, we find a reliance on the orderly grid. Once established this grid permits the sure distribution of the individuals to be disciplined and supervised; this procedure facilitates the reduction of dangerous multitudes, or wandering vagabonds, and docile individuals. (Rabinow and Dreyfus, 1982, 154) (Rabinow and Dreyfus, 1982, 154) Arguably, the contemporary 'amusement culture' no longer requires the disciplining power of a rigid organisation and segregation of space, given the ideological power of spectacle. However, this culture has also, ironically, witnessed the evolution of new types of disciplinary space - managed, surveilled, and sometimes, although not always enclosed. These include spaces like the mall which are arranged to better facilitate the efficient turnover of capital through the selling of banalised leisure. 21 \| P a g e A third, contemporary, element can be added to this debate. The last few decades, the period of the rise of global and disorganised capital, the era of deregulation, the free market and the erosion of the welfare state, the era of entrepreneurialism, have seen a profound shift in the social conditions of many late capitalist cities (Castells, 1989). Symptomatically, this shift has included 21 \| P a g e 21 \| P a g e increased social discontent, a rise in street crime, a polarisation of incomes and working conditions, an increase in the number of homeless on the streets of many cities and more begging. This has meant that those spatial strategies associated with the nineteenth century disciplinarian institutions such as surveillance and spatial management, are being revisited, with the aid of new technologies such digital surveillance. The entrepreneurial city So, perhaps the dominant narrative today which has a powerful influence in decisions about the design, regulation, retrofitting and management of city spaces is that of the entrepreneurial city. It is here where all the strategies of control cohere and are articulated together to produce public space as the space of neo-liberalism – that is, the space of economically efficient performance. It is also here that the use of remote electronic surveillance, principally but not exclusively in the form of CCTV is most evident. The concept or category of the entrepreneurial city is not a very recent one. David Harvey (1987) sketched out the idea in an article in Antipode in the 1980s, at a time when many cities were having to repurpose themselves following their decline as centres of production, in an increasingly competitive global market. In effect, the making of commodities became replaced by the making or offering of services, and was – and continues to be – associated with places as settings for leisure consumption, and as attractors for foot-loose capital. In this context, cities have had to market themselves through discursive strategies of image making (Ashworth and Voogd (1990), Knox (1993)), requiring the product of place to correspond with the projected and metonymised image. In this context, there has been a powerful and ineluctable tendency for cities to develop or create a particular ‘look’ or aesthetic in order to attract visitors, as well as investors. Coleman (2007) has argued persuasively that this entrepreneurial drive, in which places need to be visually pleasing, can be directly linked to surveillance and other mechanisms of control and exclusion – managerial, and regulatory. Setting the scene, he says that, Setting the scene, he says that, As the latest trope in a reinvigorated pursuit of urban spatial order, camera surveillance in the UK is a normal feature of city development that propagates the idea of ‘capable guardian’s’, and symbolizes the state imaginary with respect to urban order in the UK. (232) He goes on to suggest that He goes on to suggest that The notion of the visually pleasing space is tied to aesthetic considerations concerning the moral probity and appropriateness of behaviour in public space. (234-5) The notion of the visually pleasing space is tied to aesthetic considerations concerning the moral probity and appropriateness of behaviour in public space. (234-5) 22 \| P a g e 22 \| P a g e And finally that And finally that The visible ‘differences’ that homeless people, the poor, street traders and youth cultures bring to the city, undermine the hegemonic notions of ‘public’ spatial utility and function. Indeed, for these groups, it is often merely their visibility alone and not their behaviour that is deemed problematic. (239) Several decades ago, my own research work (Reeve, (1996, 1998) and Reeve and Simmonds (2000)) demonstrated that there has been a growing convergence between conceptions and expectations of the High Street and that of the shopping mall which points up Coleman’s argument. There is no reason to believe this convergence has ended. The shopping Mall, in a sense, has always been a space apart from the everyday and from the fully formed public space of the city – a single minded space, to use Walzer’s term (see Fig 4). Its techniques of control and surveillance have been well understood, and to a degree accepted because of the difference of such space to the civic space of the street. The use of private security guards, the aestheticisation, even theming, of the interior, along with the use of CCTV to monitor users simply revealed its difference, and normalised it as liminal space which applied these strategies in order to be economically viable, and to deliver a special experience – these are places of spectacle, in which the user becomes a spectator of the ‘gift’ (Maus, 1969), and therefore, again, assumes a different identity to that in the real world of the public street In this context CCTV, to use Coleman’s term, offered a form of visible guardianship, the paternal eye. Fig. 4 The mall as a liminal and aestheticized space Fig. And finally that 4 The mall as a liminal and aestheticized space 23 \| P a g e However, it can evidenced and argued that precisely the same strategies of guardianship, in which consumer identity is privileged over other identities, have found their way onto the street – and have been reinforced through such things as Town Centre Management, Business Improvement Districts (see Reeve, 2008), and a raft of legislation from ASBOs (Anti-social Behaviour Orders) to Public Space Protection Orders brought in under the UKs Anti-Social Behaviour, Crime and Policing Act 2014,which effectively criminalise historically normal modes of conduct in public space (Fig 5) As an aside, these strategies also include the use of such designed elements as twenty-minute seating, seating so designed that it becomes uncomfortable to sit on after a short while, and impossible to lie down on (Fig 6). I would argue that the increasing monitoring of city streets, along with the license given under the 2014 Act in UK town and city centres, represents increasingly desperate efforts to maintain the aesthetic mythos of town centres as spaces of safe leisure in a context in which real poverty, homelessness and the other visible consequences of the failure of the state are becoming more and more evident and intrusive. Fig 5 Seating as a form of control Fig 5 Seating as a form of control 24 \| P a g e 24 \| P a g e Fig 6 Rules of use as a form of spatial control Fig 6 Rules of use as a form of spatial control What is perhaps particularly alarming for those on the liberal left, is the degree to which this situation has become normal: particularly the extensive use of CCTV to surveil public space (Fig 7). In Ground Control, Anna Minton (2009) suggested that ‘when asked, most people like the idea of CCTV and agree that its introduction would make them feel safer’ (168). However, she goes on to say that whilst there is little evidence that it has had any impact on crime figures, its use has resulted in a ‘retreat’ from ‘collective and individual responsibility to self-interest and fear’ (169). In other words, the much celebrated tendency for animated spaces to offer natural surveillance has been eroded by the presence of the technology of surveillance. And finally that In addition, the normalisation of CCTV as an aspect of everyday life is underpinned by the extent of its use – in 2009 4.2 million 25 \| P a g e cameras in the UK according to Minton, and by implication the vested commercial interest in finding reasons to justify its continued use.3 Fig. 7 CCTV in public space – Oxford Fig. 7 CCTV in public space – Oxford Fig. 7 CCTV in public space – Oxford As an aside, the logic of economic neo-liberalism in the city, also finds expression in the suburban and residential setting, principally in the form of gated communities. These are exclusive enclaves, often heavily CCTV’d, which literally turn their back on the larger public realm, and whose privately owned dwelling attract a premium because of their emphasis on both safety and exclusive community. 3 No one knows how many cameras now operate in public space, but estimates have put the figure at around six million. 4'Rightsism' refers to what might be termed an informal, moral movement, the main characteristic of which is to define the individual in terms of the rights that they might make claim to within society, thus emphasising the centrality of the individual over the collective, or community. Transgressions in the city – appropriating the gaze Having explored surveillance in the contemporary city as an expression of control, social, political, economic and so forth – and the way in which the various modes of control are articulated together as an expression of the current neo-liberal hegemony – we can now examine surveillance from the complementary or opposite perspective; that of the individual situated in public space. 3 No one knows how many cameras now operate in public space, but estimates have put the figure at around six million 26 \| P a g e 26 \| P a g e This is a highly complex theme, which can only be briefly outlined here; but in essence it incorporates the idea of the user as increasingly private, but one whose choices in terms of identity in particular have become increasingly aestheticized in a culture of over-production of competing signs and signifiers of the self. The individual, as a body situated within urban space (ie located historically and geographically), moreover has today not only the possibility of exercising his or her gaze at the other in their immediate and unmediated physical context, they also have the means via digital technology of locating and expressing themselves to others who are physically distant, and thus appropriating new and potentially empowering narratives and identities in the juncture between the real and the virtual. At the same time, the new technology allows the environment to respond at an individual level to the apparent gendered and other identity characteristics of users in real space - for instance, in the responsiveness of advertising screens that change their content according to the dominant characteristics of those present within their space. The final sections thinks through the idea of the counter-hegemonic strategies of individuals as occupiers of the city by drawing on concepts of appropriation, critical making, walking as narrative, and the flaneur as a now technologically empowered focus of experience and identity. The key sources here include Walter Benjamin’s The Arcades Project, begun in 1927 (1999), Lefebvre’s The Production of Space (1991), and De Certeau’s The Practice of Every Day Life (1984). The aestheticized self of public space In order to appreciate the nature of the surveilled and the surveilling self in the contemporary city, it is important to grasp how this self in post-industrial and culture after modenism is different to its predecessors. A number of cultural theorists have identified two recent cultural changes which mark out the present state of public space from the past, in their extreme forms at least: first the privatisation and aestheticisation of all aspects of social existence, at the level of the individual. Second, and connected with the first, a general loss of the meaning of the collective and the community which so powerfully underpinned the political assumptions of the modernist period. This was represented historically by the welfare state (at least in Britain from the late 1940s to the 1990s) and the division of public from private responsibilities, duties and rights. These values have been displaced by the 'rightsism'4 of the individual, who has become the apparent arbiter of social values and goods, a situation in which identity politics have displaced the politics of the collective, 4'Rightsism' refers to what might be termed an informal, moral movement, the main characteristic of which is to define the individual in terms of the rights that they might make claim to within society, thus emphasising the centrality of the individual over the collective, or community. 27 \| P a g e where the interests of the individual were arguably secondary to those of his or her class, nation, or other construct. With different emphases a number of social scientists, writing at the beginning of this moment of change (Featherstone (1992), Bauman (1992, 1994), Shields (1991, 1992), Giddens (1991), and Harvey (1990), stressed that one of the cultural characteristics of post modernism is the rise of the individual as a solipsistic, or at best family-centred, project, situated in a culture where there are no longer any objective and external guarantees of value. Under this view, the individual is private in a new sense: not simply a subject occupying a separate social territory in his or her feelings and actions (the home in particular), as distinct from the polis, the (urban) world of collective actions and responsibilities. The individual was now seen as a subjective project, even in the heart of the polis, of the social. The aestheticized self of public space Taking this position in one direction, identity is guaranteed not so much by class or structural social allegiances, but increasingly by reference to unstable taste or life-style categories. In this sense, the individual finds his or her sense of self, in its public dimension, with great difficulty and often only through its apparent opposition to other taste groups, its aesthetic difference. In an important sense, then, the self as an object of surveillance in the public domain, is the subject whose intention is to be surveilled and therefore to be visible on the basis of their predominantly aesthetically expressed characteristics – whether of gender, sexuality, life-style group, race or other visual aspects of being. In a sense, the individual can be seen as subject to the identity giving claims of a relatively new and aggressive consumer culture in which 'life-styles' become the anchor for the manufacture of self, both the foundation and arbiter of projects for the individual. The claims of collective responsibility, the sense of the other as being there in their own right and demanding some moral recognition because of that are, according to this view, under increasing threat (Bauman, 1992, 1994). The argument is that both as a consequence of the techniques of consumer persuasion (seduction according to Baudrillard, 1990) through product advertising in particular, and as a resource for its continuing manipulation, the self is what it consumes. 28 \| P a g e The counter view is that the whole of the public domain of contemporary free market societies is not saturated by consumerism, and its attempted effect of instrumentalising the self as a subject of consumption. If this were the case, we would have to cynically accept that the neo-liberal economic project, whose aim is to reduce individuals to politically neutered vectors of consumption, had succeeded. The opposing position is that individuals creatively appropriate the possibilities for self- expression, the expression of identities which are not given by consumer culture (gender based, tribe based, sexuality based etc. etc.) by re-engineering or repurposing the products of consumer 28 \| P a g e 28 \| culture to their own ends. Fiske (1989) after Gramsci, refers to this as excorporation, a process in which elements of the dominant culture – in this case free market consumerism – enter the domain of the popular and emerge transformed. The aestheticized self of public space This is a form of appropriation not dissimilar to Lefebvre’s use of the term, and particularly in his argument regarding spaces of representation and representations of space. The distinction he makes here is between the imposed identity of urban space which is manufactured by certain political or economic interests – through image, aesthetics and regulation (representations of space at their most obvious in environments such as shopping malls) – and the created identity of urban space made through the appropriation of users collectively and individually, where the given identity is transgressed or contradicted as a form of spectacle. Along with De Certeau (1984) Lefebvre uses the term ‘lived space’, to designate that space which is appropriated by individuals and groups for their own ends, as opposed to space which is simply a setting for the instrumentalised expression of manufactured and inauthentic and alienated identity. However, Fiske (1989) reminds us that what he calls the ‘guerrila tactics’ and the micro-political appropriation or ‘critical making’ of public space can only achieve occasional, irregular and often illusory, or compensatory change: such appropriation affects the situation of place, but rarely the underlying structures of power or economic inequality. In terms of the notion of surveillance, the discussion of appropriation and excorporation, particularly via the notion of the spectacle, (Reeve 1995) reminds us that the city and its public spaces provide a setting or stage set, in some sense, for the performance of individuals either as the cyphers of consumption, or as at least partial agents of their own identity, who can be the object of the gaze of others. In addition, it can be argued that acts of appropriation and excoropration provide opportunities for connecting with others who share at least some of the same provisional identities, and in that sense transgress the strangerhood of the urban. Bauman (1984) sees this alienating character of cities – what he calls ‘privatism’ – as the inevitable product of an historical process integral to urbanisation. In urban cultures where almost every encounter is an encounter with a stranger we have learned not to recognise others: 'societas' has replaced 'communitas'. This failure to see the other means that we occupy social space as if we were for the most part alone, seeing only the surface of the other and therefore not recognising any moral responsibility towards the other. The aestheticized self of public space This section briefly explores the idea of the individual as an active appropriator of the city, and how the gaze of the individual is a key aspect of this mode of being, enhanced by the new technologies of the mobile phone and other personal technologies of surveillance. The discussion draws on three or four themes relatively well rehearsed in the philosophical and sociological literature of the city The previous section presented the idea of the surveilled self as both an object and subject of the city – both controlled through surveillance by CCTV, for example, and controlling via the expression of at least negotiated and temporary identities (often centred on the apparent or visble characteristics of the body) via transgressive appropriation of given stylistic tropes of appearance. – from Walter Benjamin, Lefebvre (again), De Certeau and John Urry (1990, 1995)– which again include the notion of the flaneur, the idea of the urban as a setting for ‘desire’, the idea of the city street as the raw material for the creation of ‘scripts’ of the self, and the concept of ‘gaze’ of the outsider – the tourist or visitor. I want to argue that these themes are linked together not only by their common setting – the urban – but also through the view that surveillance for these experiences and acts is a form of mediation in which the world as observed is interpreted or (and sometimes creatively) made through the act of surveillance, in which the facticity of the street is given particular personal, affective or social meaning in real time. In a sense, De Certeau’s essay, and particularly within this the chapter Walking in the City provides the starting point for understanding the potential of the individual as an appropriator of the city at the personal level, and the function of what can be seen or surveilled within this. Walking in the City begins with a brief account of a view of Manhattan from the top of the World Trade Centre (ironically, of course, since destroyed by an act of terror broadcast and gazed upon by a global audience) , which De Certeau presents as a kind of metaphor for seeing as an act of fiction, or of myth making: His elevation transfigures (the spectator) into a voyeur. It puts him at a distance. The aestheticized self of public space I want to argue that, paradoxically, the fact of the urban as a site of surveillance – albeit that of the individual - provides a place for at least a provisional and momentary overcoming of the alienation of the urban. 29 \| P a g e The surveilling self of the city The surveilling self of the city The previous section presented the idea of the surveilled self as both an object and subject of the city – both controlled through surveillance by CCTV, for example, and controlling via the expression of at least negotiated and temporary identities (often centred on the apparent or visble characteristics of the body) via transgressive appropriation of given stylistic tropes of appearance. This section briefly explores the idea of the individual as an active appropriator of the city, and how the gaze of the individual is a key aspect of this mode of being, enhanced by the new technologies of the mobile phone and other personal technologies of surveillance. The discussion draws on three or four themes relatively well rehearsed in the philosophical and sociological literature of the city – from Walter Benjamin, Lefebvre (again), De Certeau and John Urry (1990, 1995)– which again include the notion of the flaneur, the idea of the urban as a setting for ‘desire’, the idea of the city street as the raw material for the creation of ‘scripts’ of the self, and the concept of ‘gaze’ of the outsider – the tourist or visitor. I want to argue that these themes are linked together not only by their common setting – the urban – but also through the view that surveillance for these experiences and acts is a form of mediation in which the world as observed is interpreted or (and sometimes creatively) made through the act of surveillance, in which the facticity of the street is given particular personal, affective or social meaning in real time. The previous section presented the idea of the surveilled self as both an object and subject of the city – both controlled through surveillance by CCTV, for example, and controlling via the expression of at least negotiated and temporary identities (often centred on the apparent or visble characteristics of the body) via transgressive appropriation of given stylistic tropes of appearance. The aestheticized self of public space It transforms the bewitching world by which one was ‘possessed’ into a text that lies before one’s eyes. It allows one to read it, to be a solar eye, looking down like a god. The exaltation of a scopic and gnostic drive: the fiction of knowledge is related to this lust to be a viewpoint and noting more. 92 92 He sets this privileged, surveilling perspective against the experience of the practice of walking the city, which he takes as an essentially creative act like speech: He sets this privileged, surveilling perspective against the experience of the practice of walking the city, which he takes as an essentially creative act like speech: 30 \| P a g e The act of walking is to the urban system what the speech act is to language or to the statements uttered. …it is a process of appropriation of the topographical system on the part of the pedestrian. De Certeau – 97-98 The act of walking is to the urban system what the speech act is to language or to the statements uttered. …it is a process of appropriation of the topographical system on the part of the pedestrian. De Certeau – 97-98 And, If it is true that a spatial order organizes an ensemble of possibilities (e.g. by a place in which one can move) and interdictions (e.g., by a wall that prevents one from going further), then the walker actualizes some of these possibilities. In that way he makes them exist as well as emerge. But he also moves them about and he invents others, since the crossing drifting away, or improvisation of walking privilege, transform or abandon spatial elements. 99 One reading of De Certeau’s argument is that there is an essential difference between the god-loke perspective of the spectator – the one who surveills the city from a distance or on high – and that of the individual engaged in the city as a practice, mediated solely by the ‘narrative’ skills and linguistic (as a metaphor) competencies of the individual pedestrian. Looking or surveilling in this sense is a simple act of pragmatic observation engaged in way-finding and way making. The aestheticized self of public space It is a position adopted in more recent approaches to understanding the relationship of the individual to place in non-representational geography (Thrift, 2010) – and theories of place as affect (Bohme, 1993) in which the individual becomes the focus of sensation, and what they see – the world out there - is mediated only through it’s ‘atmospheric’ conditions. However, it is clear that other conceptions of the individual as appropriator or practitioner of the city acknowledge that that appropriation, and how the individual sees place as having meaning and their own relationship to that meaning, is conditioned by aspects of identity and power which they bring to the experience and seeing of place, and therefore is bound up with their own authentic or inauthentic intentions towards it. To use De Certeau’s terms, the individual in space creates a meaningful narrative by imposing or interpreting that space in relation to or by means of their own ‘voyeurism’, or set of essentially personal, political and mythic constructs. Ironically, the flaneur, gets us somewhat closer to an understanding of this relationship between the city as a setting for personal narrative and self-making, The term was used most evocatively by the 19th Century French poet Baudellaire: The crowd is his element, as the air is that of birds and water of fishes. His passion and his profession are to become one flesh with the crowd. For the perfect flâneur, for the passionate spectator, it is an immense joy to set up house in the heart of the multitude, amid the ebb 31 \| P a g e 31 \| and flow of movement, in the midst of the fugitive and the infinite. To be away from home and yet to feel oneself everywhere at home; to see the world, to be at the centre of the world, and yet to remain hidden from the world—impartial natures which the tongue can but clumsily define. The spectator is a prince who everywhere rejoices in his incognito. The lover of life makes the whole world his family, just like the lover of the fair sex who builds up his family from all the beautiful women that he has ever found, or that are or are not—to be found; or the lover of pictures who lives in a magical society of dreams painted on canvas. The aestheticized self of public space Thus the lover of universal life enters into the crowd as though it were an immense reservoir of electrical energy. Or we might liken him to a mirror as vast as the crowd itself; or to a kaleidoscope gifted with consciousness, responding to each one of its movements and reproducing the multiplicity of life and the flickering grace of all the elements of life. Quoted in Benjamin (1964) In his essays on Baudellaire, Walter Benjamin - The Painter of Modern Life (1964) and Charles Baudellaire, A Lyric Poet in the Era of High Capitalism (1983) saw the flaneur as both urban spectator, and as participant in urban life, whose projects of urban exploration were allowed by his appearance of economic independence, and directed by a desire for a separateness of identity to the crowd, and the intention to demonstrate, by their disguised visibility, a critical disposition towards the anonymity of modern urban life. De Certeau’s naïve idealizing – that the city can be appropriated as narrative through the simple act of walking- is perhaps highlighted if we acknowledge that the flaneur was always a male figure. As Elizabeth Wilson (1991) in the Sphinx in the City provocatively suggests, the female equivalent to the flaneur in 19th Century Paris was the prostitute – whose visibility was likewise a theatrical one, but whose power or autonomy was constrained entirely by, for the most part, her lack of economic choice, in which her body became a resource for the exploitation – both visually and in other ways – of the male gaze (another form of surveillance). Lefebvre’s work is again helpful in allowing us to think about another dimension of the appropriation of public space through the surveillance by the individual, which is linked to the foregoing discussion: the distinction between needs and desires: Specific needs have specific objects. Desire, on the other hand, has no particular object, except a space where it has full play: a beach, a place of festival, the space of the dream. (Lefebvre, 1991, 353) 32 \| P a g e It is at least arguable that urban spaces, precisely because of the aestheticisation necessitated in the recent and current neo-liberal economic and competitive context, are increasingly experienced as ‘dream spaces’. This is clearly evident in environments such as the shopping mall 32 \| P a g e – which, as we have seen, are often through a combination of styling, management, function and image – are set apart from the space of the everyday street. But it is increasingly evident in the everyday street of the city, with their increasingly aestheticized qualities. Quoted in Benjamin (1964) In this context, at least for the consumer, if not for those who service that consumption (who in Disney World are referred to as ‘cast members’ in relation to users who are seen as ‘guests’). John Urry’s seminal text - the Tourist Gaze ( 1990 ), further amplifies the point that users surveill the city intentionally- either as expression of desire, or through expectations already constructed for them on the basis of advertising or place promotion. Urry’s argument is that, in essence, users as tourists gaze on the spectacle of place in order to recognise its conformity to the stereotyped or metonimised representations experienced before the reality is encountered. In order to do this –as in Lefebvre’s notion of the leisure space set aside from the everyday – certain conditions have to be in place, such as the presence of a guide, the schedule in which elements of the tourist object (the city) are parcelled up in accordance with a structured timetable etc. etc. Finally, some consideration has to be given to the way in which the relatively new technologies of surveillance – mobile phones with apps to guide the visitor, mapping, GIS, pop up informatics etc. – both mediate what is seen, and offer on the face of it, opportunities for a new kind of flanerie, voyeurism or flirting, and even sexual assignations in real time, which would previously have required the actual presence of the other. Remote or digital surveillance is most commonly associated with CCTV, particularly in public settings such as malls, high streets, and even third spaces, as we have seen. However, the use of technology at the personal level, and for private ends within public space has become common place and indeed ubiquitous. This has implications in terms of how users inhabit space directly – ie in their physical engagement or disengagement with the street, but also in terms of how they access it’s virtual or physically un-immediate risks and opportunities. Twenty years ago, the sight of pedestrians – and even drivers and cyclists – on their mobile phones whilst navigating the street would have been highly unusual. There are now cities which are retrofitting streets with slow lanes for mobile phone users. 33 \| P a g e However, more relevant to this chapter are the modes of use of technology to observe and interface with the city and beyond, via the virtual. Quoted in Benjamin (1964) Clearly, at a pragmatic level, apps such as google maps allow individuals to navigate the city, along with downloadable city guides, more conveniently than other media – and place the user in real space. But even this functionality is mediated for the user through adverts for local facilities and amenities – effectively creating an edited or electronically 33 \| P a g e manufactured discursive identity of place. The counter side to this, of course, is that the phone provides access to other, blogged, readings of the city which users can personalise, or self-create, and share with others to add to or even create new communities of taste. The new technology can also extend the gaze of the users – so that it becomes possible to surveill the city and beyond, in order, for example, to locate and track other users ( eg Snapchat). The technology can also be employed, with the right apps or platforms (Tinder is notorious) to seek out others for ends once highly private, and again to create a private and exclusive communities of, say, erotic interest in the context of the public domain: again in non-deferred real time. In terms of the opportunities for surveillance – both surveilling and being surveilled - the new(ish) technology is, then, capable of being both transgressive, and of reinforcing normative spatial practices. Conclusion This chapter has attempted to capture some of the key issues and theories around the problematic of surveillance in public space, informed from the perspective of both sociological and political theory, as well as from my own view point as an academic researching and teaching in the field of urban design. The chapter began with a review of the nature of public space by setting the notion of the public, more generally, against the idea of the private – and by implication the concept of privacy. It sketched out a theory of social spatialisation, in which three aspects of the making of public space – form, image and regulation – were seen to be articulated together, particularly in spaces increasingly given over to consumption – the entrepreneurial city and its neo-liberal tendencies. In this I sought to contrast what the seminal literature highlights with respect to two key modes of surveillance, control and safety – the animated place in which natural surveillance / eyes on the street, is taken as the preferred option, and the panotpticon place, in which surveillance happens remotely and without the consent of the surveilled. The chapter began with a review of the nature of public space by setting the notion of the public, more generally, against the idea of the private – and by implication the concept of privacy. It sketched out a theory of social spatialisation, in which three aspects of the making of public space The chapter began with a review of the nature of public space by setting the notion of the public, more generally, against the idea of the private – and by implication the concept of privacy. It sketched out a theory of social spatialisation, in which three aspects of the making of public space – form, image and regulation – were seen to be articulated together, particularly in spaces increasingly given over to consumption – the entrepreneurial city and its neo-liberal tendencies. – form, image and regulation – were seen to be articulated together, particularly in spaces increasingly given over to consumption – the entrepreneurial city and its neo-liberal tendencies. Conclusion In this I sought to contrast what the seminal literature highlights with respect to two key modes of surveillance, control and safety – the animated place in which natural surveillance / eyes on the street, is taken as the preferred option, and the panotpticon place, in which surveillance happens remotely and without the consent of the surveilled. – form, image and regulation – were seen to be articulated together, particularly in spaces increasingly given over to consumption – the entrepreneurial city and its neo-liberal tendencies. In this I sought to contrast what the seminal literature highlights with respect to two key modes of surveillance, control and safety – the animated place in which natural surveillance / eyes on the street, is taken as the preferred option, and the panotpticon place, in which surveillance happens remotely and without the consent of the surveilled. The chapter concluded with a consideration of the user as appropriator of space via their own gaze, and use of space; but also through the emerging technologies of personal surveillance in which certain aspects of the private are experienced and reproduced in public settings. Finally, surveillance is an inevitable condition of public space – the question is, to what ends and for what ends is the gaze of the other exercised in such settings: and to what degree are designers, managers, and society more generally complicit when such surveillance is used to restrict liberty and identity as opposed to creating the possibility for authentic self-expression and enjoyment? 34 \| P a g e References: Anderson, S. (1986) On Streets, M.I.T. press, Mass. Anderson, S. (1986) On Streets, M.I.T. press, Mass. Ashworth, J. & Voogd, H. (1990) Selling the City, Belhaven Press, London Baudrillard, J. (1990) Seduction, Macmillan Educational, Basingstoke. Baudrillard, J. (1990) Seduction, Macmillan Educational, Basingstoke. Baudrillard, J. (1990) Seduction, Macmillan Educational, Basingstoke. Bauman, Z. (1992) Intimations of Post-Modernity, Routledge, London. Bauman, Z. (1994) Post-Modern Ethics, Blackwell, Oxford. Benjamin, W (1964) Charles Baudelaire, "The Painter of Modern Life", New York: Da Capo Press Benjamin, W. (1983) Charles Baudelaire, A Lyric Poet in the Era of High Capitalism, Verso, London. Benjamin, W (1999) The Arcades Project, Belknapp Press, Cambridge Bentley, I. et al. (1985) Responsive Environments, A Manual for Designers, Architectural Press, London. Bohme, G (1993) Atmosphere as the Fundamental Concept of a New Aesthetics in Thesis Eleven, Number 36, 113-126 Carmona, M et al (2010) Public Places – Urban Spaces, \|Architectural Press, London Campbell, C. (1987) The Romantic Ethic and the Spirit of Modern Consumerism, Blackwell, London. Castells, M. (1989) The Informational City: Information Technology, Economic Restructuring and the Urban Restructuring Process. Blackwell, Oxford. Certeau, de, M. (1984) The Practice of Everyday Life, University of California Press, Berkley. Coleman, R, (2007) Surveillance in the City, inn Hier, PS and Greenberg, G eds. The Surveillance Studies Reader, Chapter16, 231-24, McGraw Hill/ Open University Press, Maidenhead English Partnerships (2000) Urban Design Compendium, English Partnerships Marcus, CC and Francis, C eds (1998) People Places, Van Nostrand Reinhold, New York Daunton, M.J. (1983) House and Home in the Victorian City, Edward Arnold, London. Debord, G. (1973) Society of the Spectacle, Black and Red, Detroit. Debord, G. (1973) Society of the Spectacle, Black and Red, Detroit. Featherstone, M. (1983) 'Consumer Culture: An Introduction', in Theory, Culture and Society, 3, 4- 9. Featherstone, M. (1983) 'Consumer Culture: An Introduction', in Theory, Culture and Society, 3, 4- 9. 35 \| P a g e Featherstone, M. (1991) Consumer Culture and Post-Modernism, Sage, London. 35 \| P a g e Featherstone, M. (1991) Consumer Culture and Post-Modernism, Sage, London. 35 \| P a g e Featherstone, M. (1991) Consumer Culture and Post Modernism, Sage, London. 35 \| P a g e Featherstone, M. (1992) 'Post-Modernism and the Aestheticisation of Everyday Life', in Modernity and Identity ed. Fieldman and Lash, Blackwell, Oxford. Ferguson, H. (1992), 'Watching the World go Round, Atrium Culture and The Psychology of Shopping', in Lifestyle Shopping, ed., Shields, Routledge, London. (1992) 'Shopping Malady', in Designer's Journal, January, 32-33. Financial Times (1992) 'EuroDisney', June 5, 21 Finklestein, J. (1991) The Fashioned Self, Polity, Cambridge. Fiske, J (1989) Understanding popular Culture, Routledge, London Fiske, J (1989) Understanding popular Culture, Routledge, London Ford, L. Lyon, D (1994) The Electronic Eye: The rise of surveillance society, Polity Press, cambridge Baudrillard, J. (1990) Seduction, Macmillan Educational, Basingstoke. (1994) Cities and Buildings, Skyscrapers, Skid Rows and Suburbs, Johns Hopkins Foucault, M. (1973) Discipline and Punish, Pantheeon Books, London Foucault, M. (1986) 'Other Spaces: The Principles of Heretopia', Lotus International, 48/49, 9-17. Giddens, A. (1992) Transformations of Intimacy, Cambridge, Polity. oucault, M. (1986) 'Other Spaces: The Principles of Heretopia', Lotus International, 48/49, 9- Giddens, A. (1990) The Consequences of Modernity, Polity Press, Cambridge. Goffman, E. (1959) The Presentation of Self in Everyday Life, New York, 1959. Mass. Golby, JM, and Purdue, AW (1984) Civilization of the Crowd, Popular Culture in England from 1700- 1900, Batsford Academic and Educational, London Golby, JM, and Purdue, AW (1984) Civilization of the Crowd, Popular Culture in England from 1700- 1900, Batsford Academic and Educational, London Gold, RG and Revill, G (2000) Landscapes of Defence, Prentice hall, Harlow Harvey, D (1987) Flexible Accumulation through Urbanization, in Antipode, 19:3 260-287 Harvey, D. (1990) The Condition of Post-Modernity, Blackwell, Oxford. Heller, A. & Feher, F. (1988) The Postmodern Political Condition, Polity Press, Cambridge. Jackson, P. (1990) Maps of Meaning: An Introduction to Cultural Geography, Routledge, London. Jacobs, J. (1984) The Death and Life of Great American Cities,Peregrine Books, Harmondsworth Jameson, F. (1991) Post-Modernism, or the Cultural Logic of Late Capitalism, Verso, London. Jackson, P. (1990) Maps of Meaning: An Introduction to Cultural Geography, Routledge, London. Jacobs, J. (1984) The Death and Life of Great American Cities,Peregrine Books, Harmondsworth Jameson, F. (1991) Post-Modernism, or the Cultural Logic of Late Capitalism, Verso, London. Knox, P. ed. (1993) The Restless Urban Landscape, Prentice Hall, New York. Lefebvre, H. (1991) The Production of Space, Blackwell, Oxford. Lefebvre, H. (1991) The Production of Space, Blackwell, Oxford. Lyon, D (1994) The Electronic Eye: The rise of surveillance society, Polity Press, cambridge 36 \| P a g e 36 \| P a g e Markus, T. (1993) The Power of Buildings, Routledge, London. Maus. M. (1969) The Gift, Cohen and West, London. Minton, A (2009) round Control, fear and happiness in the twenty-first century city, Penguin Books, London Morris, AEJ, ( 1979) History of Urban Form, George Goodwin, London Mumford, L (1987 ) The City in History, London: Penguin Newman, O (1973) Defensible Space, Architectural Press, London Rabinow and Dreyfus. (1982) Michel Foucault, Beyond Structuralism and Hermeneutics, Chicago University Press, Chicago. Vidler, A. (1978) 'The Scenes of the Street', in On Streets, ed. Anderson,S., MIT Press, Cambridge, Mas. Walzer, M. (1986) 'Pleasures and Costs of Urbanity', in Dissent, Fall, 470-475. Baudrillard, J. (1990) Seduction, Macmillan Educational, Basingstoke. Reeve AR (1995) Urban Design and Places of Spectacle, unpublished PhD thesis, Oxford Brookes University Reeve, AR (1996) 'The Private Realm of the Managed Town Centre', in Urban Design International , March, Vol 1 No 1 Reeve AR (1998) The Panopticisation of Shopping, in Norris, C et al Surveillance, Closed Circuit Television and Social Control, Ashgate, Aldershot Reeve, AR (2008) ‘Town Centre Management, towards a research agenda’ in Business Improvement Districts: Research, theories and controversies. Morçöl, G editor MIT Press Reeve AR, Simmonds, R., (2000) ‘Hyperreality in the shire: Bicester Village and the village of Bicester’ in Urban Design International, Vol 5 No. 2 pp 141 – 154 Rojek, C. (1993) 'Disney Culture', in Leisure Studies, Vol 12, 121-135. ustin, M. (1986) 'The Fall and Rise of Public Space' in Dissent, Fall, 486-494. ds, R. (1991) Places on the Margin: Alternative Geographies of Modernity, Routledge, London Shields, R. ed. (1992) Lifestyle Shopping: The Subject of Consumption, Routledge, London. Thrift, N ed (2010) Taking-Place: Non-Representational Theories and Geography, Ashgate, Farnham Urry, J. (1990) The Tourist Gaze: Leisure and Travel in Contemporary Societies, Sage, London. Urry J (1995) Consuming Places Routledge London Urry, J. (1990) The Tourist Gaze: Leisure and Travel in Contemporary Societies, Sage, London. (1995) Consuming Places, Routledge, London. 37 \| P a g e Vidler, A. (1978) 'The Scenes of the Street', in On Streets, ed. Anderson,S., MIT Press, Cambridge, Mas. Walzer, M. (1986) 'Pleasures and Costs of Urbanity', in Dissent, Fall, 470-475. Whyte, W. (1980) The Social Life of Small Urban Spaces, Washington D.C. Conservation Foundation, Washington. Wilson, E (1991 ) The Shpinx in the City, London, Virago Vidler, A. (1978) 'The Scenes of the Street', in On Streets, ed. Anderson,S., MIT Press, Cambridge, Mas. Vidler, A. (1978) 'The Scenes of the Street', in On Streets, ed. Anderson,S., MIT Press, Cambridge, Mas. Walzer, M. (1986) 'Pleasures and Costs of Urbanity', in Dissent, Fall, 470-475. Whyte, W. (1980) The Social Life of Small Urban Spaces, Washington D.C. Conservation Foundation, Washington. Wilson, E (1991 ) The Shpinx in the City, London, Virago Wilson, E (1991 ) The Shpinx in the City, London, Virago 38 \| P a g e
https://openalex.org/W2791331105	https://europepmc.org/articles/pmc5802573?pdf=render	English	null	Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition	Blood cancer journal	2,018	cc-by	3,455	Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition Gabriela Galicia-Vázquez1,2,3, Sarah Smith1,2 and Raquel Aloyz1,2,3,4 Gabriela Galicia-Vázquez1,2,3, Sarah Smith1,2 and Raquel Aloyz1,2,3,4 del11q-positive, which represent occurrence of del11q cases in the clinical setting (~20%), while del17p cases were excluded (Supplementary Table 1). CLL lympho- cytes were maintained in a physiological glucose con- centration, since our group noticed that CLL cells exposed to high or limited glucose levels display different metabolic responses8. Under basal conditions, no sig- niﬁcant difference was found in glucose or glutamine uptake, total or reduced glutathione, and ROS levels (Supplementary Fig. 1a–d) between del11q-positive (hereafter del11q) and del11q-negative (hereafter wild- type) CLL lymphocytes, suggesting that del11q CLL lymphocytes are adapted to reduced ATM expression. Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of malignant B cells, and their accumulation in the blood stream and homing tissues. In the CLL context, the chromosomal aberrations del17p and del11q, spanning TP53 and ATM locus, respectively, are considered poor outcome predictors1,2. As well, CD38, ZAP70, and unmutated status of the IgVH locus represent bad prognosis markers1. Despite the outstanding clinical results yielded by ibrutinib (mainly used in relapsed or refractory settings), no complete remission is guaranteed due to the arising of resistance and relapse upon treat- ment discontinuation. The lack of tailored therapies upon ibrutinib failure represents a major challenge, particularly in del11q and del17p cases refractory to conventional therapies3. Recently, the analysis of metabolic repro- gramming has uncovered tumor cell vulnerabilities, leading to the development of novel therapeutic approa- ches; such as the use of ritonavir (glucose uptake inhi- bitor) and metformin (OxPhos inhibitor) for multiple myeloma treatment4. CLL lymphocyte metabolism was explored by challen- ging these lymphocytes with a panel of metabolic inhibi- tors (Supplementary Table 2). The disturbance of glycolysis (2DG), glucose uptake (ritonavir) or OxPhos (oligomycin) decreased all CLL cell viability. Del11q CLL cells displayed enhanced sensitivity for the three treat- ments. Inhibiting fatty acid oxidation (etomoxir) was equally cytotoxic to all CLL lymphocytes; conversely, the inhibition of the pentose phosphate pathway (PPP) (DHEA) or the one-carbon metabolism (AMPA) was not cytotoxic to CLL cells (Fig. 1a). The aim of this work was to deﬁne targetable metabolic features in CLL lymphocytes with respect to their del11q status, since ATM—a reactive oxygen species (ROS) sensor and metabolic regulator5—and miR125—meta- bolic regulator6—genes are comprised within del11q. Also, del11q has been linked to increased insulin receptor expression7. 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/. Galicia-Vázquez et al. Blood Cancer Journal (2018) 8:13 DOI 10.1038/s41408-017-0039-2 Galicia-Vázquez et al. Blood Cancer Journal (2018) 8:13 DOI 10.1038/s41408-017-0039-2 Blood Cancer Journal O p e n A c c e s s C O R R E S P O N D E N C E Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition We used a panel of 26 CLL primary samples donated from affected patients, 23% of which were Curtailing the ﬁrst step of glutamine metabolism, via glutaminase (GLS1) inhibition (compound 968), was cytotoxic only to del11q CLL cells (Fig. 1a). Since gluta- mine and glutamate uptake were similar between del11q and wild-type CLL cells (Supplementary Fig. 1e, f), it is likely that differential utilization of these metabolites is associated with del11q status. A distinctive characteristic of del11q CLL cells was the consumption of ammonia under basal conditions, while wild-type CLL lymphocytes © The Author(s) 2018 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/. Blood Cancer Journal Galicia-Vázquez et al. Blood Cancer Journal (2018) 8:13 Page 2 of 5 Fig. 1 Del11q CLL lymphocytes display glutamine metabolic alterations. a, g Survival fraction (relative to non-treated control (NT)) of CLL lymphocytes treated with metabolic inhibitors for 48 h. a n = 23, g n = 19. b Ammonia uptake after 24 h compound 968 treatment (n = 14). c GS (n = 13) and d GDH (n = 13). Western blot of samples treated with compound 968 for 24 h. Representative images (upper panel) and quantiﬁcation (lower panel) are shown. e Relative ROS values after 24 h treatment (n = 19, n (968 + NAC) = 8). f Population median value of total glutathione after 24 h of compound 968 treatment (n = 12). NS, not signiﬁcant, p < 0.05, p < 0.001 Galicia-Vázquez et al. Blood Cancer Journal (2018) 8:13 Page 2 of 5 Fig. 1 Del11q CLL lymphocytes display glutamine metabolic alterations. a, g Survival fraction (relative to non-treated control (NT)) of CLL lymphocytes treated with metabolic inhibitors for 48 h. a n = 23, g n = 19. b Ammonia uptake after 24 h compound 968 treatment (n = 14). Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition However, 2DG plus DHEA- induced cytotoxicity is not driven by oxidative stress, since the drugs (alone or in combination) did not sig- niﬁcantly affect ROS levels in the CLL clones tested (Supplementary Fig. 3b). Of note, similar cytotoxicity was obtained upon 2DG and ritonavir treatment in del11q CLL lymphocytes, making it possible that GLUT4 is their main glucose transporter (Fig. 1a). Unexpectedly, AMPA antagonized 2DG-induced cytotoxicity regardless of del11q status (Fig. 2c). It is possible that one-carbon metabolism drives glucose-derived carbons out of glyco- lysis in basal conditions, and AMPA treatment allows these carbons back to glycolysis. In this scenario, serine accumulation upon SHMT1/2 inhibition could contribute to PKM2 activation, enhancing glycolysis ﬂux, down- stream of hexokinase. secrete ammonia (Fig. 1b). This suggests enhanced amino-acid catabolism in wild-type CLL cells. Accord- ingly, glutamine synthetase (GS) expression was higher in del11q compared to wild-type CLL lymphocytes, while glutamate dehydrogenase (GDH) expression followed the opposite trend (Fig. 1c, d). The former suggests that glutamine is synthesized de novo via GS and the latter that transaminase reactions using α-ketoglutarate for glutamate synthesis is favored in del11q CLL cells. As well, reduced oxidative deamination of glutamate via GDH could account for decreased extracellular ammonia accumulation in del11q CLL lymphocytes. Glutamine synthesis is favored when ammonia detoxiﬁcation is required, and for tricarboxylic acid cycle cataplerosis in rich nutrient conditions9; however, glutamine uptake and GLS1 expression were similar between subsets (Supple- mentary Fig. 1e, g). The latter raises the possibility that differences in energetic or biosynthetic requirements exist between wild-type and del11q CLL lymphocytes. GLS1 inhibition increased extracellular ammonia in del11q CLL lymphocytes (Fig. 1b). However, glutamine and glutamate uptake were not affected by compound 968, suggesting that CLL lymphocytes are poised to maintain constant extracellular concentrations of these metabolites (Sup- plementary Fig. 1e, f). Our ﬁndings suggest that amino- acid catabolism contributes to glutamate production in CLL lymphocytes. Also, we propose that GDH contribu- tion to glutamate pools is limited under GLS1 inhibition, since GDH expression was signiﬁcantly reduced after compound 968 treatment, regardless of del11q status (Fig. 1d). ROS levels increased upon GLS1 inhibition, a possible consequence of decreased total glutathione—a tri-peptide formed by glutamate, glycine, and cysteine (Fig. 1e, f). Noteworthy, the cysteine precursor N-Acetyl-L-cysteine (NAC) reduced ROS levels without affecting survival (Fig. 1e, g), and promoted the accumulation of extracellular glutamate (Supplementary Fig. 2a). Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition c GS (n = 13) and d GDH (n = 13). Western blot of samples treated with compound 968 for 24 h. Representative images (upper panel) and quantiﬁcation (lower panel) are shown. e Relative ROS values after 24 h treatment (n = 19, n (968 + NAC) = 8). f Population median value of total glutathione after 24 h of d 968 t t t ( 12) NS t i iﬁ t < 0 05 ** < 0 001 Fig. 1 Del11q CLL lymphocytes display glutamine metabolic alterations. a, g Survival fraction (relative to non-treated control (NT)) of CLL lymphocytes treated with metabolic inhibitors for 48 h. a n = 23, g n = 19. b Ammonia uptake after 24 h compound 968 treatment (n = 14). c GS (n = 13) and d GDH (n = 13). Western blot of samples treated with compound 968 for 24 h. Representative images (upper panel) and quantiﬁcation (lower panel) are shown. e Relative ROS values after 24 h treatment (n = 19, n (968 + NAC) = 8). f Population median value of total glutathione after 24 h of compound 968 treatment (n = 12). NS, not signiﬁcant, p < 0.05, p < 0.001 Blood Cancer Journal Page 3 of 5 Galicia-Vázquez et al. Blood Cancer Journal (2018) 8:13 CLL lymphocytes, in the context of glycolysis inhibition, DHEA potentiated 2DG cytotoxicity (Fig. 2a, Combina- tion Index = 0.67), especially in del11q CLL clones. Such an effect has been previously reported for other cancer cell lines11, and is suggestive of the dependence of CLL lymphocytes on the coordinated use of glycolysis and PPP to survive. In line with differential glucose metabolic reprogramming, 2DG decreased glucose uptake only in del11q CLL cells (Fig. 2b), suggesting increased depen- dency on glycolysis for survival. Contrariwise, DHEA increased glucose uptake and metabolic activity (detected by C-12 Resazurin reduction) only in wild-type CLL clones (Fig. 2b and Supplementary Fig. 3a), suggesting that glucose ﬂux through the PPP is favored in this subset, and that DHEA triggers the redirection of glucose carbons to glycolysis, promoting mitochondrial activity. The shunting of glucose from glycolysis to PPP was previously documented in red blood cells under 2DG treatment and it is believed to occur in quiescence in response to high NADPH demands12,13. Blood Cancer Journal Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition Since CLL lympho- cytes express the cysteine/glutamate xCT antiporter, we propose that NAC uptake limits glutamate-dependent processes and reduces nutrient usage plasticity in CLL lymphocytes, as recently reported for other cancer cells10. Accordingly, NAC enhanced compound 968 cytotoxicity regardless of del11q status (Fig. 1g), which could not be compensated by increased glutamine uptake (Supple- mentary Fig. 2b). Furthermore, compound 968 triggered an increase in glucose uptake in wild-type CLL lympho- cytes only (Supplementary Fig. 2c). Based on our results, we infer that wild-type cells but not del11q lymphocytes can efﬁciently rewire their amino-acid and glucose metabolism to compensate GLS1 inhibition. Recently, it was reported that enhanced OxPhos is associated with unmutated IgVH and advanced Rai stage, but not with del11q or del17p in CLL lymphocytes (on hyperglycemic conditions). Furthermore, in contrast to our results below using ibrutinib, pharmacological tar- geting of the PI3K pathway in primary CLL lymphocytes decreased OxPhos without affecting glutamine/glucose uptake or ROS levels14. Bruton's Tyrosine Kinase (BTK) inﬂuence on metabo- lism was assessed by ibrutinib treatment. Ibrutinib increased glucose uptake, glutamine uptake, and ammo- nia secretion, without disturbing glutamate secretion (Supplementary Fig. 4a–d), regardless of del11q status. In line with enhanced ROS levels upon ibrutinib treatment (Fig. 2d), total and reduced glutathione levels were decreased by 60% and 90%, respectively, as well as NADP/ NADPH ratio (Supplementary Fig. 4e–g). Ibrutinib- induced cytotoxicity was inﬂuenced by oxidative stress, since NAC could decrease ROS levels along with To further explore differences in glucose metabolism by del11q, we made drug combination treatments with 2DG and DHEA. Although PPP inhibition was not cytotoxic to Galicia-Vázquez et al. Blood Cancer Journal (2018) 8:13 Page 4 of 5 Fig. 2 Effect of glucose metabolic inhibitors and ibrutinib on CLL metabolism. a, c, e, f Survival fraction (relative to non-treated control (NT)) of CLL lymphocytes treated with metabolic inhibitors for 48 h. b n = 21, c n = 12, e n = 19, f n = 23. b Glucose uptake after 24 h compound treatment (n = 15). d Relative ROS median values of CLL cells after 24 h of ibrutinib and/or NAC treatment (n = 20). p < 0.05, *p < 0.001. g Model for basal wild- type and del11q CLL cell metabolism. The proposed metabolic ﬂux in CLL lymphocytes is indicated with brown (wild-type) and pink (del11q) arrows. Del11q CLL lymphocytes promote glutamine synthesis, while decreasing GDH reaction. Blood Cancer Journal References 1. Oscier, D. G. et al. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 100, 1177–1184 (2002). 1. Oscier, D. G. et al. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 100, 1177–1184 (2002). p p g , ( ) 2. Amin, N. A. et al. Cell-intrinsic determinants of ibrutinib-induced apoptosis in h l h l k l 2. Amin, N. A. et al. Cell-intrinsic determinants of ibrutinib-induced apoptosis in chronic lymphocytic leukemia. Clin. Cancer Res. 23, 1049–1059 (2017). 2. Amin, N. A. et al. Cell-intrinsic determinants of ibrutinib-induced apoptosis in chronic lymphocytic leukemia. Clin. Cancer Res. 23, 1049–1059 (2017). y p y , ( ) 3. Woyach, J. A. & Johnson, A. J. Targeted therapies in CLL: mechanisms of resistance and strategies for management. Blood 126, 471–477 (2015). 3. Woyach, J. A. & Johnson, A. J. Targeted therapies in CLL: mechanisms of resistance and strategies for management. Blood 126, 471–477 (2015). Author details 1 1Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal Quebec, Canada. 2Segal Cancer Center, Jewish General Hospital, Montreal, Quebec, Canada. 3Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada. 4Department of Oncology, McGill University, Montreal, Quebec, Canada 7. Saiya-Cork, K. et al. A pathobiological role of the insulin receptor in chronic lymphocytic leukemia. Clin. Cancer Res. 17, 2679–2692 (2011). 7. Saiya-Cork, K. et al. A pathobiological role of the insulin receptor in chronic lymphocytic leukemia. Clin. Cancer Res. 17, 2679–2692 (2011). 8. Martinez Marignac, V. L., Smith, S., Toban, N., Bazile, M. & Aloyz, R. Resistance to Dasatinib in primary chronic lymphocytic leukemia lymphocytes involves AMPK-mediated energetic re-programming. Oncotarget 4, 2550–2566 (2013). 8. Martinez Marignac, V. L., Smith, S., Toban, N., Bazile, M. & Aloyz, R. Resistance to Dasatinib in primary chronic lymphocytic leukemia lymphocytes involves AMPK-mediated energetic re-programming. Oncotarget 4, 2550–2566 (2013). 9. Suarez, I., Bodega, G. & Fernandez, B. Glutamine synthetase in brain: effect of ammonia. Neurochem. Int. 41, 123–142 (2002). Acknowledgements h k 4. Dalva-Aydemir, S. et al. Targeting the metabolic plasticity of multiple myeloma with FDA-approved ritonavir and metformin. Clin. Cancer Res. 21, 1161–1171 (2015). 4. Dalva-Aydemir, S. et al. Targeting the metabolic plasticity of multiple myeloma with FDA-approved ritonavir and metformin. Clin. Cancer Res. 21, 1161–1171 (2015). g This work was supported by a Leukemia & Lymphoma Society of Canada operating grant (360235) to R.A. G.G.-V. was a recipient of The Rosenberg/ Unger/Schwarzbard/Kallchman Hematology Research Award, Faculty of Medicine, McGill University. 5. Kruger, A. & Ralser, M. ATM is a redox sensor linking genome stability and carbon metabolism. Sci. Signal. 4, pe17 (2011). 5. Kruger, A. & Ralser, M. ATM is a redox sensor linking genome stability and carbon metabolism. Sci. Signal. 4, pe17 (2011). 6. Tili, E. et al. The down-regulation of miR-125b in chronic lymphocytic leuke- mias leads to metabolic adaptation of cells to a transformed state. Blood 120, 2631–2638 (2012). 6. Tili, E. et al. The down-regulation of miR-125b in chronic lymphocytic leuke- mias leads to metabolic adaptation of cells to a transformed state. Blood 120, 2631–2638 (2012). Competing interests The authors declare that they have no competing interests. 10. Shin, C. S. et al. The glutamate/cystine xCT antiporter antagonizes glutamine metabolism and reduces nutrient ﬂexibility. Nat. Commun. 8, 15074 (2017). 11. Li, L., Fath, M. A., Scarbrough, P. M., Watson, W. H. & Spitz, D. R. Combined inhibition of glycolysis, the pentose cycle, and thioredoxin metabolism selectively increases cytotoxicity and oxidative stress in human breast and prostate cancer. Redox Biol. 4, 127–135 (2015). stress, due to reduced metabolic plasticity (Fig. 2g). This study opens the door for the development of personalized medicine for CLL del11q-positive patients, especially in cases presenting relapse to chemotherapy or ibrutinib treatment. Moreover, the availability of GLS1 inhibitors such as CB-839—currently in clinical trials for AML and ALL (clinicaltrials.gov)—paves the way for the targeting of glutamine metabolism in CLL. Blood Cancer Journal Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition Blood Cancer Journal (2018) 8:13 Page 5 of 5 Page 5 of 5 Page 5 of 5 Page 5 of 5 Del11q-positive CLL lymphocytes exhibit altered glutamine metabolism and differential response to GLS1 and glucose metabolism inhibition Glutamate production might be maintained via amino-acid catabolism through transaminase reactions, while glycolysis could provide the necessary metabolites to feed the TCA, PPP, and one-carbon metabolism. Wild-type CLL lymphocytes have a signiﬁcant ﬂux of glucose-derived carbons through PPP, while their amino-acid metabolism tends to generate ammonia Fig. 2 Effect of glucose metabolic inhibitors and ibrutinib on CLL metabolism. a, c, e, f Survival fraction (relative to non-treated control (NT)) of CLL lymphocytes treated with metabolic inhibitors for 48 h. b n = 21, c n = 12, e n = 19, f n = 23. b Glucose uptake after 24 h compound treatment (n = 15). d Relative ROS median values of CLL cells after 24 h of ibrutinib and/or NAC treatment (n = 20). p < 0.05, **p < 0.001. g Model for basal wild- type and del11q CLL cell metabolism. The proposed metabolic ﬂux in CLL lymphocytes is indicated with brown (wild-type) and pink (del11q) arrows. Del11q CLL lymphocytes promote glutamine synthesis, while decreasing GDH reaction. Glutamate production might be maintained via amino-acid catabolism through transaminase reactions, while glycolysis could provide the necessary metabolites to feed the TCA, PPP, and one-carbon metabolism. Wild-type CLL lymphocytes have a signiﬁcant ﬂux of glucose-derived carbons through PPP, while their amino-acid metabolism tends to generate ammonia lymphocytes (Fig. 2f), in line with increased sensitivity to GLS1 inhibition. Noteworthy, the simultaneous inhibition of BTK and glucose uptake was highly cytotoxic to CLL lymphocytes, especially to del11q CLL cells, underscoring the role of BTK in metabolic homeostasis (Fig. 2f). We presume that in addition to its effects on BCR signaling, ibrutinib disturbs glutamine metabolism possibly by decreasing glutamine/glutamate availability. ibrutinib-induced cytotoxicity in CLL lymphocytes (Fig. 2d, e). ROS levels increased upon GLS1or glutamine deprivation (Supplementary Fig. 4h), suggesting that the contribution of glutamine metabolism to ROS control is not compensated by other NADPH-generating pathways, and that ibrutinib affects glutamine metabolism by reducing glutamine pools. This is supported by the observation of decreased extracellular accumulation of glutamate upon GLS1 inhibition in ibrutinib treated CLL lymphocytes (Supplementary Fig. 4d). Importantly, ibrutinib-induced cytotoxicity was higher on del11q CLL Based on our results, we propose that the differences in glutamine metabolism displayed by del11q CLL lympho- cytes could account for their oversensitivity to metabolic Blood Cancer Journal Galicia-Vázquez et al. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 12. Oburoglu, L. et al. Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage speciﬁcation. Cell. Stem Cell. 15, 169–184 (2014). 12. Oburoglu, L. et al. Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage speciﬁcation. Cell. Stem Cell. 15, 169–184 (2014). Supplementary Information accompanies this paper at https://doi.org/ 10.1038/s41408-017-0039-2. 13. Lemons, J. M. et al. Quiescent ﬁbroblasts exhibit high metabolic activity. PLoS. Biol. 8, e1000514 (2010). 13. Lemons, J. M. et al. Quiescent ﬁbroblasts exhibit high metabolic activity. PLoS. Biol. 8, e1000514 (2010). Received: 15 September 2017 Revised: 31 October 2017 Accepted: 29 November 2017 14. Vangapandu, H. V. et al. B-cell receptor signaling regulates metabolism in chronic lymphocytic leukemia. Mol. Cancer Res. 15, 1692–1703 (2017). Blood Cancer Journal
https://openalex.org/W4245187108	https://www.qeios.com/read/E9MDHA/pdf	English	null	Pelger-Huet Anomaly	Definitions	2,020	cc-by	78	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Pelger-Huet Anomaly National Cancer Institute National Cancer Institute Qeios ID: E9MDHA · https://doi.org/10.32388/E9MDHA Source National Cancer Institute. Pelger-Huet Anomaly. NCI Thesaurus. Code C85002. An autosomal dominant inherited condition caused by mutations in the lamin B receptor gene. It is characterized by defects in the neutrophil lobulation, resulting in the presence of dumbbell-shaped neutrophils with bilobed nuclei in the peripheral blood smear. Qeios ID: E9MDHA · https://doi.org/10.32388/E9MDHA 1/1
https://openalex.org/W3128543044	https://europepmc.org/articles/pmc7863471?pdf=render	English	null	Seasons, weather, and device-measured movement behaviors: a scoping review from 2006 to 2020	The international journal of behavioural nutrition and physical activity	2,021	cc-by	19,473	© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. REVIEW Open Access Seasons, weather, and device-measured movement behaviors: a scoping review from 2006 to 2020 Taylor B. Turrisi1, Kelsey M. Bittel1, Ashley B. West1, Sarah Hojjatinia2, Sahar Hojjatinia3, Scherezade K. Mama4, Constantino M. Lagoa3 and David E. Conroy1,5* Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 https://doi.org/10.1186/s12966-021-01091-1 https://doi.org/10.1186/s12966-021-01091-1 Abstract * Correspondence: conroy@psu.edu 1Department of Kinesiology, The Pennsylvania State University, University Park, PA 16802, USA 5Department of Preventive Medicine, Northwestern University, Chicago, IL, USA Full list of author information is available at the end of the article USA Full list of author information is available at the end of the article Abstract Background: This scoping review summarized research on (a) seasonal differences in physical activity and sedentary behavior, and (b) specific weather indices associated with those behaviors. Methods: PubMed, CINAHL, and SPORTDiscus were searched to identify relevant studies. After identifying and screening 1459 articles, data were extracted from 110 articles with 118,189 participants from 30 countries (almost exclusively high-income countries) on five continents. Results: Both physical activity volume and moderate-to-vigorous physical activity (MVPA) were greater in summer than winter. Sedentary behavior was greater in winter than either spring or summer, and insufficient evidence existed to draw conclusions about seasonal differences in light physical activity. Physical activity volume and MVPA duration were positively associated with both the photoperiod and temperature, and negatively associated with precipitation. Sedentary behavior was negatively associated with photoperiod and positively associated with precipitation. Insufficient evidence existed to draw conclusions about light physical activity and specific weather indices. Many weather indices have been neglected in this literature (e.g., air quality, barometric pressure, cloud coverage, humidity, snow, visibility, windchill). Conclusions: The natural environment can influence health by facilitating or inhibiting physical activity. Behavioral interventions should be sensitive to potential weather impacts. Extreme weather conditions brought about by climate change may compromise health-enhancing physical activity in the short term and, over longer periods of time, stimulate human migration in search of more suitable environmental niches. vironment, Seasons, Meteorological concepts, Rain, Sunlight, Temperature, Wind, Exercise, Screen time Keywords: Environment, Seasons, Meteorological concepts, Rain, Sunlight, Temperature, Wind, Exe The global prevalence of insufficient physical activity is approximately 28% but exceeds 40% in some regions [1]. Physical activity promotion efforts have targeted determi- nants at multiple levels of the socio-ecological model, in- cluding the person (e.g., motivation), social environment (e.g., family, peers), and built environment (e.g., access to equipment, neighborhood walkability) [2–4]. The natural environment includes a number of factors that influence physical activity, including seasons and weather. Weather is often cited as a perceived barrier to participation in movement behaviors [5–9]. Although weather conditions are not acutely modifiable, they are important to under- stand because of their ability to alter opportunities for physical activity and moderate the effectiveness of inter- ventions targeting determinants at other levels. * Correspondence: conroy@psu.edu 1Department of Kinesiology, The Pennsylvania State University, University Park, PA 16802, USA 5Department of Preventive Medicine, Northwestern University, Chicago, IL, USA Full list of author information is available at the end of the article Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 2 of 26 Page 2 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity explain seasonal differences and facilitate the develop- ment of just-in-time interventions that leverage infor- mation from short-term weather forecasts. Second, weather and physical activity data were aggregated into person-level summary measures, yet both weather and physical activity are dynamic. Understanding the timing of and changes in weather conditions is essen- tial because current weather conditions are likely to have a more immediate influence on physical activity than average weather conditions. Third, both reviews focused on a general physical activity outcome. Phys- ical activity can be quantified as total volume (to rep- resent energy expended) or the duration of activities completed at specific intensities (to represent time al- located to different effort levels). Sedentary behavior may also be impacted as weather conditions alter people’s activity choices. Additional reviews published since these seminal reviews have been limited by in- complete search strategies and a focus on narrow seg- ments of the population [14, 22]. In light of these limitations and the broader context described earlier (increasing public interest in climate, advances in mo- bile technology, the emergence of sedentary behavior), an updated review of weather and movement behav- ior, including both physical activity and sedentary be- havior, would be a valuable contribution. Two seminal reviews of research on seasonality, weather, and physical activity across the lifespan were published over a decade ago [10, 11]. Physical activity was typically greatest during spring and summer and lowest during winter, but regions with more extreme weather conditions sometimes yielded different con- clusions. For example, a study conducted in Galves- ton, Texas, where the average temperature during summer months is over 28 °C (82 °F), revealed lower levels of physical activity in the summer than in win- ter [12]. However, the general pattern of seasonal trends demonstrated that people accumulated greater levels of physical activity in warmer and more arid conditions. Weather conditions were also found to have differing impacts on physical activity between sub-groups in the population [10, 13, 14]. For in- stance, physical activity was not associated with wind gusts for most individuals, but individuals with lower body mass were less active in the presence of stron- ger wind gusts than individuals with higher body mass [15]. The social context for research on weather and physical activity changed in three significant ways around the time of the seminal Tucker and Gilliland review [10]. First, public interest in weather and health increased following the 2007 Nobel Peace Prize that was awarded jointly to the Intergovernmental Panel on Climate Change and former US Vice Presi- dent Al Gore [16]. As Earth’s surface temperature rises, extreme weather conditions will increase air pollution and ultraviolet radiation exposure, increasing risk for cardiovascular and lung diseases as well as cancer [17]. Second, the mobile and wearable technol- ogy industries experienced major disruptions in 2007 due to the launch of the Apple iPhone and the founding of Fitbit [18, 19]. The widespread adoption of mobile technologies such as smartphones and wearable activity monitors enabled researchers to monitor both physical activity and location-specific weather indices in real-time. Third, sedentary behav- ior – waking activity conducted in a seated or re- clined posture involving low energy expenditure – has emerged as a distinct behavior that has important health consequences independent of physical activity levels [20, 21]. A scoping review was conducted to examine associ- ations between device-based measures of physical ac- tivity, sedentary behavior and a broad array of weather-related phenomena at different levels of spe- cificity, ranging from seasons (e.g., spring, summer, fall, winter) to specific weather indices (e.g., humidity, precipitation, temperature). A scoping review was se- lected over a systematic review or meta-analysis based on the breadth of weather indices available and the need to both analyze the available evidence and iden- tify existing knowledge gaps [23]. Search strategy PubMed, the Cumulative Index of Nursing and Allied Health Literature (CINAHL), and SPORTDiscus elec- tronic databases were searched from January 1, 2006 to October 31, 2020. This date range was chosen to capture all research since the end of the search period used by Tucker and Gilliland [10]. Three data- bases were selected based on their likelihood of in- cluding both environmental and health behavior data. Three main subject categories were included in our searches: movement-related behavior, movement- related behavior measurement (i.e., intensity, volume), and weather. Movement-related behavior search terms related to physical activity or sedentary behavior were combined with “or” statements. These terms mirrored Despite summarizing over 60 studies with over 300, 000 participants from approximately 18 countries, these seminal reviews possess several limitations. First, although they both examined seasonal differences in physical activity, most findings at the time focused on a relatively narrow range of specific weather indices with temperature and precipitation being most com- mon. Understanding associations between additional weather indices and physical activity could both Page 3 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Page 3 of 26 Page 3 of 26 the search terms used to compile the literature for the 2018 Physical Activity Guidelines Advisory Com- mittee [24]. Measurement search terms related to technologies commonly used to measure physical ac- tivity were combined with “or” statements (e.g., accel- erometer or pedometer). Weather search terms related to seasonality or specific weather indices were combined with “or” statements (e.g., seasons or temperature or humidity or precipitation). The search strategies for each database used are available in Ap- pendices 1, 2, and 3. The searches were restricted to articles that (a) were written in English, (b) examined human subjects only, and (c) included empirical stud- ies only (i.e., no review papers). This search strategy was constructed in consultation with a trained refer- ence librarian and search specialist at The Pennsylva- nia State University. The review protocol was not preregistered. The coders then met to compare codes and discuss disagreements to promote consistency for final extrac- tion. After this calibration exercise, each coder inde- pendently extracted data from the remaining articles using the standardized coding guide. Prior to analysis, the first author reviewed each article to ensure that the necessary data was extracted in a manner consist- ent with the coding guide. Data extraction l Articles containing studies that met inclusion criteria during full-text review were advanced for data extrac- tion. Prior to data extraction, the first author and four trained coders used a standardized coding guide to code three identical papers that were eligible for data extraction. The coding guide was developed to ensure that sufficient descriptive information was col- lected to properly characterize associations of interest. Search strategy A copy of the coding guide is available in supplemen- tary online files. Extracted sample characteristics in- cluded age, sex, education, race, and the country where the data were collected. If countries included multiple diverse climate zones, the region of the country was also collected. Physical activity, sedentary behavior, weather indices (temperature, precipitation, wind speed, photo- period, snow, cloud coverage, humidity, visibility, baro- metric pressure, windchill, and air quality; see Appendix 4 for definitions), and seasonality were all characterized by assessment timeframe and method of measurement. Research designs were classified as cross-sectional or longitudinal. The following statistics were extracted when available: t-scores, F-scores, correlation coeffi- cients, β coefficients, p-values, effect sizes, and odds ra- tios. Significance thresholds were kept consistent with the author’s prespecified level of significance, and all re- sults were coded having a positive or negative associ- ation, or failing to reach significance. Evidence grading ll f h l b All of the available evidence from independent samples was observational so all studies were deemed to have a high risk of bias. Instead of rating study-level bias, evi- dence was graded based on the quantity and consistency of findings when five of more studies were available for a specific comparison or association. A “strong” grade was assigned when a conclusion was based on highly consistent findings related to the direction of a differ- ence or association (or moderately consistent findings when a large number of studies was available). A “mod- erate” grade was assigned when a conclusion was based on mixed findings but the preponderance of evidence pointed to a consistent direction of a difference or asso- ciation. A “limited” grade was assigned when a conclu- sion could not be drawn because of equivocal evidence related to the direction of the difference or association. When fewer than five studies were available, we noted that a grade was not assignable. This grading system was drawn from criteria used by the 2018 Physical Activity Guidelines Advisory Committee and adapted to match the state of this literature [24]. Titles and abstracts were independently reviewed in a blinded manner by coders trained by the first author using the eligibility criteria described above. The first au- thor exported citations for each article found in the PubMed, CINAHL, and SPORTDiscus searches, and uploaded these citations into Rayyan. Rayyan is a web application that is used to facilitate collaborative screen- ing of titles and abstracts for reviews [25, 26]. Each coder accessed their assigned articles via the Rayyan web interface, reviewed titles and abstracts, and recorded de- cisions to include or exclude each article. Figure 1 sum- marizes study selection [27]. Selection process Articles were included if (a) physical activity or seden- tary behavior was an outcome variable of interest, (b) physical activity data were collected using device-based measurements (e.g., accelerometer, pedometer), and (c) results involved associations or differences between ei- ther movement-related behavior and either seasonality or weather indices. Articles were excluded if (a) studies were not published in English, (b) samples included non-human subjects, (c) results were limited to preva- lence rates or other descriptive data, or (d) physical ac- tivity data were collected using self-report measures exclusively. Results A total of 1459 unique articles were identified during the initial search. Following title and abstract screening, Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 4 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Page 4 of 26 Fig. 1 Process of Identification and Screening for Included Articles Fig. 1 Process of Identification and Screening for Included Articles 1208 articles were excluded. Full-text review of the remaining 251 articles led to an additional 141 exclu- sions. A total of 110 articles reporting 144 studies of in- dependent samples were identified as eligible for inclusion in this review. Among those 110 articles, 26 re- ported two independent samples, one reported three in- dependent samples, and two reported four independent samples to examine physical activity behaviors between sex, age group, or region, or examined both seasonality and weather [28–44]. sampling density across the globe. Most data represented western countries such as the United Kingdom, United States, Norway, Australia, Denmark, and Canada. Studies were primarily conducted in countries that the World Bank currently (July 2020) classifies as high income (27/ 30, 90.0%) and three were upper-middle income (3/30, 10.0%) [45]. None were from low- or middle-income countries. Approximately equal numbers of studies examined weather (77/144, 53.5%) and seasonality (67/144, 46.5%). Studies sampled youth (persons under age 18; 73/144, 50.7%), adults (44/144, 30.6%), and older adults (typically defined as over age 65; 27/144, 18.8%). Study designs were longitudinal (97/144, 67.4%) and cross-sectional (47/144, 32.6%). The modal Table 1 summarizes participant characteristics and study designs for all included articles. In total, articles included 118,189 participants (62.0% female participants, median N = 272, IQR = 85–722) from 30 unique coun- tries on five continents. Figure 2 summarizes the Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 5 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Page 5 of 26 Table 1 Sample and design characteristics of eligible articles Reference Country N Female (%) Age Group Age (M ± SD [years]) Race Monitoring Period Design Environmen Measure Aadland et al. (2018) Norway 465 51.6 Youth 10.9 ± 0.3 NA 7 days Longitudinal Season Aebi et al. Results International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 6 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 6 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Table 1 Sample and design characteristics of eligible articles (Continued) Reference Country N Female (%) Age Group Age (M ± SD [years]) Race Monitoring Period Design Environmen Measure Cepeda et al. (2018) Netherlands 1166 44.4 Adults & older adults Middle aged: Median = 59.1 [IQR = 56.1–62.4]; Young elderly: 71.3 [67.3–72.8]; Old elderly: 78.9 [77.0–81.4] NA 7 days Cross- sectional Season Chang et al. (2020) China 53 49.1 Youth 4.9 ± 0.2 NA 7 days Longitudinal Season Clemes et al. (2011) United Kingdom 96 53.2 Adults 40.7 ± 12.5 NA 4 weeks Longitudinal Season Collings et al. (2020) United Kingdom 342 48.8 Youth 3.4 ± 0.8 White: 40.9%; South Asian: 59.1% 6 days Longitudinal Season Colom et al. (2019) Spain 218 51.0 Adults Range: 55–75 NA 9 days Cross- sectional Weather Cooper et al. (2010) United Kingdom 1010 53.3 Youth 11.0 ± 0.4 NA 4 days Cross- sectional Season Cradock et al. (2009) United States 152 41.0 Youth 13.7 ± 0.7 White: 57.0%; Black/ African-American: 10.0%; Hispanic: 13.0%; Asian: 12.0%; Other Race/Ethnicity: 9.0% 4 days Cross- sectional Weather Crowley et al. (2016) United States 510 55.0 Adults 43.5 ± 9.2 NA 12 months Longitudinal Season Cullen et al. (2017) United States 342 100 Youth Range: 8–10 African-American: 100% 7 days Cross- sectional Weather Davis et al. (2011) United Kingdom 230 49.1 Older adults 78.1 ± 5.8 7 days Longitudinal Season, Weather de Vries et al. (2019) Netherlands 10 50.0 Youth 12.5 NA 14 days Longitudinal Season, Weather Declòs-Alió et al. (2019) Spain 227 56.0 Older adults NA NA 7 days Longitudinal Season Deng and Fredriksen (2018) Norway 2123 49.6 Youth 9 ± 1.5 NA 7 days Cross- sectional Season Dias et al. (2019) Switzerland, Belgium, United Kingdom, United States 1052 49.8 Youth 3.7 ± 0.4 NA 3 days Longitudinal Season, Weather Diaz et al. (2016) United States 2096 54.2 Adults Range: 45–75 Black: 31.6% 7 days Cross- sectional Season Dill et al. (2014) United States 353 NA Adults NA 3 days Longitudinal Weather Duncan et al. Results (2020) Switzerland 1314 48.7 Adults 67.9 ± 7.9 NA 8 days Cross- sectional Season Aibar Solana et al. (2015) France, Spain 646 58.8 Youth 14.3 ± 0.7 NA 7 days Longitudinal Weather Akande et al. (2019) Canada 272 43.8 Adults 34.9 ± 12.6 Inuit: 74.6%; Other: 25.4% 7 days Longitudinal Albrecht et al. (2020) Germany 577 52.0 Older adults Range: 65–75 NA 7 days Longitudinal Al- Mohannadi et al. (2016) Qatar 2088 33.4 Adults 41.6 ± 10.7 Eastern Mediterranean: 52.3%; South East Asian: 30.7%; Western Pacific:8.0%; African: 3.4%; North/South American: 3.1%; European: 2.5% 24 months Longitudinal Arnardottir et al. (2017) Iceland 138 60.1 Older adults 80.3 ± 4.9 NA 7 days Cross- sectional Season Aspvik et al. (2018) Norway 1219 51.2 Older adults 72.4 ± 2.1 NA 7 days Longitudinal Weather Atkin et al. (2016) United Kingdom, Wales, Scotland, Northern Ireland 704 52.6 Youth 7.6 ± 0.3 Caucasian: 92.9%; Other: 7.1% 7 days Longitudinal Weather Badland et al. (2011) Australia 1754 59.3 Adults 39.9 ± 11.8 NA 7 days Longitudinal Season, Weather Balish et al. (2017) Canada 190 45.3 Adults NA NA 7 days Longitudinal Season, Weather Barkley and Herrmann (2017) United States 16 NA Older adults NA NA 7 days Longitudinal Season Beighle et al. (2013) United States 321 52.0 Youth 9.1 ± 1.5 NA 7 days Longitudinal Season Bejarano et al. (2019) United States 26 42.3 Youth 16 ± 1.6 Caucasian: 69.2%; Native American: 15.4%; Hispanic: 11.5%; Asian: 3.8% 20 days Longitudinal Weather Boutou et al. (2019) United Kingdom, Belgium, Greece, Netherlands 157 75.8 Adults 67.2 ± 7.8 NA 7 days Longitudinal Weather Brandon et al. (2009) Canada 48 75.0 Older adults 77.4 ± 4.7 NA 7 days Longitudinal Weather Bremer et al. (2019) Canada 110 51.8 Youth 10.2 ± 1.7 NA 7 days Longitudinal Weather Bringolf-Isler et al. (2009) Switzerland 164 52.0 Youth Range: 6–14 NA 7 days Longitudinal Season Brychta et al. (2016) Iceland 70 64.3 Older adults 79.5 ± 4.8 NA 7 days Cross- sectional Weather Buchowski et al. (2009) United States 57 100 Adults 36.5 ± 9.2 NA 7 days Longitudinal Season Button et al. (2020) Canada 90 61.1 Youth 10.6 ± 1.4 White: 56.7%; Indigenous & Visible Minority: 43.3% 8 days Longitudinal Weather Carr et al. (2016) United States 132 100 Adults 41.6 ± 10.1 Hispanic/Latino: 100% 12 months Longitudinal Season Turrisi et al. Results (2008) New Zealand 1115 51.9 Youth Range: 5–16 Caucasian: 49.2%; Polynesian: 30.0%; Asian: 16.5%; Other: 4.3% 5 days Cross- sectional Season Edwards et al. (2015) United States 372 48.0 Youth 3.4 ± 0.3 Caucasian: 88.0% African-American: 22.0% 3 days Longitudinal Season Feinglass et al. (2011) United States 241 74.7 Adults < 50 years: (26.1%); 50–65 years: (39.3%); 66– 75 years: (22.2%); > 75 years: (12.5%) Caucasian/ Other: 75.1%; African- American: 17.8%; Hispanic/ Latino: 7.1% 7 days Longitudinal Weather Goodman et al. (2012) United Kingdom 325 52.3 Youth Range: 8–11 NA 4 days Longitudinal Weather Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 7 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 7 of 26 Table 1 Sample and design characteristics of eligible articles (Continued) Reference Country N Female (%) Age Group Age (M ± SD [years]) Race Monitoring Period Design Environmen Measure Goodman et al. (2014) Australia, Brazil, Denmark, England, Estonia, Spain, Norway, Switzerland, United States 23, 188 62.0 Youth Range: 5–16 NA 7 days Longitudinal Weather Gracia- Marco et al. (2013) Sweden, Greece, Italy, Spain, Hungary, Belgium, France, Germany, Austria 2173 54.0 Youth 15 ± 1.2 NA 7 days Longitudinal Season Griew et al. (2010) United Kingdom 1307 51.8 Youth Range: 10–11 NA 5 days Longitudinal Weather Hagströmer et al. (2014) Sweden 1172 54.0 Adults 45 ± 15 NA 7 days Longitudinal Season Hamilton et al. (2009) United Kingdom 96 51.1 Adults 41.0 ± 12.3 NA 4 weeks Longitudinal Weather Harrison et al. (2011) United Kingdom 1794 55.0 Youth Range: 9–10 NA 7 days Longitudinal Weather Harrison et al. (2015) United Kingdom 283 55.1 Youth Range: 9–14 NA 7 days Longitudinal Weather Harrison et al. (2017) United Kingdom, Switzerland, Belgium, Australia, Denmark, Estonia, Norway, Madeira, United States 23, 451 62.0 Youth Range: 3–18 NA 7 days Longitudinal Weather Hjorth et al. (2013) Denmark 730 48.5 Youth 10.0 ± 0.6 NA 7 days Longitudinal Weather Hoaas et al. (2019) Norway, Denmark, Australia 168 42.9 Adults Range: 60–73 NA 7 days Longitudinal Season Hopkins et al. (2011) United States 145 59.3 Youth 10.7 ± 0.3 NA 7 days Longitudinal Season Hoppmann et al. (2017) Canada 126 64.0 Older adults 71.9 ± 5 European: 62.0%; Asian: 36.0%; Other: 1.0%; Missing: 1.0% 10 days Longitudinal Weather Hunter et al. Results (2019) Canada 149 47.0 Adults 19 ± 1.9 White: 59.7%; Other: 40.3% 7 days Longitudinal Weather Jehn et al. (2014) Germany 15 40.0 Adults 66.7 ± 5.2 NA 6 months Longitudinal Season Jones et al. (2017) Canada 42 24.0 Older adults 77.4 ± 4.7 NA 7 days Cross- sectional Weather Jones et al. (2017) Canada 42 NA Older adults 77.4 ± 4.7 NA 7 days Longitudinal Season, Weather Katapally et al. (2016) Canada 331 49.8 Youth 11.6 ± 1.1 NA 7 days Longitudinal Weather Kharlova et al. (2020) Norway 2015 50.6 Youth 9.5 ± 1.8 NA 6 days Longitudinal Season Kimura et al. (2015) Japan 39 56.0 Older adults 70.7 ± 3.2 NA 2 weeks Longitudinal Weather King et al. (2011) United Kingdom 480 49.1 Youth NA NA 7 days Longitudinal Season Kolle et al. Norway 1824 48.5 Youth Range: 9–15 NA 4 days Cross- Weather Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 8 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Page 8 of 26 Table 1 Sample and design characteristics of eligible articles (Continued) Reference Country N Female (%) Age Group Age (M ± SD [years]) Race Monitoring Period Design Environmental Measure (2009) sectional Kong et al. (2020) South Korea 555 43.8 Adults 61.1 ± 8.9 NA 7 days cross- sectional Weather Koolhaas et al. (2017) Netherlands 1200 52.3 Older adults 77.5 ± 5.0 NA 7 days Cross- sectional Season Larouche et al. (2019) Canada 1699 55.0 Youth 10.2 ± 1.0 NA 8 days Cross- sectional Season Lewis et al. (2016) Australia and Canada 953 57.0 Youth 10.6 ± 0.4 NA 7 days Cross- sectional Season Ma et al. (2018) Hong Kong 210 33.8 Adults 26.1 ± 8.7 NA 35 days Longitudinal Season Martins et al. (2015) Brazil 16 50.0 Adults Range: 18–35 NA 6 days Longitudinal Weather McCrorie et al. (2015) Scotland 33 54.5 Youth 12.2 ± 0.3 NA 8 days Cross- sectional Weather McKee et al. (2012) Northern Ireland 85 38.8 Youth Range: 4–5 NA 6 days Longitudinal Weather McMurdo et al. (2012) United Kingdom 547 54.0 Older adults 79 ± 8 NA 7 days Cross- sectional Weather Mitchell et al. (2018) United States 575 34.3 Adults 38.6 ± 0.1 Hispanic: 100.0% 1 day Cross- sectional Weather Mitsui et al. Results (2010) Japan 50 0.0 Adults 43.6 ± 10.8 NA 7 days Longitudinal Weather Nagy et al. (2019) United Kingdom 108 51.0 Youth 7.5 ± 0.5 Caucasian: 59.0%; South Asian: 41.0% 7 days Cross- sectional Season Nakashima et al. (2019) Japan 22 86.4 Older adults 75.1 ± 7.3 NA 7 days Longitudinal Season Newman et al. (2009) United States 500 100.0 Adults 57 ± 2.9 African-American: 13.2% 7 days Cross- sectional Season Nilsen et al. (2019) Norway 1154 49.0 Youth 4.7 ± 0.9 NA 14 days Longitudinal Season O’Connell et al. (2013) United Kingdom 46 72.0 Adults 41.7 ± 14.4 NA 7 days Longitudinal Season Ogawa et al. (2018) Japan 35 65.7 Adults 69.3 ± 5.3 NA 1 month Longitudinal Season Oliver et al. (2011) New Zealand 135 60.0 Youth Range: 5–6 NA 8 days Cross- sectional Season Pagels et al. (2016) Sweden 179 48.6 Youth 11.1 ± 2.1 NA 5 days Longitudinal Season Patnode et al. (2010) United States 294 49.3 Youth 15.4 ± 1.7 Caucasian: 93.5%; Other: 6.5% 7 days Cross- sectional Season, weather Pearce et al. (2012) United Kingdom 482 52.0 Youth Range: 8–10 NA 7 days Longitudinal Season Pechova et al. (2019) Czech Republic, Slovakia & Poland 83 89.7 Adults 65 ± 6 NA 8 days Longitudinal Season, Weather Pelclova et al. (2010) Czech Republic 13 84.6 Youth 15.6 ± 0.5 NA 10 months Longitudinal Weather Prins and van Lenthe (2015) Netherlands 43 52.5 Adults NA NA 7 days Longitudinal Weather Rahman et al. (2019) Canada 972 58.0 Youth 10.9 ± 0.4 NA 7 days Cross- sectional Season, weather Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 9 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Page 9 of 26 Table 1 Sample and design characteristics of eligible articles (Continued) Reference Country N Female (%) Age Group Age (M ± SD [years]) Race Monitoring Period Design Environmental Measure Remmers et al. (2017) Australia 307 52.0 Youth 11.1 ± 0.7 NA 7 days Longitudinal Season, Weather Ridgers et al. (2015) Australia 326 50.3 Youth 10 ± 0.7 NA 7 days Cross- sectional Season Ridgers et al. (2018) Australia 326 50.3 Youth Range: 8–11 NA 7 days Longitudinal Season Robbins et al. Results (2013) Canada 38 26.3 Adults 54 ± 7.0 NA 7 days Cross- sectional Weather Rosenthal et al. (2020) United States 266 58.4 Adults 52.1 ± 14 NA NA Cross- sectional Weather Rowlands et al. (2009) United Kingdom 64 50.0 Youth 9.9 ± 0.3 NA 6 days Longitudinal Season Sartini et al. (2017) United Kingdom 1361 0.0 Older adults 78.5 ± 4.6 NA 7 days Longitudinal Weather Schepps et al. (2018) United States 16, 741 100.0 Older adults 72.0 ± 5.7 NA 7 days Cross- sectional Weather Sewell et al. (2010) United Kingdom 95 41.0 Adults 65.5 ± 8.5 NA 2 days Cross- sectional Season Shen et al. (2013) United States 46 56.5 Youth 4.2 ± 0.2 Caucasian: 68.0%; Other: 32.0% 5 days Cross- sectional Weather Silva et al. (2011) Portugal 24 50.0 Youth 11.0 ± 1.5 NA 7 days Longitudinal Season Sit et al. (2019) Hong Kong 270 40.0 Youth NA NA 3 days Longitudinal Season Stabell et al. (2020) United States 38 10.5 Adults Range: 50–75 Caucasian:81.6%; Other: 18.4% 24 weeks Longitudinal Weather Sugino et al. (2012) Japan 9 0.0 Adults 71.7 ± 8.3 NA 2 weeks Cross- sectional Season Sumukadas et al. (2008) Scotland 127 71.0 Older adults 78.6 ± 6.5 NA 7 days Longitudinal Season Van Kann et al. (2016) Netherlands 520 55.6 Youth 10.1 ± 0.7 NA 7 days Longitudinal Weather Wang et al. (2017) China 34 38.2 Adults 31 ± 10 NA 7 days Longitudinal Weather Witham et al. (2014) United Kingdom 547 54.3 Older adults 78.5 ± 7.7 NA 7 days Longitudinal Weather Wong et al. (2020) Mexico 559 49.0 Youth 4.8 ± 0.5 NA 7 days Longitudinal Weather Wu et al. (2017) United Kingdom 4051 55.7 Adults 67.4 ± 9.5 NA 7 days Cross- sectional Weather Yildrim et al. (2012) Belgium, Greece, Hungary, Netherlands, Switzerland 722 53.0 Youth 11.6 ± 0.9 NA 6 days Cross- sectional Weather Zheng et al. (2019) Hong Kong 740 40.9 Youth 14.7 ± 1.6 Chinese: 100.0% 7 days Longitudinal Weather Table 1 Sample and design characteristics of eligible articles (Co Reference Country N Female (%) Age Group Age (M ± SD [years]) Remmers et al. (2017) Australia 307 52.0 Youth 11.1 ± 0.7 Ridgers et al. (2015) Australia 326 50.3 Youth 10 ± 0.7 Ridgers et al. (2018) Australia 326 50.3 Youth Range: 8–11 Robbins et al. (2013) Canada 38 26.3 Adults 54 ± 7.0 Rosenthal et al. Activity monitoring methods Almost all studies measured physical activity (138/144, 95.2%) and fewer measured sedentary behavior (50/144, 34.7%). Table 2 summarizes characteristics of the devices used to measure physical activity and sedentary behav- ior. Physical activity was measured with research-grade accelerometers (121/144, 84.0%), pedometers (22/144, 15.3%), or global positioning system logger (1/144, 0.7%). Although most studies with accelerometer-based measurements used wearable devices, one study mea- sured physical activity using the accelerometer contained within a smartphone [46]. Results 2 Geographic distribution of participants included in this review Fig. 2 Geographic distribution of participants included in this review 800 vector magnitude counts per minute [72], less than 820 vector magnitude counts per minute [73, 74], less than 1110 vector magnitude counts per minute [75, 76], falling below activity registering at 1.0 [77] or 1.5 [78– 80] metabolic equivalents to task (METs). Four studies in three papers did not report how sedentary behavior was defined [30, 34, 81]. duration of activity monitoring for movement behav- ior was 7 days (82/144, 56.9%) but ranged from 1 day to 2 years. Seasonal differences in physical activity and movement- related behaviors Table 3 summarizes seasonal differences in a variety of movement-related behaviors. Studies either compared two seasons (29/67, 43.3%), three seasons (9/67, 13.4%), or four seasons (25/67, 37.3%). Four studies did not re- port their definition of season (4/67, 6.0%) [86, 87, 103]. Winter (64/67, 95.5%) and summer (51/67, 76.1%) were studied most frequently, with spring (40/67, 59.7%) and autumn (36/67, 53.7%) receiving less attention. Studies comparing physical activity and sedentary behavior be- tween seasons sampled youth (40/67, 59.7%), adults (17/ 67, 25.4%) and older adults (10/67, 14.9%). Studies of seasonal differences measured volume (47/67, 70.1%), moderate-to-vigorous intensity physical activity (MVPA) duration (37/67, 55.2%), light-intensity physical activity (LPA) duration (19/67, 28.4%), and sedentary behavior (25/67, 37.3%). Physical activity measures included volume (i.e., step counts, total accelerometer counts) and intensity-specific durations. Volume measures represent the total amount of energy expended in physical activity whereas intensity-specific durations represent how individuals al- locate their time to more and less effortful forms of physical activity. Figure 3 summarizes the frequency of studies using volume and intensity-specific duration measures in samples of youth, adults, and older adults. Seasonal differences in physical activity were more fre- quently estimated in youth than adults or older adults. Weather indices associated with physical activity were studied most frequently in youth, and approximately equally in adults and older adults. Results (2020) United States 266 58.4 Adults 52.1 ± 14 Rowlands et al. (2009) United Kingdom 64 50.0 Youth 9.9 ± 0.3 Sartini et al. (2017) United Kingdom 1361 0.0 Older adults 78.5 ± 4.6 Schepps et al. (2018) United States 16, 741 100.0 Older adults 72.0 ± 5.7 Sewell et al. (2010) United Kingdom 95 41.0 Adults 65.5 ± 8.5 Shen et al. (2013) United States 46 56.5 Youth 4.2 ± 0.2 Silva et al. (2011) Portugal 24 50.0 Youth 11.0 ± 1.5 Sit et al. (2019) Hong Kong 270 40.0 Youth NA Stabell et al. (2020) United States 38 10.5 Adults Range: 50–75 Sugino et al. (2012) Japan 9 0.0 Adults 71.7 ± 8.3 Sumukadas et al. (2008) Scotland 127 71.0 Older adults 78.6 ± 6.5 Van Kann et al. (2016) Netherlands 520 55.6 Youth 10.1 ± 0.7 Wang et al. (2017) China 34 38.2 Adults 31 ± 10 Witham et al. (2014) United Kingdom 547 54.3 Older adults 78.5 ± 7.7 Wong et al. (2020) Mexico 559 49.0 Youth 4.8 ± 0.5 Wu et al. (2017) United Kingdom 4051 55.7 Adults 67.4 ± 9.5 Yildrim et al. (2012) Belgium, Greece, Hungary, Netherlands, Switzerland 722 53.0 Youth 11.6 ± 0.9 Zheng et al. (2019) Hong Kong 740 40.9 Youth 14.7 ± 1.6 nued) Race Monitoring Period Design Environmenta Measure NA 7 days Longitudinal Season, Weather NA 7 days Cross- sectional Season NA 7 days Longitudinal Season NA 7 days Cross- sectional Weather NA NA Cross- sectional Weather NA 6 days Longitudinal Season NA 7 days Longitudinal Weather NA 7 days Cross- sectional Weather NA 2 days Cross- sectional Season Caucasian: 68.0%; Other: 32.0% 5 days Cross- sectional Weather NA 7 days Longitudinal Season NA 3 days Longitudinal Season Caucasian:81.6%; Other: 18.4% 24 weeks Longitudinal Weather NA 2 weeks Cross- sectional Season NA 7 days Longitudinal Season NA 7 days Longitudinal Weather NA 7 days Longitudinal Weather NA 7 days Longitudinal Weather NA 7 days Longitudinal Weather NA 7 days Cross- sectional Weather NA 6 days Cross- sectional Weather Chinese: 100.0% 7 days Longitudinal Weather Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 10 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 10 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Fig. Physical activity volume (2016) Pedometer Jawbone UP Wris Cullen et al. (2017) Accelerometer ActiGraph GT3X+ NA Davis et al. (2011) Accelerometer ActiGraph GT1M Wais de Vries et al. (2019) Accelerometer 1. ActiWatch 2; 2. GENEActiv Original; 3. Sensewear Mini 1. W Arm Declòs-Alió et al. (2019) Accelerometer ActiGraph GT3X NA Deng and Fredriksen (2018) Accelerometer ActiGraph wGT3X-BT Hip Dias et al. (2019) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M; 3. ActiGraph 71256 Wais Diaz et al. (2016) Accelerometer Actical Wais Dill et al. (2014) Accelerometer ActiGraph GT3X Hip Duncan et al. (2008) Pedometer New Lifestyles 2000 Wais Edwards et al. (2015) Accelerometer RT3 Hip Feinglass et al. (2011) Accelerometer ActiGraph GT1M Hip Goodman et al. (2012) Accelerometer RT3 Wais Table 2 Characteristics of devices used to measure physical activity and sedentary behavior Reference Type of Device Specific Device Wear Location Measure(s) of Activity Aadland et al. (2018) Accelerometer ActiGraph GT3X+ NA SB, LPA, MVPA, V Aebi et al. (2020) Accelerometer ActiGraph wGT3X-BT Hip SB, LPA, MVPA Aibar Solana et al. (2015) Accelerometer ActiGraph GT3X NA SB Alande et al. (2019) Pedometer Kaden G-Sport Pocket Pedometer 793 Waist V Albrecht et al. (2020) Accelerometer ActiGraph wGT3X-BT Wrist V Al-Mohannadi et al. (2016) Pedometer Omron HJ-720 ITC NA V Arnardottir et al. (2017) Accelerometer ActiGraph GT3X+ Hip SB, LPA, MVPA, V Aspvik et al. (2018) Accelerometer ActiGraph GT3X+ Waist V Atkin et al. (2016) Accelerometer ActiGraph GT1M Waist SB, MVPA Badland et al. (2011) Pedometer Yamax Digiwalker SW-200 Hip V Balish et al. (2017) Pedometer Yamax Digiwalker SW-200 Hip V Barkley and Herrmann (2017) Accelerometer ActiGraph GT3X Waist LPA, MVPA Beighle et al. (2013) Pedometer MLS 205 Waist V Bejarano et al. (2019) Accelerometer ActiGraph wActi Sleep-BT Wrist SB, MVPA Boutou et al. (2019) Accelerometer ActiGraph GT3X Hip MVPA, V Brandon et al. (2009) Accelerometer ActiGraph GT1M Torso V Bremer et al. (2019) Pedometer PiezoRX Hip V Bringolf-Isler et al. (2009) Accelerometer ActiGraph AM7164 Waist V Brychta et al. (2016) Accelerometer ActiGraph GT3X Hip V Buchowski et al. (2009) Accelerometer Tritrac-R3D NA SB, V Button et al. (2020) Accelerometer Actical Z Hip LPA, MVPA Carr et al. (2016) Pedometer Omron HJ-720 ITC NA V Cepeda et al. (2018) Accelerometer GENEActiv Wrist SB, LPA, MVPA Chang et al. (2020) Accelerometer ActiGraph GT3X Hip SB, LPA, MVPA, V Clemes et al. (2011) Pedometer Yamax Digiwalker SW-200 Waist V Collings et al. Physical activity volume Studies comparing volume between seasons sampled youth (29/47, 61.7%), adults (13/47, 27.7%) and older adults (5/47, 10.6%). The most common seasonal com- parisons of volume involved winter vs. summer (30/47, 63.8%), winter vs. spring (22/47, 46.8%), spring vs. au- tumn (12/47, 25.5%), and winter vs. autumn (12/47, 25.5%). Sedentary behavior was typically classified using accel- erometer cut-points that included < 50, < 100, ≤100, < 150, < 175, and < 200 counts per minute [33, 38, 39, 47– 70]. Other sedentary behavior classifications were based on accumulating less than 122 counts per minute, less than or equal to 328 counts per minute [71], less than Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 11 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 11 of 26 Table 2 Characteristics of devices used to measure physical activity and sedentary behavior Reference Type of Device Specific Device Wea Aadland et al. (2018) Accelerometer ActiGraph GT3X+ NA Aebi et al. (2020) Accelerometer ActiGraph wGT3X-BT Hip Aibar Solana et al. (2015) Accelerometer ActiGraph GT3X NA Alande et al. (2019) Pedometer Kaden G-Sport Pocket Pedometer 793 Wais Albrecht et al. (2020) Accelerometer ActiGraph wGT3X-BT Wris Al-Mohannadi et al. (2016) Pedometer Omron HJ-720 ITC NA Arnardottir et al. (2017) Accelerometer ActiGraph GT3X+ Hip Aspvik et al. (2018) Accelerometer ActiGraph GT3X+ Wais Atkin et al. (2016) Accelerometer ActiGraph GT1M Wais Badland et al. (2011) Pedometer Yamax Digiwalker SW-200 Hip Balish et al. (2017) Pedometer Yamax Digiwalker SW-200 Hip Barkley and Herrmann (2017) Accelerometer ActiGraph GT3X Wais Beighle et al. (2013) Pedometer MLS 205 Wais Bejarano et al. (2019) Accelerometer ActiGraph wActi Sleep-BT Wris Boutou et al. (2019) Accelerometer ActiGraph GT3X Hip Brandon et al. (2009) Accelerometer ActiGraph GT1M Tors Bremer et al. (2019) Pedometer PiezoRX Hip Bringolf-Isler et al. (2009) Accelerometer ActiGraph AM7164 Wais Brychta et al. (2016) Accelerometer ActiGraph GT3X Hip Buchowski et al. (2009) Accelerometer Tritrac-R3D NA Button et al. (2020) Accelerometer Actical Z Hip Carr et al. (2016) Pedometer Omron HJ-720 ITC NA Cepeda et al. (2018) Accelerometer GENEActiv Wris Chang et al. (2020) Accelerometer ActiGraph GT3X Hip Clemes et al. (2011) Pedometer Yamax Digiwalker SW-200 Wais Collings et al. (2020) Accelerometer ActiGraph GT3X+ Hip Colom et al. (2019) Accelerometer GENEActiv Wris Cooper et al. (2010) Accelerometer ActiGraph GT1M Wais Cradock et al. (2009) Accelerometer TriTac-R3D Hip Crowley et al. Physical activity volume (2017) Accelerometer ActiGraph GT1M Waist V Katapally et al. (2016) Accelerometer Actical Waist SB Kharlova et al. (2020) Accelerometer ActiGraph wGT3X-BT Hip SB, MVPA Kimura et al. (2015) Pedometer Kenz Lifecorder EX Waist V King et al. (2011) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M Hip SB, V Kolle et al. (2009) Accelerometer ActiGraph MTI 7164 Waist V Kong et al. (2020) Accelerometer Fitbit Flex Wrist MVPA, V Koolhaas et al. (2017) Accelerometer GENEActiv Wrist SB, LPA, MVPA Larouche et al. (2019) Pedometer SC-Step Rx NA MVPA, V Lewis et al. (2016) Accelerometer ActiGraph GT3X+ Hip SB, MVPA Ma et al. (2018) Accelerometer 1. iPhones 5S ; 2. iPhone 6 NA V Martins et al. (2015) Accelerometer ActiGraph GT3X+ Hip MVPA McCrorie et al. (2015) Accelerometer activPAL Thigh V McKee et al. (2012) Pedometer DigiWalker DW-2000 NA V McMurdo et al. (2012) Accelerometer RT3 Hip V Mitchell et al. (2018) Accelerometer Actical NA V Mitsui et al. (2010) Pedometer Yamasa EM-180 NA V Nagy et al. (2019) Accelerometer ActiGraph GT3X+ Hip SB, MVPA, V Nakashima et al. (2019) Accelerometer LifeLyzer05 Coach Back LPA, MVPA, V Newman et al. (2009) Pedometer Accusplit AE120 Hip V Nilsen et al. (2019) Accelerometer ActiGraph GT3X+ Hip SB, LPA, MVPA, V O’Connell et al. (2013) Accelerometer ActiGraph GT1M Hip SB, LPA, MVPA Ogawa et al. (2018) Accelerometer Kenz Lifecorder GS Waist MVPA, V Oliver et al. (2011) Accelerometer Mini-mitter NA MVPA Pagels et al. (2016) Accelerometer ActiGraph GT3X+ NA MVPA P t d t l (2010) A l t A tiG h 7164 Hi MVPA acteristics of devices used to measure physical activity and sedentary behavior (Continued) Table 2 Characteristics of devices used to measure physical activity and sedentary behav Reference Type of Device Specific Device Goodman et al. (2014) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M; 3. ActiGraph 712 Gracia-Marco et al. (2013) Accelerometer ActiGraph GT1M Griew et al. (2010) Accelerometer ActiGraph GT1M Hagströmer et al. (2014) Accelerometer ActiGraph 7164 Hamilton et al. (2009) Pedometer New Lifestyles Digi-Walker SW-200 Harrison et al. (2011) Accelerometer ActiGraph GT1M Harrison et al. (2015) Accelerometer ActiGraph GT1M Harrison et al. (2017) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M Hjorth et al. (2013) Accelerometer ActiGraph GT3X+ Hoaas et al. (2019) Accelerometer SenseWear Pro Hopkins et al. (2011) Accelerometer ActiGraph GT1M Hoppmann et al. (2017) Accelerometer ActiGraph GT3X Hunter et al. (2019) Accelerometer ActiGraph wGT3X-BT Jehn et al. Physical activity volume (2020) Accelerometer ActiGraph GT3X+ Hip SB, LPA, MVPA, V Colom et al. (2019) Accelerometer GENEActiv Wrist MVPA Cooper et al. (2010) Accelerometer ActiGraph GT1M Waist V Cradock et al. (2009) Accelerometer TriTac-R3D Hip MVPA Crowley et al. (2016) Pedometer Jawbone UP Wrist V Cullen et al. (2017) Accelerometer ActiGraph GT3X+ NA V Davis et al. (2011) Accelerometer ActiGraph GT1M Waist LPA de Vries et al. (2019) Accelerometer 1. ActiWatch 2; 2. GENEActiv Original; 3. Sensewear Mini 1. Wrist; 2. Wrist; 3. Arm SB, LPA, MVPA, V Declòs-Alió et al. (2019) Accelerometer ActiGraph GT3X NA V Deng and Fredriksen (2018) Accelerometer ActiGraph wGT3X-BT Hip MVPA Dias et al. (2019) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M; 3. ActiGraph 71256 Waist SB, V, MVPA Diaz et al. (2016) Accelerometer Actical Waist SB Dill et al. (2014) Accelerometer ActiGraph GT3X Hip V, MVPA Duncan et al. (2008) Pedometer New Lifestyles 2000 Waist V Table 2 Characteristics of devices used to measure physical activity and sedentary behavior Hip Waist Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 12 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 12 of 26 (2021) 18:24 Table 2 Characteristics of devices used to measure physical activity and sedentary behavior (Continued) Reference Type of Device Specific Device Wear Location Measure(s) of Activity Goodman et al. (2014) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M; 3. ActiGraph 71256 Waist V Gracia-Marco et al. (2013) Accelerometer ActiGraph GT1M Back SB, MVPA, V Griew et al. (2010) Accelerometer ActiGraph GT1M Hip V Hagströmer et al. (2014) Accelerometer ActiGraph 7164 Hip SB, MVPA, V Hamilton et al. (2009) Pedometer New Lifestyles Digi-Walker SW-200 NA V Harrison et al. (2011) Accelerometer ActiGraph GT1M Hip SB, V, MVPA Harrison et al. (2015) Accelerometer ActiGraph GT1M Hip SB, MVPA, V Harrison et al. (2017) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M NA V Hjorth et al. (2013) Accelerometer ActiGraph GT3X+ Hip SB, MVPA, V Hoaas et al. (2019) Accelerometer SenseWear Pro Arm SB, LPA, MVPA, V Hopkins et al. (2011) Accelerometer ActiGraph GT1M Hip V Hoppmann et al. (2017) Accelerometer ActiGraph GT3X Hip MVPA, V Hunter et al. (2019) Accelerometer ActiGraph wGT3X-BT Waist SB, LPA, MVPA, V Jehn et al. (2014) Accelerometer AiperMotion Hip V Jones et al. (2017) Accelerometer ActiGraph GT1M Waist V Jones et al. Physical activity volume (2014) Accelerometer AiperMotion Jones et al. (2017) Accelerometer ActiGraph GT1M Jones et al. (2017) Accelerometer ActiGraph GT1M Katapally et al. (2016) Accelerometer Actical Kharlova et al. (2020) Accelerometer ActiGraph wGT3X-BT Kimura et al. (2015) Pedometer Kenz Lifecorder EX King et al. (2011) Accelerometer 1. ActiGraph 7164; 2. ActiGraph GT1M Kolle et al. (2009) Accelerometer ActiGraph MTI 7164 Kong et al. (2020) Accelerometer Fitbit Flex Koolhaas et al. (2017) Accelerometer GENEActiv Larouche et al. (2019) Pedometer SC-Step Rx Lewis et al. (2016) Accelerometer ActiGraph GT3X+ Ma et al. (2018) Accelerometer 1. iPhones 5S ; 2. iPhone 6 Martins et al. (2015) Accelerometer ActiGraph GT3X+ McCrorie et al. (2015) Accelerometer activPAL McKee et al. (2012) Pedometer DigiWalker DW-2000 McMurdo et al. (2012) Accelerometer RT3 Mitchell et al. (2018) Accelerometer Actical Mitsui et al. (2010) Pedometer Yamasa EM-180 Nagy et al. (2019) Accelerometer ActiGraph GT3X+ Nakashima et al. (2019) Accelerometer LifeLyzer05 Coach Newman et al. (2009) Pedometer Accusplit AE120 Nilsen et al. (2019) Accelerometer ActiGraph GT3X+ O’Connell et al. (2013) Accelerometer ActiGraph GT1M Ogawa et al. (2018) Accelerometer Kenz Lifecorder GS Oliver et al. (2011) Accelerometer Mini-mitter Pagels et al. (2016) Accelerometer ActiGraph GT3X+ Patnode et al. (2010) Accelerometer ActiGraph 7164 Pearce et al. (2012) Accelerometer ActiGraph GT1M Pechova et al. (2019) Accelerometer ActiGraph GT1M Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 13 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 13 of 26 (2021) 18:24 Table 2 Characteristics of devices used to measure physical activity and sedentary behavior (Continued) Reference Type of Device Specific Device Wear Location Measure(s) of Activity Pelclova et al. (2010) Pedometer Omron HJ-105 Hip V Prins and van Lenthe (2015) GPS Logger QStarz BT-Q1000XT Hip V Rahman et al. (2019) Pedometer Omron HJ-720 ITC Hip V Remmers et al. (2017) Accelerometer ActiGraph GT3X+ Hip SB, LPA, MVPA Ridgers et al. (2015) Accelerometer ActiGraph GT3X+ Hip MVPA Ridgers et al. (2018) Accelerometer ActiGraph GT3X+ Hip LPA, MVPA Robbins et al. (2013) Accelerometer ActiGraph GT1M Hip MVPA, V Rosenthal et al. (2020) Accelerometer NA NA V Rowlands et al. (2009) Accelerometer ActiGraph GT1M Hip MVPA, V Sartini et al. (2017) Accelerometer ActiGraph GT3X Hip SB, LPA, MVPA, V Schepps et al. (2018) Accelerometer ActiGraph GT3X+ Hip SB, LPA, MVPA, V Sewell et al. (2010) Accelerometer Gaehwiler Electronic Z80-32 k V1 Waist V Shen et al. Physical activity volume (2013) Accelerometer ActiGraph GT1M Hip LPA, MVPA Silva et al. (2011) Accelerometer ActiGraph MTI/CSA 7164 Hip SB, MVPA Sit et al. (2019) Accelerometer ActiGraph GT3X Hip SB, MVPA Stabell et al. (2020) Pedometer Omron HJ-324 U Waist V Sugino et al. (2012) Accelerometer 1. Actimarker; 2. Dynaport Activity Monitor (DAM) Waist V Sumukadas et al. (2008) Accelerometer RT3 Hip V Van Kann et al. (2016) Accelerometer Actiheart Chest LPA, MVPA Wang et al. (2017) Accelerometer ActiGraph GT3X Waist MVPA, V Witham et al. (2014) Accelerometer RT3 Waist V Wong et al. (2020) Accelerometer ActiGraph GT3X+ Hip SB Wu et al. (2017) Accelerometer ActiGraph GT1M Wrist SB, V Yildrim et al. (2012) Accelerometer 1. ActiGraph ActiTrainer; 2. ActiGraph GT1M; 3. ActiGraph GT3X Waist SB, LPA, MVPA, V Zheng et al. (2019) Accelerometer activPAL Thigh SB, MVPA Note. V Physical activity volume, LPA light-intensity physical activity duration, MVPA moderate-to-vigorous intensity physical activity duration, SB sedentary Fig. 3 Movement behavior measures in studies examining seasonality and weather indices across the lifespan Fig. 3 Movement behavior measures in studies examining seasonality and weather indices across the lifespan Fig. 3 Movement behavior measures in studies examining seasonality and weather indices across the lifespan Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 14 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Turrisi et al. Physical activity volume International Journal of Behavioral Nutrition and Physical Activity Page 14 of 26 Table 3 Seasonal differences in movement-related behaviors Season Reference Season Physical Activity Volume Light-Intensity Physical Activity Moderate-to-Vigorous Intensity Physical Activity Sedentary Behavior Winter > Summer 1 [82] 0 1 [79] 10 [30, 47, 51, 52, 58, 67, 72, 74, 75, 79] = Summer 6 [65, 72, 74, 80, 83, 84] 6 [52, 55, 79, 80, 85] 7 [65, 67, 74, 80, 85] 3 [65, 74, 80] < Summer 23 [28, 31, 44, 52, 74–77, 86– 99] 6 [53, 67, 74, 89, 100] 11 [31, 51–53, 58, 72, 89, 100–102] 0 Evidence Grade Strong Moderate Strong Strong > Spring 0 0 0 11 [51, 52, 54, 55, 67–69, 72, 74, 79] = Spring 7 [29, 49, 65, 68, 80, 84, 98] 7 [30, 52, 74, 80, 100] 10 [49, 53, 55, 65, 67, 68, 79, 80] 5 [53, 65, 68, 77, 80] < Spring 15 [29, 52, 54, 68, 72, 74, 89, 90, 103–107] 7 [53–55, 64, 67, 79, 89] 12 [33, 52, 54, 68, 72, 74, 89, 100, 101, 105] 0 Evidence Grade Strong Moderate Strong Strong > Autumn 0 2 [79, 100] 1 [79] 3 [52, 67, 74] = Autumn 7 [65, 68, 72, 74, 80, 84, 98] 4 [52, 55, 74, 80] 12 [49, 52, 65–68, 72, 74, 80, 101] 8 [65, 66, 68, 72, 74, 79, 80] < Autumn 5 [40, 52, 66, 74, 104] 5 [32, 66, 67, 74] 4 [32, 40, 100] 0 Evidence Grade Moderate Limited Strong Moderate Spring > Autumn 5 [29, 52, 65, 90] 1 [67] 5 [33, 52, 65, 79] 0 = Autumn 6 [29, 49, 80, 84, 98, 105] 5 [30, 52, 64, 79, 80] 7 [30, 49, 55, 67, 80, 105] 5 [53, 55, 65, 79, 80] < Autumn 1 [104] 1 [30] 0 4 [52, 63, 67, 77] Evidence Grade Moderate Limited Moderate Limited > Summer 2 [52, 82] 1 [79] 2 [33, 79] 0 = Summer 6 [65, 80, 84, 98, 105, 108] 7 [30, 52, 64, 67, 80, 108] 10 [30, 49, 52, 55, 65, 67, 80, 105, 108] 8 [53, 55, 65, 67, 69, 79, 80, 108] < Summer 2 [87, 104] 0 1 [41] 2 [52, 77] Evidence Grade Limited Moderate Strong Moderate Summer > Autumn 1 [87] 1 [67] 0 0 = Autumn 5 [52, 65, 80, 84, 98] 2 [52, 79] 5 [52, 65, 67, 79, 80] 4 [52, 65, 69, 80] < Autumn 1 [82] 0 0 2 [67, 79] Evidence Grade Limited Not Assignable Moderate Limited Moderate evidence indicated mixed findings for phys- ical activity volume between spring and autumn with most studies revealing either greater volume in spring than autumn (5/12, 41.7%) or no differences in volume (6/12, 50.0%). Sedentary behavior St di i Studies comparing sedentary behavior between seasons sampled youth (14/23, 60.9%), adults (7/23, 30.4%) and older adults (2/23, 8.7%). The most common seasonal comparisons of volume involved winter vs. spring (16/ 23, 69.6%), winter vs. summer (13/23, 56.5%), winter vs. autumn (11/23, 47.8%), spring vs. summer (10/23, 43.5%), spring vs. autumn (9/23, 39.1%), and summer vs. autumn (6/23, 26.1%). Studies comparing sedentary behavior between seasons sampled youth (14/23, 60.9%), adults (7/23, 30.4%) and older adults (2/23, 8.7%). The most common seasonal comparisons of volume involved winter vs. spring (16/ 23, 69.6%), winter vs. summer (13/23, 56.5%), winter vs. autumn (11/23, 47.8%), spring vs. summer (10/23, 43.5%), spring vs. autumn (9/23, 39.1%), and summer vs. autumn (6/23, 26.1%). Strong evidence indicated either greater MVPA in spring than winter (12/22, 54.5%) or no difference be- tween these seasons (10/22, 44.5%). No studies found greater MVPA duration in winter than spring. Based on moderate evidence, findings about LPA were mixed: half of the studies reported greater LPA in spring than winter (7/14, 50.0%) and half reported no difference in LPA be- tween spring and winter (7/14, 50.0%). Strong evidence indicated that MVPA duration was generally greater in summer than winter (11/19, 57.9%). Six studies found no difference between these seasons (6/19, 20%) and one study found winter activity greater than summer (1/30, 3.3%). Based on moderate evidence, studies indicated ei- ther greater LPA in summer than winter (6/12, 50.0%) or no difference (6/12, 50.0%). Strong evidence found mixed findings for seasonal differences in MVPA be- tween autumn and winter, either indicating no difference (12/17, 70.6%) or greater MVPA in autumn than winter (4/17, 23.5%). One study demonstrated greater MVPA duration in autumn compared to winter (1/17, 5.9%). Based on limited evidence, LPA was either greater in Strong evidence indicated that sedentary behavior was generally greater in winter than spring (11/16, 68.8%) al- though some studies indicated no difference (5/16, 31.3%). Strong evidence indicated that sedentary behav- ior was generally greater in winter than summer (10/13, 76.9%) with a few studies indicating no difference (3/13, 23.1%). Moderate evidence indicated that sedentary be- havior did not vary between autumn and winter (8/11, 72.7%) but a few studies indicated greater sedentary be- havior in winter than autumn (3/11, 27.3%). Moderate evidence indicated that sedentary behavior generally did not differ between spring and summer (8/10, 80%) but two studied indicated greater sedentary behavior during summer (2/10, 20%). Physical activity volume One study reported greater volume in au- tumn than spring (1/12, 8.3%). Moderate evidence indi- cated mixed findings for physical activity volume between autumn and winter with some studies indicat- ing no difference (7/12, 58.3%) and others indicating great volume in autumn than winter (5/12, 41.7%). No Strong evidence indicated that physical activity volume was greater in summer than winter (23/30, 76.7%), al- though one study found it was greater in winter than summer (1/30, 3.3%). Six studies (20.0%) comparing vol- ume in winter and summer yielded null results. Strong evidence also indicated that physical activity volume was consistently greater in spring than winter (15/22, 68.2%), but seven studies indicated no difference (7/22, 31.8%). No studies found evidence of greater volume in winter than spring. Page 15 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 15 of 26 (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity studies found evidence of greater physical activity vol- ume in winter than autumn. autumn than winter (5/11, 45.5%), not different between these seasons (4/11, 36.6%), or greater in winter than au- tumn (2/11, 18.2%). Strong evidence indicated that MVPA duration typically did not differ between summer and spring (10/13, 76.9%), but two studies revealed greater MVPA durations in summer (2/13, 15.3%) and another revealed greater MVPA duration in spring (1/ 13, 7.7%). Moderate evidence indicated that LPA gener- ally did not differ between summer than spring (7/8, 87.5%) but one study indicated greater LPA in spring than summer (1/8, 12.5%). Limited evidence compared physical activity volume in spring and summer were equivocal: six studies indicated no difference in volume between summer and spring (6/ 10, 60%) but the remaining studies were evenly split be- tween greater volume in summer (2/10, 20.0%) and spring (2/10, 20.0%). Limited evidence compared phys- ical activity volume between autumn and summer. Find- ings generally indicated no difference (5/7, 71.4%) with the exception of one study indicating greater volume in autumn than summer (1/7, 14.3%) and another study in- dicating greater volume in summer than autumn (5/7, 14.3%). Moderate evidence indicated mixed findings from comparisons of MVPA between spring and autumn, ei- ther indicating no differences (7/12, 58.3%) or greater MVPA in spring than autumn (5/12, 41.7%). No study demonstrated greater MVPA duration in autumn com- pared to spring. Physical activity volume Based on limited evidence, LPA gener- ally did not differ between spring and autumn (5/7, 71.4%); however, one study indicated greater LPA in spring than autumn (1/7, 14.3%) and another study indi- cated greater LPA in autumn than spring (1/7, 14.3%). Moderate evidence indicated that MVPA duration did not differ between summer and autumn in five studies (5/5, 100%). A grade was not assignable for LPA com- parisons between summer and autumn. Intensity-specific physical activity duration Seasonal comparisons more frequently involved the dur- ation of MVPA (37/37, 100.0%) than LPA (20/37, 54.1%). Studies comparing MVPA duration between sea- sons sampled youth (25/37, 67.6%), adults (6/37, 16.2%), and older adults (6/67 16.7%). Seasonal comparisons of LPA duration sampled youth (11/20, 55.0%), older adults (6/20, 30.0%), and adults (3/20, 15.0%). Seasonal comparisons more frequently involved the dur- ation of MVPA (37/37, 100.0%) than LPA (20/37, 54.1%). Studies comparing MVPA duration between sea- sons sampled youth (25/37, 67.6%), adults (6/37, 16.2%), and older adults (6/67 16.7%). Seasonal comparisons of LPA duration sampled youth (11/20, 55.0%), older adults (6/20, 30.0%), and adults (3/20, 15.0%). As shown in Table 3, the most common seasonal com- parisons of intensity-specific behavior durations involved winter vs. spring (overall: 22/37 [59.5%]; MVPA: 22/22 [100%]; LPA: 14/22 [63.6%]; winter vs. summer (overall: 19/37 [51.4%]; MVPA: 19/19 [%]; LPA: 12/19 [63.2%]), winter vs. autumn (overall: 17/37 [45.9%]; MVPA: 17/17 [100%]; LPA: 11/17 [64.7%]), spring vs. summer (overall: 13/37 [35.1%]; MVPA: 13/13 [100%]; LPA: 8/13 [61.5%]), and spring vs. autumn (overall: 12/37 [32.4%]; MVPA: 12/12 [100%]; LPA: 7/12 [58.3%]). Sedentary behavior St di i Limited evidence also indicated that sedentary behavior either did not differ between spring and autumn (5/9, 55.6%) or was greater in au- tumn than spring (4/9, 44.4%). Limited evidence indi- cated that sedentary behavior either did not differ Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 16 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 16 of 26 Turrisi et al. Sedentary behavior St di i International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 17 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (20 Page 17 of 26 (2021) 18:24 Table 4 Associations between movement-related behaviors and specific weather indices (Continued) Weather Index Finding Physical Activity Volume Light-Intensity Physical Activity Moderate-to-Vigorous Intensity Physical Activity Sedentary Behavior Associations Evidence Grade Moderate Not Assignable Moderate Limited Visibility Positive Associations 1 [73] 0 0 0 Null Associations 0 1 [34] 0 2 [34] Negative Associations 1 [127] 1 [34] 1 [34] 0 Evidence Grade Not Assignable Not Assignable Not Assignable Not Assignable Barometric Pressure Positive Associations 1 [127] 0 0 0 Null Associations 0 0 0 0 Negative Associations 0 0 0 0 Evidence Grade Not Assignable Not Assignable Not Assignable Not Assignable Wind Chill Positive Associations 1 [73] 0 0 0 Null Associations 0 0 0 0 Negative Associations 0 0 0 0 Evidence Grade Not Assignable Not Assignable Not Assignable Not Assignable Air Quality Positive Associations 0 0 0 0 Null Associations 1 [120] 0 1 [120] 0 Negative Associations 1 [138] 0 0 0 Evidence Grade Not Assignable Not Assignable Not Assignable Not Assignable ssociations between movement-related behaviors and specific weather indices (Continued) between summer and autumn (4/6, 66.7%) or was greater in autumn than summer (2/6, 33.3%). humidity (16/77, 20.8%), snow (12/77, 15.6%), and cloud coverage (7/77, 9.1%). No grade was assigned to research on barometric pressure, visibility, wind chill, or air qual- ity due to insufficient evidence. Specific weather indices associated with movement- related behaviors Weather indices were typically collected from regional weather stations or national institutes (73/77, 94.8%); few studies used self-reported weather indices (3/77, 3.9%) or did not specify where weather indices were col- lected (1/77, 1.3%). Associations between specific indices and behavior examined in five or more studies are dis- cussed below; all available results are summarized in Table 4. The weather indices are defined in Appendix 2. The most frequently-reported weather indices included temperature (60/77, 77.9%), precipitation (58/77, 75.3%), photoperiod (33/77, 42.9%), wind speed (20/77, 26.0%), Sedentary behavior St di i International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Table 4 Associations between movement-related behaviors and specific weather indices Weather Index Finding Physical Activity Volume Light-Intensity Physical Activity Moderate-to-Vigorous Intensity Physical Activity Sedentary Behavior Temperature Positive Associations 19 [35–38, 40, 43, 47, 70, 88, 107–114] 4 [47, 64, 70, 115] 11 [35, 39, 42, 47, 57, 73, 79, 81, 110, 116] 2 [38, 81] Null Associations 13 [28, 35–37, 61, 117–122] 4 [34, 56, 116] 14 [34, 35, 40, 42, 56, 61, 70, 120, 121, 123, 124] 7 [34, 63, 70, 73, 81, 115] Negative Associations 6 [38, 46, 125–128] 0 3 [81, 122, 124] 9 [38, 39, 47, 60–63, 71, 109] Evidence Grade Strong Limited Strong Moderate Precipitation Positive Associations 1 [28] 0 2 [79, 123] 12 [38, 39, 48, 60, 61, 63, 71, 73, 81, 109, 129] Null Associations 5 [37, 70, 114, 118] 3 [34, 56, 70] 15 [35, 39, 42, 56, 70, 81, 121, 122, 124, 130, 131] 7 [34, 39, 62, 63, 70, 81] Negative Associations 25 [28, 36–38, 40, 43, 46, 48, 57, 61, 88, 109, 111–113, 119, 121, 122, 125, 127, 131, 132] 3 [34, 57, 116] 15 [34, 40, 48, 57, 61, 73, 81, 109, 116, 124, 129, 133, 134] 0 Evidence Grade Strong Limited Strong Strong Wind Speed Positive Associations 0 0 1 [39] 2 [48, 60] Null Associations 6 [28, 36, 40, 43] 2 [34] 3 [34, 40] 4 [34, 39] Negative Associations 6 [36, 46, 48, 109, 113, 127] 0 3 [39, 48, 135] 0 Evidence Grade Moderate Not Assignable Limited Limited Photoperiod Positive Associations 15 [38, 46–48, 50, 72, 78, 88, 109, 114, 125, 127, 134, 136, 137] 4 [34, 47, 64, 78] 9 [34, 48, 50, 72, 73, 78, 133, 135] 1 [63] Null Associations 6 [43, 113, 118, 121, 122] 1 [34] 6 [47, 79, 81, 121, 122] 3 [34, 81] Negative Associations 0 0 0 10 [38, 47, 48, 50, 63, 71–73, 78, 81] Evidence Grade Strong Limited Strong Strong Snow Positive Associations 0 0 0 0 Null Associations 3 [111, 114, 125] 1 [116] 4 [39, 42, 116] 0 Negative Associations 4 [38, 88, 93] 1 [64] 1 [40] 0 Evidence Grade Limited Not Assignable Limited Not Assignable Cloud Coverage Positive Associations 1 [127] 0 0 0 Null Associations 2 [36, 37] 0 0 0 Negative Associations 2 [36, 37] 0 2 [41, 135] 0 Evidence Grade Limited Not Assignable Not Assignable Not Assignable Humidity Positive Associations 0 0 0 2 [78, 81] Null Associations 0 1 [34] 6 [39, 81, 124] 5 [34, 39, 81] Negative 5 [78, 107, 126, 127] 2 [34, 78] 6 [34, 78, 79, 81, 124] 0 Table 4 Associations between movement-related behaviors and specific weather indices Turrisi et al. Wind speed Associations between precipitation and movement- related behaviors were estimated in youth (28/58, 48.3%), adults (21/58, 36.2%) and older adults (9/58, 15.5%). Precipitation was most frequently studied in relation with physical activity volume (31/58, 53.4%) followed by intensity-specific physical activity dura- tions (overall: 32/58 [55.2%]; MVPA: 32/32 [100%]; LPA: 6/32 [18.8%]) and sedentary behavior (19/32, 32.8%). Associations between wind speed and movement-related behaviors were estimated in youth (8/20, 40.0%), older adults (7/20, 35.0%), and adults (5/20, 25.0%). These studies reported associations between wind speed and total physical activity volume (12/20, 60.0%) intensity- specific physical activity durations (overall: 7/20 [35.0%]; MVPA: 7/7 [100%]; LPA: 3/7 [42.9%]), and sedentary be- havior (6/20, 35.0%). Strong evidence indicated that associations between precipitation and physical activity volume were largely negative (25/31, 80.6%) with a few null (5/31, 16.1%) and one positive (1/31, 3.2%) association. Strong evi- dence indicated that the association between precipi- tation and MVPA durations were mixed with negative (15/32, 46.9%) and null (15/32, 46.9%) results. Two studies reported a positive association between pre- cipitation and MVPA duration (2/32, 6.3%). Limited evidence indicated that precipitation and LPA had ei- ther a negative (3/6, 50.0%) or null (3/6, 50.0%) asso- ciation. Moderate evidence indicated a mix of positive (12/19, 63.2%) and null associations (7/19, 36.8%) be- tween precipitation and sedentary behavior. No studies reported a negative association between pre- cipitation and sedentary behavior. Moderate evidence indicated that associations between wind speed and physical activity volume were a mix of null (6/12, 50.0%), and negative associations (6/12, 50.0%). No study reported a positive association between wind speed and volume. Limited evidence indicated that wind speed and MVPA durations demonstrated mostly null (3/7, 42.9%) or negative associations (3/7, 42.9%]), although one study found a positive association (1/7, 14.3%). A grade was not assignable for evidence on wind speed and LPA. Limited evidence indicated that wind speed and seden- tary behavior primarily displayed null (4/6, 66.7%) or positive associations (2/6, 33.3%); no studies reported a negative association. Temperature Associations between temperature and movement- related behaviors were estimated in youth (24/60, 40.0%), adults (23/60, 38.3%), and older adults (13/60, 21.7%). The most common behavioral measure in these studies was physical activity volume (38/60, 61.7%), followed by intensity-specific physical activity duration (overall: 30/60 [50.0%]; MVPA: 28/30 [93.3%]; LPA: 8/30 [26.7%]) and sedentary behavior (18/60, 30.0%). Page 18 of 26 Page 18 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 18 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity photoperiod and measures of physical activity volume (21/33, 63.6%), intensity-specific physical activity dur- ation (overall: 19/33 [57.6%]; MVPA: 15/19 [78.9%], LPA: 5/19 [26.3%]) and sedentary behavior (14/33, 42.4%). Associations between temperature and physical activity volume were more often positive (19/38, 50.0%) than null (13/38, 34.2%) or negative (6/38, 15.8%). No studies explicitly tested for curvilinear relations, but four studies noted an inverted-U pattern in which volume was lower during normatively warmer or colder days [38, 78, 117, 125]. Additional negative associations with volume were found when extreme temperatures were present [126, 127]. This evidence was graded as strong in favor of an inverted-U relation between temperature and physical activity volume. Strong evidence indicated that associations between photoperiod and physical activity volume were mostly positive (15/21, 71.4%) with a few null associations (6/21, 28.6%). No studies reported a negative associ- ation between photoperiod and physical activity vol- ume. Strong evidence indicated that associations between the photoperiod and MVPA duration were mostly positive (9/15, 60.0%) or null (6/15, 40.0%). No studies reported a negative association between photoperiod and MVPA duration. Limited evidence suggested a positive association between photoperiod and LPA duration (4/5, 80.0%) with a single study in- dicating a null association (1/5, 20.0%). Strong evi- dence indicated that the photoperiod and sedentary behavior had a negative association (10/14, 71.4%), with a few studies indicating either null (3/14, 21.4%) or positive associations (1/14, 7.1%). Moderate evidence indicated that associations between temperature and MVPA duration were more often null (14/28, 50.0%) or positive (11/28, 39.3%) than negative (3/28, 10.7%). Limited evidence suggested that LPA ex- hibited a similar pattern with studies showing either a null (4/8, 50.0%) or positive (4/8, 50.0%) association with temperature. Moderate evidence indicated that sedentary behavior exhibited more negative associations with temperature (9/18, 50.0%) than null (7/18, 38.9%) or positive associations (2/18, 11.1%). Humidity Associations between humidity and movement-related behaviors were estimated in adults (7/16, 43.8%), youth (5/16, 31.3%), and older adults (4/16, 25.0%). Studies including humidity frequently examined mea- sures of physical activity volume (5/16, 31.3%), followed in frequency by intensity-specific physical Snow Associations between snow and movement-related be- haviors were estimated in youth (5/12, 41.7%), adults (3/ 12, 25.0%), and older adults (4/12, 33.3%). Studies in- cluding snow frequently examined measures of physical activity volume (7/12, 58.3%), followed in frequency by intensity-specific physical activity duration (overall: 6/12 [50.0%]; MVPA: 5/6 [83.3%], LPA: 2/6 [33.3%]). Limited evidence on snow and physical activity volume was split between negative (4/7, 57.1%) and null (3/7, 42.9%) associations. No study examining snow and vol- ume demonstrated a positive association. Limited evi- dence on snow and MVPA duration was split between null (4/5, 80.0%) and negative (1/5, 20.0%) associations. A grade was not assignable to evidence linking snow with either LPA or sedentary behavior. One contribution of the present review was that it ex- amined a broad spectrum of movement behaviors that contribute to physical activity volume. MVPA duration has enjoyed a privileged status in the scientific literature because it has the strongest connections with health benefits [24]. MVPA also exhibited the clearest pattern of relations with seasonality and specific weather indices, including temperature, precipitation, and photoperiod. Thus, MVPA may provide one pathway by which the natural environment gets “under the skin” to affect health [139, 140]. Cloud coverage Associations between cloud coverage and movement- related behaviors were estimated in youth (4/7, 57.1%), adults (1/7, 14.3%), and older adults (2/7, 28.6%). Studies including cloud coverage frequently examined measures of physical activity volume (5/7, 71.4%), followed in fre- quency by intensity-specific physical activity duration (overall: 2/7 [28.6%]; MVPA: 2/2 [100%], LPA: 0/2 [0.0%]). Moving the needle on population-level MVPA has proven to be difficult [141, 142]. Far more time is spent in LPA than MVPA and LPA is a greater contributor to physical activity volume for most people [143]. Recent work has established unique health benefits from LPA after adjusting for MVPA [144]. As a consequence, LPA is a desirable substitute for prolonged sedentary behavior when MVPA is not feasible. Although a trend for in- creased LPA in summer and spring compared to winter was observed in the literature, LPA in the spring did not differ from LPA in the summer or autumn. In the inter- est of understanding how the natural environment facili- tates or inhibits movement, this common form of physical activity should be a priority measure in future research examining seasonal differences and weather correlates. Limited evidence indicated that cloud coverage and physical activity volume exhibit a negative (2/5, 40.0%) or null associations (2/5, 40.0%). One study found a positive association between cloud coverage and physical activity volume (1/5, 40.0%). A grade was not assignable to evidence linking cloud coverage and durations of MVPA, LPA, or sedentary behavior. Photoperiod Associations between the photoperiod and movement- related behaviors were estimated in youth (14/33, 42.4%), adults (10/33, 30.3%) and older adults (9/33, 27.3%). These studies reported associations between the Page 19 of 26 Page 19 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity activity duration (overall: 12/16 [75.0%]; MVPA: 12/12 [100%]; LPA: 3/12 [25.0%], sedentary behavior: 7/16 [43.8%]). activity duration (overall: 12/16 [75.0%]; MVPA: 12/12 [100%]; LPA: 3/12 [25.0%], sedentary behavior: 7/16 [43.8%]). participants from approximately 18 countries [10, 11]. In those reviews, studies on seasonal differences in physical activity greatly outnumbered studies on wea- ther correlates. Over the past decade, attention has been divided more equally between seasonal differ- ences in and weather correlates of movement behav- iors. This review also extended the prior reviews by capturing the spectrum of movement behaviors ran- ging from physical activity volume to intensity- specific durations to sedentary behavior. Collectively, these reviews establish how key features of the nat- ural environment are linked with a variety of move- ment behaviors. Moderate evidence indicated a consistently negative association between humidity and physical activity volume (5/5, 100.0%). Moderate evidence indicated a mix of negative (6/12, 50.0%) and null (6/12, 50.0%) associations between humidity and MVPA. A grade was not assignable to evidence linking humidity and LPA. Limited evidence suggested a null association between humidity and sedentary behavior (5/7, 71.4%) although some studies found a positive association (2/ 7, 28.6%). Physical activity volume and MVPA duration were the most frequent measures of movement behavior. Consistent with prior work, winter and summer were marked by the lowest and greatest movement behav- ior, respectively [10, 11]. The present review extended those conclusions by documenting a trend in favor of greater physical activity volume and MVPA in spring than autumn. Comparisons of spring vs summer and summer vs autumn largely revealed no differences. Overall, the pattern of seasonal differences in physical activity volume and MVPA duration resembled a si- nusoidal pattern and corresponded with both fluctua- tions in temperature and the waxing and waning photoperiod across the calendar year. Discussion The present review summarized 144 studies from 110 articles with 118,189 participants from 30 countries. It updates conclusions from two seminal reviews based on a total of over 60 studies on over 300,000 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 Page 20 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity This review had limitations as well. The search was conducted using three databases and limited to English language publications. It is possible that studies were missed if they were published in journals not indexed by PubMed, CINAHL, or SPORTDiscus, or were written in languages other than English. All data were observa- tional so strong causal inferences are not possible. Re- sults were obtained from 30 countries on five continents but western and high-income countries were overrepre- sented in the data. Low- and middle-income countries are home to over 80% of the world’s population and four times as many deaths are attributed to physical inactivity in those countries than in high-income countries [147, 148]. Conclusions may not generalize equally well to all regions, climatic zones, or economic strata. Most studies did not report on race or ethnicity so it is unclear how physical, social or cultural differences influence seasonal differences or relations between weather and movement behaviors. Devices were used to obtain measures of physical activity volume and intensity-specific duration – primarily MVPA - but some activity types do not lend themselves to accurate measurement with devices (e.g., cycling, swimming) or are sub-optimal with devices at- tached at the waist (e.g., sedentary behavior). Devices also provide no insights into the specific domains of physical activity (e.g., occupational, transport-related, oc- cupational, domestic) or sedentary behavior (e.g., read- ing, screen time, socializing, eating). The available literature has focused almost exclusively on aggregated weather summaries during monitoring periods. Yet wea- ther is dynamic so those summaries may not generalize to within-person change processes [149]. Additionally, physiological or psychological differences in environ- mental tolerances likely exist. Some people, for example, will be more heat tolerant or may simply enjoy running in the rain. Person-specific models of physical activity under different weather conditions could shed light on these dynamics [145]. Finally, the review focused on 11 common weather indices but other indices may also be relevant. Sedentary behavior was consistently greater in winter than spring or summer. Discussion Weather has been cited as a bar- rier to physical activity that facilitates sedentary behavior [8, 9]. This study extended previous work by reviewing how specific weather indices, as opposed to perceptions of the weather, are associated with sedentary behavior. People engage in more sedentary behavior on shorter days (photoperiod), when precipitation is greater, and when temperatures are lower. These findings revealed that weather is likely to be a third variable influencing the entire spectrum of movement-related behaviors. These findings have two major implications. First, al- though the studies reviewed here were necessarily obser- vational, they can inform behavioral interventions. Weather conditions that may serve as actual, as well as perceived, barriers to physical activity (e.g., too hot, cold, rainy or snowy, windy, or shorter days). Current or fore- casted weather conditions may be useful for providing contextual information about opportunities for activity that could inform just-in-time interventions for move- ment behaviors. For example, users could be prompted to develop coping plans for exercise in adverse weather conditions and then reminded of those plans when ad- verse weather conditions were expected. Digital tools could also extend work on person-specific physical activ- ity interventions by learning how to identify user- specific preferred weather patterns for movement behav- iors and prompt users to ensure they capitalize on their preferred conditions to be active [145]. At a more general level, the impact of seasonal differ- ences in movement behaviors on the implementation and evaluation of physical activity promotion programs should be considered when interpreting ambulatory be- havior changes. Lifestyle physical activity intervention evaluations often last 1–6 months, and few last 12 months, so baseline and follow-up assessments are often conducted during different seasons. Seasonal influences on activity levels are typically allocated to the error term of statistical models but may be informative to include as a covariate or moderator of intervention effects. For example, including season as a moderator could reveal if interventions work better when days are lengthening (winter to summer) or when days are shortening (sum- mer to winter). Conclusions In sum, this review established consistent patterns of seasonal and weather-specific differences in physical ac- tivity and sedentary behavior. People tend to be most ac- tive in summer and least active in winter. This pattern coincides with temperature and photoperiod cycles. Weather may also influence comfort with physical activ- ity as indicated by associations with precipitation and wind speed. These findings can inform physical activity promotion initiatives in the short-term and may have long-term implications for environmental influences on human health during the climate crisis. Second, climate change is increasing the frequency of extreme weather conditions. Climatic zones that are cur- rently favorable for movement behaviors may become inhospitable, inhibit physical activity, and contribute to health disparities. Some have speculated that climate change will increase migration as people seek to preserve an adaptive environmental niche [146]. Population-level data on movement behaviors could be investigated as a leading indicator of future health or migration due to climate change. Page 21 of 26 (2021) 18:24 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Appendix A Table 5 Search Methods \| PubMed Authors’ contributions ABW, SKM, CML, and DEC made substantial contributions to the conception or design of the work; TBT, KMB, SahH, SarH made substantial contributions to the acquisition, analysis and interpretation of data for the work. All authors contributed to drafting or revising the work critically for important intellectual content, approved the final version to be published, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ABW, SKM, CML, and DEC made substantial contributions to the conception or design of the work; TBT, KMB, SahH, SarH made substantial contributions to the acquisition, analysis and interpretation of data for the work. All authors contributed to drafting or revising the work critically for important intellectual content, approved the final version to be published, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. (DE “WEATHER”) OR (DE “TEMPERATURE”) OR (DE “HUMIDITY”) OR (DE “ICE”) OR (DE “ULTRAVIOLET radiation”) OR (DE “SUNSHINE”) OR (DE “ATMOSPHERIC pressure”) OR (DE “SNOW”) OR (DE “COLD (Temperature)”) OR TI (“WEATHER”) OR (“TEMPERATURE”) OR (“HUMIDITY”) OR (“ICE”) OR (“ULTRAVIOLET radiation”) OR (“SUNSHINE”) OR (“ATMOSPHERIC pressure”) OR (“SNOW”) OR (“COLD (Temperature)”) OR (“ATMOSPHERE”) OR (“METEOROLOGICAL FACTORS”) OR (“EXTREME WEATHER”) OR (“CLIMATE”) OR AB (“WEATHER”) OR (“TEMPERATURE”) OR (“HUMIDITY”) OR (“ICE”) OR (“ULTRAVIOLET radiation”) OR (“SUNSHINE”) OR (“ATMOSPHERIC pressure”) OR (“SNOW”) OR (“COLD (Temperature)”) OR (“ATMOSPHERE”) OR (“METEOROLOGICAL FACTORS”) OR (“EXTREME WEATHER”) OR Funding h (“ATMOSPHERE”) OR (“METEOROLOGICAL FACTORS”) OR (“EXTREME WEATHER”) OR (“CLIMATE”) g Research reported in this publication was supported in part by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award number R01HL142732 and the Division of Electrical, Communications, and Cyber Systems of the National Science Foundation under award number 1808266. Scherezade Mama is supported in part by a National Cancer Institute career development award (K07 CA222335). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the National Science Foundation. The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript should be declared. Limiters: (“2006/01/01”[Date - Publication]: “2020/10/31”[Date - Publication]) AND (English[Language]) Filters: Humans Filters: Humans Consent for publication Not applicable. Consent for publication Not applicable. Author details 1 1Department of Kinesiology, The Pennsylvania State University, University Park, PA 16802, USA. 2Advanced Safety & User Experience, Aptiv, Troy, MI, USA. 3Department of Electrical Engineering & Computer Science, The Pennsylvania State University, University Park, PA, USA. 4Department of Health Disparities Research, The University of Texas MD Anderson Cancer Center, Houston, TX, USA. 5Department of Preventive Medicine, Northwestern University, Chicago, IL, USA. Received: 17 June 2020 Accepted: 22 January 2021 Received: 17 June 2020 Accepted: 22 January 2021 Received: 17 June 2020 Accepted: 22 January 2021 Acknowledgements Not applicable. AND (DE “PEDOMETERS”) OR (DE “ACCELEROMETERS”) OR (“PEDOMETERS”) OR (“ACCELEROMETERS”) OR (“STEP”) OR (“FITNESS TRACKERS”) OR (“WEARABLE ELEC TRONIC DEVICES”) OR AB (“PEDOMETERS”) OR (“ACCELEROMETERS”) OR (“STEP”) OR (“FITNESS TRACKERS”) OR (“WEARABLE ELECTRONIC DEVICES”) AND Additional file 1. Additional file 1. Table 6 Search Methods \| CINAHL International Journal of Behavioral Nutrition and Physical Activity (2021) 18: Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity Table 6 Search Methods \| CINAHL Table 6 Search Methods \| CINAHL Search terms: ((“Exercise”[MeSH] OR “Exercises”[tiab] OR “Exercising”[MeSH] OR “Exercising”[tiab] OR “Physical activities”[MeSH] OR “Physical activities”[tiab] OR “Physical activity”[MeSH] OR “Physical activity”[tiab] OR “Physical fitness”[MeSH] OR “Physical fitness”[tiab] OR “Recreation”[MeSH] OR “Recreation”[tiab] OR “Run”[MeSH] OR “Run”[tiab] OR “Running”[MeSH] OR “Running”[tiab] OR “Walk”[MeSH] OR “Walk”[tiab] OR “Walking”[MeSH] OR “Walking”[tiab]) OR (“Computer game”[MesH] OR “Computer game”[tiab] OR “Computer games”[MeSH] OR “Computer games”[tiab] OR “Computer usage”[MeSH] OR “Computer usage”[tiab] OR “Computer use”[MeSH] OR “Computer use”[tiab] OR “Physically inactive”[MeSH] OR “Physically inactive”[tiab] OR “Screen time”[MeSH] OR “Screen time”[tiab] OR “Sedentarism”[MeSH] OR “Sedentarism”[tiab] OR “Sedentary behavior”[MeSH] OR “Sedentary behavior”[tiab] OR “TV viewing”[MeSH] OR “TV viewing”[tiab] OR “TV watching”[MeSH] OR “TV watching”[tiab] OR “Video game”[MeSH] OR “Video game”[tiab] OR “Video games”[MeSH] OR “Video games”[tiab] OR “Video gaming”[MeSH] OR “Video gaming”[tiab])) AND Search terms: (MH “Exercise”) OR (MH “Physical Fitness”) OR (MH “Cardiorespiratory Fitness”) OR (MH “Physical Activity”) OR (MH “Recreation”) OR (MH “Running, Distance”) OR (MH “Running”) OR (MH “Walking”) OR (MH “Life Style, Sedentary”) OR (MH “Television”) OR (MH “Video Games”) OR (MH “Computers, Portable”) OR (MH “Computers, Hand-Held”) OR (MH “Screen Time”)OR TI “Exercise” OR “Physical Fitness” OR “Cardiorespiratory Fitness” OR “Physical Activity” OR “Recreation” OR “Running, Distance” OR “Running” OR “Walking” OR “Life Style, Seden- tary” OR “Television” OR “Video Games” OR “Computers, Portable” OR “Computers, Hand-Held” OR “Screen Time” OR AB “Exercise” OR “Physical Fitness” OR “Cardiorespiratory Fitness” OR “Physical Activity” OR “Recre- ation” OR “Running, Distance” OR “Running” OR “Walking” OR “Life Style, Sedentary” OR “Television” OR “Video Games” OR “Computers, Portable” OR “Computers, Hand-Held” OR “Screen Time” AND (MH “Accelerometers”) OR (MH “Pedometers”) OR (MH “Step”) OR (MH “Fitness Trackers”) OR TI “Accelerometers” OR “Pedometers” OR “Step” OR “Fitness Trackers” OR “Wearable Electronic Devices” OR AB “Accelerometers” OR “Pedometers” OR “Step” OR “Fitness Trackers” OR “Wearable Electronic Devices” “Accelerometers” OR “Pedometers” OR “Step” OR “Fitness Trackers” OR “Wearable Electronic Devices” (“Accelerometer”[MeSH] OR “Accelerometer”[tiab] OR “Accelerometry”[MeSH] OR “Accelerometry”[tiab] OR “Pedometer”[MeSH] OR “Pedometer”[tiab] OR “Steps”[MeSH] OR “Steps”[tiab]) AND (MH “Weather”) OR (MH “Extreme Weather”) OR (MH “Seasons”) OR (MH “Temperature”) OR (MH “Humidity”) OR (MH “Heat”) OR (MH “Ice”) OR (MH “Ultraviolet Rays”) OR (MH “Sunlight”) OR (MH “Smog”) OR (MH “Light”) OR (MH “Lightning”) OR (MH “Atmospheric Pressure”) OR (MH “Rain”) OR (MH “Snow”) OR (MH “Atmosphere”) OR (MH “Meteorological Factors”) OR (MH “Climate”) OR TI “Weather” OR “Extreme Weather” OR “Seasons” OR “Temperature” OR “Humidity” OR “Heat” OR “Ice” OR “Ultraviolet Rays” OR “Sunlight” OR “Smog” OR “Light” OR “Lightning” OR “Atmospheric Pressure” OR “Rain” OR “Snow” OR “Atmosphere” OR “Meteorological Factors” OR “Climate” OR AB “Weather” OR “Extreme Weather” OR “Seasons” OR “Temperature” OR “Humidity” OR “Heat” OR “Ice” OR “Ultraviolet Rays” OR “Sunlight” OR “Smog” OR “Light” OR “Lightning” OR “Atmospheric Pressure” OR “Rain” OR “Snow” OR “Atmosphere” OR “Meteorological Factors” OR “Climate” ((“Season”[MeSH] OR “Season”[tiab] OR “Seasonal”[MeSH] OR “Seasonal”[tiab] OR “Seasons”[MeSH] OR “Seasons”[tiab]) OR (“Atmospheric Pressure”[MesH] OR “Atmospheric Pressure”[tiab] OR “Cloud”[MeSH] OR “Cloud”[tiab] OR “Cloud Cover”[MeSH] OR “Cloud Cover”[tiab] OR “Clouds”[MeSH] OR “Clouds”[tiab] OR “Cloudy”[MeSH] OR “Cloudy”[tiab] OR “Fog”[MeSH] OR “Fog”[tiab] OR “Heat index”[MeSH] OR “Fog”[tiab] OR “Humid”[MeSH] OR “Humid”[tiab] OR “Humidity”[MeSH] OR “Humidity”[tiab] OR “Ice”[MeSH] OR “Ice”[tiab] OR “Lightning”[MeSH] OR “Lightning”[tiab] OR “Overcast”[MeSH] OR “Overcast”[tiab] OR “Precipitation”[MeSH] OR “Precipitation”[tiab] OR “Rain”[MeSH] OR “Rain”[tiab] OR “Saturation”[MeSH] OR “Saturation”[tiab] OR “Severe weather”[MeSH] OR “Severe weather”[tiab] OR “Snow”[MeSH] OR “Snow”[tiab] OR “Sunlight”[MeSH] OR “Sunlight”[tiab] OR “Temperature”[MeSH] OR “Temperature”[tiab] OR “Temperatures”[MeSH] OR “Temperatures”[tiab] OR “Ultraviolet rays”[MeSH] OR “Ultraviolet rays”[tiab] OR “UV index”[MeSH] OR “UV index”[tiab] OR “UV rays”[MeSH] OR “UV rays”[tiab] OR “Visibility”[MeSH] OR “Visibility”[tiab] OR “Weather”[MeSH] OR “Weather”[tiab] OR “Wind”[MeSH] OR “Wind”[tiab] OR “Wind chill”[MeSH] OR “Wind chill”[tiab] OR “Windy”[MeSH] OR “Windy”[tiab])) Limiters: (“2006/01/01”[Date - Publication]: “2020/10/31”[Date - Publication]) AND (English[Language]) Limiters: (“2006/01/01”[Date - Publication]: “2020/10/31”[Date - Publication]) AND (English[Language]) Limiters: (“2006/01/01”[Date - Publication]: “2020/10/31”[Date - Publication]) AND (English[Language]) Filters: Humans Filters: Humans Filters: Humans Limiters: (“2006/01/01”[Date - Publication]: “2020/10/31”[Date - Publication]) AND (English[Language]) Filters: Humans Limiters: (“2006/01/01”[Date - Publication]: “2020/10/31”[Date - Publication]) AND (English[Language]) Filters: Humans Page 22 of 26 Page 22 of 26 (2021) 18:24 Turrisi et al. Appendix C Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12966-021-01091-1. pp y The online version contains supplementary material available at https://doi. org/10.1186/s12966-021-01091-1. Table 7 Search Methods \| SPORTDiscus Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. All data generated or analyzed during this study are included in this published article and its supplementary information files. Table 8 Glossary of Weather Indices Index Definition Temperature Degree or intensity of heat in the environment Precipitation Product of condensation from atmospheric water vapor (typically rain and sleet in this review) Wind Speed Atmospheric quantity resulting from air traveling from high to low pressure Photoperiod Day length, or the period of daily illumination Snow Small, frozen white ice crystals generated from atmospheric water vapor at temperatures below the freezing point Cloud Coverage The fraction of the sky obscured by clouds in a particular location Humidity Concentration of atmospheric water vapor present in the environment Visibility Distance that can be seen due to light and weather conditions Barometric Pressure Pressure within the atmosphere Windchill Quantity of air temperature that is effectively lowered due to wind Air Quality A measure of how clean or polluted the air is Note. Definitions obtained from the National Weather Service and the Oxford English Dictionary [150, 151] Table 8 Glossary of Weather Indices Ethics approval and consent to participate Not applicable. Competing interests Competing interests The authors declare that they have no competing interests. Table 7 Search Methods \| SPORTDiscus Search terms: (DE “EXERCISE”) OR (DE “PHYSICAL fitness”) OR (DE “PHYSICAL activity”) OR (DE “RECREATION”) OR (DE “RUNNING”) OR (DE “WALKING”) OR (DE “SEDENTARY behavior”) OR (DE “SUBSCRIPTION television”) OR (DE “VIDEO games”) OR (DE “COMPUTER games”) OR (DE “SEDENTARY people”) OR TI (“EXERCISE”) OR (“PHYSICAL fitness”) OR (“PHYSICAL activity”) OR (“RECREATION”) OR (“RUNNING”) OR (“WALKING”) OR (“SEDENTARY behavior”) OR (“SUBSCRIPTION television”) OR (“VIDEO games”) OR (“COMPUTER games”) OR (“SEDENTARY people”) OR AB (“EXERCISE”) OR (“PHYSICAL fitness”) OR (“PHYSICAL activity”) OR (“RECREATION”) OR (“RUNNING”) OR (“WALKING”) OR (“SEDENTARY behavior”) OR (“SUBSCRIPTION television”) OR (“VIDEO games”) OR (“COMPUTER games”) OR (“SEDENTARY people”) AND References Relationship between objective measures of physical activity and weather: a longitudinal study. Int J Behav Nutr Phys Act. 2006;3:21. 38. Wu Y-T, Luben R, Wareham N, Griffin S, Jones AP. Weather, day length and physical activity in older adults: cross-sectional results from the European prospective investigation into Cancer and nutrition (EPIC) Norfolk cohort. PLoS One. 2017;12(5):e0177767. 16. The Nobel Peace Prize Award Ceremony 2007 [Internet]. 2007; Oslo, Norway. Available from: NobelPrize.org 39. Lewis LK, Maher C, Belanger K, Tremblay M, Chaput J-P, Olds T. At the mercy of the gods: associations between weather, physical activity, and sedentary time in children. Pediatr Exerc Sci. 2016;28(1):152–63. 17. Friel S, Bowen K, Campbell-Lendrum D, Frumkin H, McMichael AJ, Rasanathan K. Climate change, noncommunicable diseases, and development: the relationships and common policy opportunities. Annu Rev Public Health. 2011;32:133–47. 40. Ogawa S, Seko T, Ito T, Mori M. Differences in physical activity between seasons with and without snowfall among elderly individuals residing in areas that receive snowfall. J Phys Ther Sci. 2019 Jan;31(1):12–6. 18. Vogelstein F. The untold story: How the iPhone blew up the wireless industry. Wired [Internet]. 2008; Available from: https://www.wired.com/2 008/01/ff-iphone/ 41. Pagels P, Raustorp A, Guban P, Fröberg A, Boldemann C. Compulsory school in- and outdoors—implications for school children’s physical activity and health during one academic year. Int J Environ Res Public Health. 2016;13(7): 699. 19. About Fitbit [Internet]. Available from: https://www.fitbit.com/us/about-us 20. Ekelund U, Steene-Johannessen J, Brown WJ, Fagerland MW, Owen N, Powell KE, et al. Does physical activity attenuate, or even eliminate, the detrimental association of sitting time with mortality? A harmonised meta- analysis of data from more than 1 million men and women. Lancet. 2016 Sep 24;388(10051):1302–10. 42. Patnode CD, Lytle LA, Erickson DJ, Sirard JR, Barr-Anderson D, Story M. The relative influence of demographic, individual, social, and environmental factors on physical activity among boys and girls. Int J Behav Nutr Phys Act. 2010 Nov 3;7(1):79. 21. Stamatakis E, Gale J, Bauman A, Ekelund U, Hamer M, Ding D. Sitting time, physical activity, and risk of mortality in adults. J Am Coll Cardiol. 2019; 73(16):2062–72. 43. Duncan JS, Hopkins WG, Schofield G, Duncan EK. Effects of weather on pedometer-determined physical activity in children. Med Sci Sports Exerc. 2008 Aug;40(8):1432–8. 22. Carson V, Spence J. Seasonal variation in physical activity among children and adolescents: a review. Pediatr Exerc Sci. 2010;22:81–92. 44. McCrorie PRW, Duncan E, Granat MH, Stansfield BW. References 1. Guthold R, Stevens GA, Riley LM, Bull FC. Worldwide trends in insufficient physical activity from 2001 to 2016: a pooled analysis of 358 population- based surveys with 1·9 million participants. Lancet Glob Health. 2018;6(10): e1077–86. 1. Guthold R, Stevens GA, Riley LM, Bull FC. Worldwide trends in insufficient physical activity from 2001 to 2016: a pooled analysis of 358 population- based surveys with 1·9 million participants. Lancet Glob Health. 2018;6(10): e1077–86. 2. McLeroy KR, Bibeau D, Steckler A, Glanz K. An ecological perspective on health promotion programs. Health Educ Q. 1988;15(4):351–77. 2. McLeroy KR, Bibeau D, Steckler A, Glanz K. An ecological perspective on health promotion programs. Health Educ Q. 1988;15(4):351–77. Page 23 of 26 Page 23 of 26 Page 23 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 3. Sallis JF, Cervero RB, Ascher W, Henderson KA, Kraft MK, Kerr J. An ecological approach to creating active living communities. Annu Rev Public Health. 2006;27:297–322. 28. Aspvik NP, Viken H, Ingebrigtsen JE, Zisko N, Mehus I, Wisløff U, et al. Do weather changes influence physical activity level among older adults? – the generation 100 study. PLoS One. 2018;13(7):e0199463. 4. Spence JC, Lee RE. Toward a comprehensive model of physical activity. Psychol Sport Exerc. 2003;4(1):7–24. Spence JC, Lee RE. Toward a comprehensive model of physical activi 29. Kolle E, Steene-Johannessen J, Andersen LB, Anderssen SA. Seasonal variation in objectively assessed physical activity among children and adolescents in Norway: a cross-sectional study. Int J Behav Nutr Phys Act. 2009 Jun 29;6:36. Psychol Sport Exerc. 2003;4(1):7–24. 5. Bopp M, Gayah VV, Campbell ME. Examining the link between public transit use and active commuting. Int J Environ Res Public Health. 2015 Apr;12(4): 4256–74. 30. Koolhaas CM, van Rooij FJA, Schoufour JD, Cepeda M, Tiemeier H, Brage S, et al. Objective measures of activity in the elderly: distribution and associations with demographic and health factors. J Am Med Dir Assoc. 2017 Oct 1;18(10):838–47. 6. Burton NW, Turrell G, Oldenburg B. Participation in recreational physical activity: why do socioeconomic groups differ? Health Educ Behav. 2003; 30(2):225–44. 31. Rowlands AV, Pilgrim EL, Eston RG. Seasonal changes in children’s physical activity: an examination of group changes, intra-individual variability and consistency in activity pattern across season. Ann Hum Biol. 2009 Jan 1; 36(4):363–78. 7. Tappe MK, Duda JL, Ehrnwald PM. Perceived barriers to exercise among adolescents. J Sch Health. References 1989 Apr 1;59(4):153–5. 8. Salmon J, Owen N, Crawford D, Bauman A, Sallis JF. Physical activity and sedentary behavior: a population-based study of barriers, enjoyment, and preference. Health Psychol. 2003;22(2):178–88. 32. Shen B, Alexander G, Milberger S, Jen K-LC. An exploratory study of seasonality and preschoolers’ physical activity engagement. J Phys Act Health. 2013 Sep;10(7):993–9. 9. O’Donoghue G, Perchoux C, Mensah K, Lakerveld J, van der Ploeg H, Bernaards C, et al. A systematic review of correlates of sedentary behaviour in adults aged 18–65 years: a socio-ecological approach. BMC Public Health. 2016;16(1):163. 33. Atkin AJ, Sharp SJ, Harrison F, Brage S, Van Sluijs EMF. Seasonal variation in children’s physical activity and sedentary time. Med Sci Sports Exerc. 2016 Mar;48(3):449–56. 10. Tucker P, Gilliland J. The effect of season and weather on physical activity: a systematic review. Public Health. 2007;121(12):909–22. 34. Cepeda M, Koolhaas CM, van Rooij FJA, Tiemeier H, Guxens M, Franco OH, et al. Seasonality of physical activity, sedentary behavior, and sleep in a middle-aged and elderly population: the Rotterdam study. Maturitas. 2018 Apr;110:41–50. 11. Chan CB, Ryan DA. Assessing the effects of weather conditions on physical activity participation using objective measures. Int J Environ Res Public Health. 2009;6(10):2639–54. 35. Larouche R, Blanchette S, Faulkner G, Riazi N, Trudeau F, Tremblay MS. Correlates of children’s physical activity: a Canadian multisite study. Med Sci Sports Exerc. 2019 Dec;51(12):2482–90. 12. Baranowski T, Thompson WO, DuRant RH, Baranowski J, Puhl J. Observations on physical activity in physical locations: age, gender, ethnicity, and month effects. Res Q Exerc Sport. 1993;64(2):127–33. 36. Albrecht BM, Stalling I, Recke C, Bammann K. Accelerometer-assessed outdoor physical activity is associated with meteorological conditions among older adults: cross-sectional results from the OUTDOOR ACTIVE study. PLoS One. 2020;15(1):e0228053. 13. Haggarty P, McNeill G, Manneh MK, Davidson L, Milne E, Duncan G, et al. The influence of exercise on the energy requirements of adult males in the UK. Br J Nutr. 1994;72(6):799–813. 14. Rich C, Griffiths LJ, Dezateux C. Seasonal variation in accelerometer- determined sedentary behaviour and physical activity in children: a review. Int J Behav Nutr Phys Act. 2012;9(1):49. 37. Rahman S, Maximova K, Carson V, Jhangri GS, Veugelers PJ. Stay in or play out? The influence of weather conditions on physical activity of grade 5 children in Canada. Can J Public Health. 2019;110(2):169–77. 15. Chan CB, Ryan DA, Tudor-Locke C. References Seasonal variation in the distribution of daily stepping in 11–13 year old school children. Int J Exerc Sci. 2015;8:4. 23. Munn Z, Peters MDJ, Stern C, Tufanaru C, McArthur A, Aromataris E. Systematic review or scoping review? Guidance for authors when choosing between a systematic or scoping review approach. BMC Med Res Methodol. 2018 Nov 19;18(1):143. 45. World Bank Country and Lending Groups [Internet]. The World Bank. 2020 [cited 2020 Jun 5]. Available from: https://datahelpdesk.worldbank.org/ knowledgebase/articles/906519-world-bank-country-and-lending-groups 24. U.S. Department of Health and Human Services. 2018 Physical Activity Guidelines Advisory Committee Scientific Report. U.S. Department of Health and Human Services; 2018 p. 779. 46. Ma BD, Ng SL, Schwanen T, Zacharias J, Zhou M, Kawachi I, et al. Pokémon GO and physical activity in Asia: multilevel study. J Med Internet Res. 2018; 20(6):e217. 25. Ouzzani M, Hammady H, Fedorowicz Z, Elmagarmid A. Rayyan—a web and mobile app for systematic reviews. Systematic Reviews. 2016;5(1):210. 47. Arnardottir NY, Oskarsdottir ND, Brychta RJ, Koster A, Van Domelen DR, Caserotti P, et al. Comparison of summer and winter objectively measured physical activity and sedentary behavior in older adults: age, gene/ environment susceptibility Reykjavik study. Int J Environ Res Public Health. 2017;14(10):1268. 26. Kellermeyer L, Harnke B, Knight S. Covidence and Rayyan. J Med Libr Assoc. 2018;106(4):580–3. 27. Moher D, Liberati A, Tetzlaff J, Altman DG. PRISMA group. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009;6(7):e1000097–7. Page 24 of 26 Page 24 of 26 Page 24 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 (2021) 18:24 48. Edwards NM, Myer GD, Kalkwarf HJ, Woo JG, Khoury PR, Hewett TE, et al. Outdoor temperature, precipitation, and wind speed affect physical activity levels in children: a longitudinal cohort study. J Phys Act Health. 2015 Aug; 12(8):1074–81. 69. Diaz KM, Howard VJ, Hutto B, Colabianchi N, Vena JE, Blair SN, et al. Patterns of sedentary behavior in US middle-age and older adults: the REGARDS study. Med Sci Sports Exerc. 2016;48(3):430–8. 70. Hunter S, Rosu A, Hesketh KD, Rhodes RE, Rinaldi CM, Rodgers W, et al. Objectively measured environmental correlates of toddlers’ physical activity and sedentary behavior. Pediatr Exerc Sci. 2019;31(4):480–7. 49. Hjorth MF, Chaput J-P, Michaelsen K, Astrup A, Tetens I, Sjödin A. Seasonal variation in objectively measured physical activity, sedentary time, cardio-respiratory fitness and sleep duration among 8-11 year-old Danish children: a repeated-measures study. References Early predictors of objectively measured physical activity and sedentary behaviour in 8-10 year old children: the Gateshead millennium study. PLoS One. 2012;7(6):e37975. 56. Van Kann DHH, de Vries SI, Schipperijn J, de Vries NK, Jansen MWJ, Kremers SPJ. Schoolyard characteristics, physical activity, and sedentary behavior: combining GPS and accelerometry. J Sch Health. 2016;86(12):913–21. 77. Buchowski MS, Choi L, Majchrzak KM, Acra S, Mathews CE, Chen KY. Seasonal changes in amount and patterns of physical activity in women. J Phys Act Health. 2009;6(2):252–61. 57. Yildirim M, Schoeni A, Singh AS, Altenburg TM, Brug J, De Bourdeaudhuij I, et al. Daily variations in weather and the relationship with physical activity and sedentary time in European 10- to 12-year-olds: the ENERGY-project. J Phys Act Health. 2014 Feb;11(2):419–25. 78. Sartini C, Morris RW, Whincup PH, Wannamethee SG, Ash S, Lennon L, et al. Association of maximum temperature with sedentary time in older British men. J Phys Act Health. 2017;14(4):265–9. 58. Sit CHP, Huang WY, Yu JJ, McKenzie TL. Accelerometer-assessed physical activity and sedentary time at school for children with disabilities: seasonal variation. Int J Environ Res Public Health. 2019;16(17):3163. 79. Remmers T, Thijs C, Timperio A, Salmon J, Veitch J, Kremerr SPJ, et al. Daily weather and children’s physical activity patterns. Med Sci Sports Exerc. 2017; 49(5):922–9. 59. Harrison F, Jones AP, Bentham G, van Sluijs EMF, Cassidy A, Griffin SJ. The impact of rainfall and school break time policies on physical activity in 9-10 year old British children: a repeated measures study. Int J Behav Nutr Phys Act. 2011 May 24;8:47. 80. Hoaas H, Zanaboni P, Hjalmarsen A, Morseth B, Dinesen B, Burge AT, et al. Seasonal variations in objectively assessed physical activity among people with COPD in two Nordic countries and Australia: a cross-sectional study. Int J Chron Obstruct Pulmon Dis. 2019;14:1219–28. 81. Zheng C, Huang WY, Wong SH-S. Associations of weather conditions with adolescents’ daily physical activity, sedentary time, and sleep duration. Applied Physiology, Nutrition & Metabolism. 2019;44(12):1339–44. 60. Katapally TR, Rainham D, Muhajarine N. The influence of weather variation, urban design and built environment on objectively measured sedentary behaviour in children. AIMS Public Health. 2016;3(4):663–81. 82. Cullen KW, Liu Y, Thompson D. Diet and physical activity in african-american girls: seasonal differences. Am J Health Behav. 2017 Mar 1;41(2):171–8. 61. Harrison F, van Sluijs EMF, Corder K, Ekelund U, Jones A. References BMC Public Health. 2013 Sep 8;13:808. 71. Wong S, Cantoral A, Téllez-Rojo MM, Pantic I, Oken E, Svensson K, et al. Associations between daily ambient temperature and sedentary time among children 4–6 years old in Mexico City. PLoS One [Internet]. 2020 Oct 30 [cited 2020 Dec 3];15(10). Available from: https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC7598506/ 50. Schepps MA, Shiroma EJ, Kamada M, Harris TB, Lee I-M. Day length is associated with physical activity and sedentary behavior among older women. Sci Rep. 2018;8(1):6602. 72. Dias KI, White J, Jago R, Cardon G, Davey R, Janz KF, et al. International comparison of the levels and potential correlates of objectively measured sedentary time and physical activity among three-to-four-year-old children. Int J Environ Res Public Health. 2019;16:11. 51. Silva P, Santos R, Welk G, Mota J. Seasonal differences in physical activity and sedentary patterns: the relevance of the PA context. J Sports Sci Med. 2011;10(1):66–72. 73. Kharlova I, Deng WH, Mamen J, Mamen A, Fredriksen MV, Fredriksen PM. The weather impact on physical activity of 6–12 year old children: a clustered study of the Health Oriented Pedagogical Project (HOPP). Sports (Basel). 2020;8:1. 52. Chang Z, Wang S, Zhang X. Seasonal variations in physical activity and sedentary behavior among preschool children in a Central China city. Am J Hum Biol. 2020 Feb;25:e23406. 53. Nagy L, Faisal M, Horne M, Collings P, Barber S, Mohammed M. Factors associated with accelerometer measured movement behaviours among White British and south Asian children aged 6–8 years during school terms and school holidays. BMJ Open. 2019;9:e025071. 74. Collings PJ, Dogra SA, Costa S, Bingham DD, Barber SE. Objectively- measured sedentary time and physical activity in a bi-ethnic sample of young children: variation by socio-demographic, temporal and perinatal factors. BMC Public Health. 2020 Jan 28;20(1):109. 54. Aadland E, Andersen LB, Anderssen SA, Resaland GK, Resaland GK. A comparison of 10 accelerometer non-wear time criteria and logbooks in children. BMC Public Health. 2018;18:1–1. 75. King AC, Parkinson KN, Adamson AJ, Murray L, Besson H, Reilly JJ, et al. Correlates of objectively measured physical activity and sedentary behaviour in English children. Eur J Pub Health. 2011 Aug;21(4):424–31. 55. Aebi NJ, Bringolf-Isler B, Schaffner E, Caviezel S, Imboden M, Probst-Hensch N. Patterns of cross-sectional and predictive physical activity in Swiss adults aged 52+: results from the SAPALDIA cohort. Swiss Med Wkly. 2020;150: w20266. 76. Pearce MS, Basterfield L, Mann KD, Parkinson KN, Adamson AJ, Reilly JJ, et al. References The changing relationship between rainfall and children’s physical activity in spring and summer: a longitudinal study. International Journal of Behavioral Nutrition & Physical Activity. 2015;12:1–9. 83. Bringolf-Isler B, Grize L, Mäder U, Ruch N, Sennhauser FH, Braun-Fahrländer C. Assessment of intensity, prevalence and duration of everyday activities in Swiss school children: a cross-sectional analysis of accelerometer and diary data. Intern J Behav Nutr Phys Act. 2009;6:10. 62. Aibar Solana A, Bois JE, Zaragoza J, Bru N, Paillard T, Generelo E. Adolescents’ sedentary behaviors in two European cities. Research Quarterly for Exercise & Sport. 2015;86(3):233–43. 84. Newman MA, Pettee KK, Storti KL, Richardson CR, Kuller LH, Kriska AM. Monthly variation in physical activity levels in postmenopausal women. Med Sci Sports Exerc. 2009 Feb;41(2):322–7. 63. Pechová J, Pelclová J, Dygryn J, Zajac-Gawlak I, Tlucakova L. Sedentary behaviour patterns and spring-autumn seasonality in older central European adults. Journal of Physical Education & Sport. 2019;19(2):1092–8. 85. Barkley SA, Herrmann SD. Seasonal variation of physical Activity in community-living vs. residential-dwelling older adults. Californian Journal of Health Promotion. 2017 Dec;15(3):37–47. 64. Davis MG, Fox KR, Hillsdon M, Sharp DJ, Coulson JC, Thompson JL. Objectively measured physical activity in a diverse sample of older urban UK adults. Med Sci Sports Exerc. 2011;43(4):647–54. 86. Brychta RJ, Arnardottir NY, Johannsson E, Wright EC, Eiriksdottir G, Gudnason V, et al. Influence of day length and physical activity on sleep patterns in older Icelandic men and women. J Clin Sleep Med. 2016;12(2): 203–13. 65. Hagströmer M, Rizzo NS, Sjöström M. Associations of season and region on objectively assessed physical activity and sedentary behaviour. J Sports Sci. 2014;32(7):629–34. 66. Nilsen AKO, Anderssen SA, Ylvisaaker E, Johannessen K, Aadland E. Physical activity among Norwegian preschoolers varies by sex, age, and season. Scand J Med Sci Sports. 2019;29(6):862–73. 87. Carr LJ, Dunsinger S, Marcus BH. Long-term surveillance of physical activity habits of latinas enrolled in a 12-month physical activity intervention. J Phys Act Health. 2016;13(7):740–6. 67. O’Connell SE, Griffiths PL, Clemes SA. Seasonal variation in physical activity, sedentary behaviour and sleep in a sample of UK adults. Ann Hum Biol. 2014;41(1):1–8. 88. Sumukadas D, Witham M, Struthers A, McMurdo M. Day length and weather conditions profoundly affect physical activity levels in older functionally impaired people. J Epidemiol Community Health. 2009 Apr 1;63(4):305. 68. Gracia-Marco L, Ortega FB, Ruiz JR, Williams CA, HagstrÖmer M, Manios Y, et al. References Deng WH, Fredriksen PM. Objectively assessed moderate-to-vigorous physical activity levels among primary school children in Norway: The Health Oriented Pedagogical Project (HOPP). Scand J Public Health. 2018; 46(21_suppl):38–47. 124. Martins RC, Reichert FF, Bielemann RM, Hallal PC. One-year stability of objectively measured physical Activity in young Brazilian adults. J Phys Act Health. 2017;14(3):208–12. 125. Feinglass J, Lee J, Dunlop D, Song J, Semanik P, Chang RW. The effects of daily weather on accelerometer-measured physical activity among adults with arthritis. J Phys Act Health. 2011;8(7):934–43. 103. Bremer E, Graham JD, Bedard C, Rodriguez C, Kriellaars D, Cairney J. The association between PLAYfun and physical activity: a convergent validation study. Res Q Exerc Sport. 2019;16:1–9. 104. Crowley O, Pugliese L, Kachnowski S. The impact of wearable device enabled health initiatives on physical activity and sleep. Cureus. 2016;8(10):e825. 126. Jehn M, Gebhardt A, Liebers U, Kiran B, Scherer D, Endlicher W, et al. Heat stress is associated with reduced health status in pulmonary arterial hypertension: a prospective study cohort. Lung. 2014;192(4):619–24. 105. Kong S, Park HY, Kang D, Lee JK, Lee G, Kwon OJ, et al. Seasonal variation in physical activity among preoperative patients with lung cancer determined using a wearable device. J Clin Med. 2020;27:9(2). 127. Al-Mohannadi AS, Farooq A, Burnett A, Van Der Walt M, Al-Kuwari MG. Impact of climatic conditions on physical Activity: a 2-year cohort study in the Arabian gulf region. J Phys Act Health. 2016;13(9):929–37. 106. McKee DP, Murtagh EM, Boreham CAG, Nevill AM, Murphy MH. Seasonal and annual variation in young children’s physical activity. Med Sci Sports Exerc. 2012;44(7):1318–24. 128. Mitchell DC, Armitage TL, Bennett DH, Schenker MB, Castro J, Tancredi DJ. Physical activity and common tasks of California farm workers: California heat illness prevention study (CHIPS). J Occupational & Environmental Hygiene. 2018;15(12):857–69. 107. Jones GR, Brandon C, Gill DP. Physical activity levels of community-dwelling older adults are influenced by winter weather variables. Arch Gerontol Geriatr. 2017;71:28–33. 129. Bejarano CM, Cushing CC, Crick CJ. Does context predict psychological states and activity? An ecological momentary assessment pilot study of adolescents. Psychology of Sport & Exercise. 2019 Mar;41:146–52. 108. de Vries PR, Janssen M, Spaans E, de Groot I, Janssen A, Smeitink J, et al. Natural variability of daily physical activity measured by accelerometry in children with a mitochondrial disease. Mitochondrion. 2019;47:30–7. 130. Colom A, Ruiz M, Wärnberg J, Compa M, Muncunill J, Barón-López FJ, et al. References UK adults exhibit higher step counts in summer compared to winter months. Ann Hum Biol. 2008 Apr;35(2):154–69. 118. Badland HM, Christian H, Giles-Corti B, Knuiman M. Seasonality in physical activity: should this be a concern in all settings? Health & Place. 2011;17(5): 1084–9. 96. Kimura T, Kobayashi H, Nakayama E, Kakihana W. Seasonality in physical activity and walking of healthy older adults. J Physiol Anthropol [Internet]. 2015 Oct 2 [cited 2020 Mar 4];34. Available from: https://www.ncbi.nlm.nih. gov/pmc/articles/PMC4591564/ 119. Delclòs-Alió X, Marquet O, Vich G, Schipperijn J, Zhang K, Maciejewska M, et al. Temperature and rain moderate the effect of neighborhood walkability on walking time for seniors in Barcelona. Int J Environ Res Public Health. 2019;18:17(1). 97. Mitsui T, Barajima T, Kanachi M, Shimaoka K. Daily walking activity among male office workers in a rural town in northern Japan. J Physiol Anthropol. 2010;29(1):43–6. 120. Wang G, Li B, Zhang X, Niu C, Li J, Li L, et al. No seasonal variation in physical activity of Han Chinese living in Beijing. International Journal of Behavioral Nutrition & Physical Activity. 2017;14:1–10. 98. Sewell L, Singh SJ, Williams JE, Morgan MD. Seasonal variations affect physical activity and pulmonary rehabilitation outcomes. J Cardiopulm Rehabil Prev. 2010 Oct;30(5):329–33. 121. Hoppmann CA. Chak man Lee J, Ziegelmann JP, Graf P, khan KM, Ashe MC. Precipitation and physical Activity in older adults: the moderating role of functional mobility and physical activity intentions. J Gerontol Series B: Psychol Sci Soc Sci. 2017;72(5):792–800. 99. Hopkins ND, Stratton G, Tinken TM, Ridgers ND, Graves LE, McWhannell N, et al. Seasonal reduction in physical activity and flow-mediated dilation in children. Med Sci Sports Exerc. 2011;43(2):232–8. 100. Ridgers ND, Salmon J, Timperio A. Seasonal changes in physical activity during school recess and lunchtime among Australian children. J Sports Sci. 2018;36(13):1508–14. 122. Boutou AK, Raste Y, Demeyer H, Troosters T, Polkey MI, Vogiatzis I, et al. Progression of physical inactivity in COPD patients: the effect of time and climate conditions – a multicenter prospective cohort study. Int J Chron Obstruct Pulmon Dis. 2019 Sep 3;14:1979–92. 101. Ridgers ND, Salmon J, Timperio A. Too hot to move? Objectively assessed seasonal changes in Australian children’s physical activity. Int J Behav Nutr Phys Act. 2015 Jun 19;12(1):77. 123. Cradock AL, Melly SJ, Allen JG, Morris JS, Gortmaker SL. Youth destinations associated with objective measures of physical activity in adolescents. J Adolesc Health. 2009 Sep 2;45(3):S91–8. 102. References Seasonal variation in physical activity and sedentary time in different European regions. The HELENA study. J Sports Sci. 2013;31(16):1831–40. 89. Nakashima D, Kimura D, Watanabe H, Goto F, Kato M, Fujii K, et al. Influence of seasonal variations on physical activity in older people living in mountainous agricultural areas. J Rural Med. 2019 Nov;14(2):165–75. Page 25 of 26 Page 25 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 (2021) 18:24 90. Pelclová J, Walid EA, Vašícková J. Study of day, month and season pedometer-determined variability of physical activity of high school pupils in the Czech Republic. J Sports Sci Med. 2010;9(3):490–8. 113. Prins RG, van Lenthe FJ. The hour-to-hour influence of weather conditions on walking and cycling among Dutch older adults. Age Ageing. 2015;44(5): 886–90. 91. Beighle A, Alderman B, Morgan CF, Le Masurier G. Seasonality in children’s pedometer-measured physical activity levels. Res Q Exerc Sport. 2008 Jun; 79(2):256–60. 114. Witham MD, Donnan PT, Vadiveloo T, Sniehotta FF, Crombie IK, Feng Z, et al. Association of day length and weather conditions with physical activity levels in older community dwelling people. PLoS One. 2014;9(1): e85331. 92. Akande VO, Ruiter RAC, Kremers SPJ. Environmental and motivational determinants of physical activity among Canadian inuit in the arctic. Int J Environ Res Public Health. 2019;09:16(13). 115. Yıldırım M, Schoeni A, Singh AS, Altenburg TM, Brug J, De Bourdeaudhuij I, et al. Daily variations in weather and the relationship with physical activity and sedentary time in European 10- to 12-year-Olds: the ENERGY-project. J Phys Act Health. 2014;11(2):419–25. 93. Clemes SA, Hamilton SL, Griffiths PL. Summer to winter variability in the step counts of normal weight and overweight adults living in the UK. J Phys Act Health. 2011 Jan 1;8(1):36–44. 116. Button BLG, Shah TI, Clark AF, Wilk P, Gilliland JA. Examining weather-related factors on physical activity levels of children from rural communities. Can J Public Health. 2020. 94. Cooper AR, Page AS, Wheeler BW, Hillsdon M, Griew P, Jago R. Patterns of GPS measured time outdoors after school and objective physical activity in English children: the PEACH project. Int J Behav Nutr Phys Act. 2010 Dec; 7(1):1–9. 117. Brandon CA, Gill DP, Speechley M, Gilliland J, Jones GR. Physical activity levels of older community-dwelling adults are influenced by summer weather variables. Applied Physiology, Nutrition & Metabolism. 2009;34(2):182–90. 95. Hamilton SL, Clemes SA, Griffiths PL. References Mediterranean built environment and precipitation as modulator factors on physical activity in obese mid-sge and old-age adults with metabolic syndrome: cross-sectional study. Int J Environ Res Public Health. 2019;16(5):854. 109. Harrison F, Goodman A, van Sluijs EMF, Andersen LB, Cardon G, Davey R, et al. Weather and children’s physical activity; how and why do relationships vary between countries? Int J Behav Nutr Phys Act. 2017;14(1):74. 110. Robbins SM, Jones GR, Birmingham TB, Maly MR. Quantity and quality of physical activity are influenced by outdoor temperature in people with knee osteoarthritis. Physiother Can. 2013;65(3):248–54. 131. Dill J, McNeil N, Broach J, Ma L. Bicycle boulevards and changes in physical activity and active transportation: findings from a natural experiment. Prev Med. 2014;69(Suppl 1):S74–8. 132. Sugino A, Minakata Y, Kanda M, Akamatsu K, Koarai A, Hirano T, et al. Validation of a compact motion sensor for the measurement of physical activity in patients with chronic obstructive pulmonary disease. Respiration. 2012;83(4):300–7. 111. Balish SM, Dechman G, Hernandez P, Spence JC, Rhodes RE, McGannon K, et al. The relationship between weather and objectively measured physical activity among individuals with COPD. J Cardiopulm Rehabil Prev. 2017; 37(6):445–9. 133. Oliver M, Schluter PJ, Schofield GM, Paterson J. Factors related to accelerometer-derived physical activity in Pacific children aged 6 years. Asia Pac J Public Health. 2011 Jan;23(1):44–56. 112. Stabell AC, Wilson M, Jankowski CM, MaWhinney S, Erlandson KM. The impact of a structured, supervised exercise program on daily step count in sedentary older adults with and without HIV. JAIDS. 2020;84(2):228–33. Page 26 of 26 Turrisi et al. International Journal of Behavioral Nutrition and Physical Activity (2021) 18:24 134. Goodman A, Page AS, Cooper AR. International Children’s Accelerometry Database (ICAD) Collaborators. Daylight saving time as a potential public health intervention: an observational study of evening daylight and objectively-measured physical activity among 23,000 children from 9 countries. Int J Behav Nutr Phys Act. 2014;11:84. 135. Goodman A, Paskins J, Mackett R. Day length and weather effects on children’s physical Activity and participation in play, sports, and active travel. J Phys Act Health. 2012;9(8):1105–16. 136. Griew P, Page A, Thomas S, Hillsdon M, Cooper AR. The school effect on children’s school time physical activity: the PEACH project. Prev Med. 2010; 51(3–4):282–6. 137. McMurdo MET, Argo I, Crombie IK, Feng Z, Sniehotta FF, Vadiveloo T, et al. Social, environmental and psychological factors associated with objective physical activity levels in the over 65s. References Vol. 2. Oxford, England: Oxford University Press (OUP); 1989. 21,728. Available from: https://www.oed.com/ 151. National Weather Service. National Weather Service Glossary [Internet]. National Oceanic and Atmospheric Administration’s National Weather Service. 2009 [cited 2020 Jun 5]. Available from: https://w1.weather.gov/ glossary/ References PLoS One. 2012;7(2):e31878. 138. Rosenthal DG, Vittinghoff E, Tison GH, Pletcher MJ, Olgin JE, Grandis DJ, et al. Assessment of accelerometer-based physical activity during the 2017- 2018 California wildfire seasons. JAMA Netw Open. 2020;3(10):e2018116–6. 139. Institute of Medicine (US). Human health and the natural environment [Internet]. Rebuilding the Unity of Health and the Environment: A New Vision of Environmental Health for the 21st Century. National Academies Press (US); 2001. Available from: http://www.ncbi.nlm.nih.gov/books/NBK995 84/. 140. Taylor SE, Repetti RL, Seeman T. Health psychology: what is an unhealthy environment and how does it get under the skin? Annu Rev Psychol. 1997; 48:411–47. 141. Barreto P. de S. Why are we failing to promote physical activity globally? Bull World Health Organ. 2013;91(6):390–390A. 142. Whitfield G, Carlson S, Ussery E, Fulton J, Galuska D, Petersen R. Trends in meeting physical activity guidelines among urban and rural dwelling adults — United States, 2008–2017. MMWR Morb Mortal Wkly Rep. 2019;68:513–8. 143. Martin KR, Koster A, Murphy RA, Van Domelen DR, Hung M, Brychta RJ, et al. Changes in daily activity patterns with age in U.S. men and women: National Health and nutrition examination survey (NHANES) 2003–04 and 2005–06. J Am Geriatr Soc. 2014 Jul 1;62(7):1263–71. 144. Amagasa S, Machida M, Fukushima N, Kikuchi H, Takamiya T, Odagiri Y, et al. Is objectively measured light-intensity physical activity associated with health outcomes after adjustment for moderate-to-vigorous physical activity in adults? A systematic review. Int J Behav Nutr Phys Act. 2018 Dec;15(1):1–13. 145. Conroy DE, Lagoa CM, Hekler E, Rivera DE. Engineering person-specific behavioral interventions to promote physical Activity. Exerc Sport Sci Rev. 2020;48(4):170–9. 146. Xu C, Kohler TA, Lenton TM, Svenning J-C, Scheffer M. Future of the human climate niche. Proc Natl Acad Sci U S A. 2020 May;4:201910114. 147. World Health Organization, editor. Global health risks: mortality and burden of disease attributable to selected major risks. Geneva, Switzerland: World Health Organization; 2009. 62 p. 148. Population estimates and projections [Internet]. DataBank. 2020 [cited 2020 Jun 5]. Available from: https://databank.worldbank.org/source/population- estimates-and-projections 149. Molenaar PCM. On the necessity to use person-specific data analysis approaches in psychology. Eur J Dev Psychol. 2013 Jan 1;10(1):29–39. 149. Molenaar PCM. On the necessity to use person-specific data analysis approaches in psychology. Eur J Dev Psychol. 2013 Jan 1;10(1):29–39. 150. Oxford English Dictionary (OED) [Internet]. Vol. 2. Oxford, England: Oxford University Press (OUP); 1989. 21,728. Available from: https://www.oed.com/ 150. Oxford English Dictionary (OED) [Internet]. 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.
https://openalex.org/W170187608	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1569424/pdf/envhper00452-0326.pdf	English	null	Report on the Consensus Workshop on Formaldehyde	Environmental health perspectives	1,984	public-domain	57,992	REPORT ON THE CONSENSUS WORKSHOP ON FORMALDEHYDE Environmental Health Perspectives Vol. 58, pp. 323-381, 1984 Report on the Consensus Workshop on Formaldehyde* The Consensus Workshop on Formaldehyde consisted of bringing together scientists from academia, government, industry and public interest groups to address some important toxicological questions concerning the health effects of formaldehyde. The participants in the workshop, the Executive Panel which coordinated the meeting, and the questions posed, all were chosen through a broadly based nomination process in order to achieve as comprehensive a consensus as possible. The subcommittees considered the toxicological problems associated with formaldehyde in the areas of exposure, epidemiology, carcinogenicity/histology/genotoxicity, immunology/sensitization/irritation, structure activity/biochemis- try/metabolism, reproduction/teratology, behavior/neurotoxicity/psychology and risk estimation. Some questions considered included the possible human carcinogenicity of formaldehyde, as well as other human health effects, and the interpretation of pathology induced by formaldehyde. These reports, plus introductory material on the procedures used in setting up the Consensus Workshop are presented here. Additionally, there is included a listing of the data base that was made available to the panel chairmen prior to the meeting and was readily accessible to the participants during their deliberations in the meeting. This data base, since it was computerized, was also capable of being searched for important terms. These materials were supplemented by information brought by the panelists. y p g p p p Additionally, there is included a listing of the data base that was made available to the panel chairmen prior to the meeting and was readily accessible to the participants during their deliberations in the meeting. This data base, since it was computerized, was also capable of being searched for important terms. These materials were supplemented by information brought by the panelists. pp y g y p The workshop has defined the consensus concerning a number of major points in formaldehyde toxicology and has identified a number of major deficits in understanding which are important guides to future research. Introduction Center for Toxicological Research (NCTR) to conduct a Consensus Workshop on Formaldehyde. This workshop is part of the NCTR Consensus Workshop Series, which continues the commitment of addressing today's impor- tant and often controversial toxicological issues. This series is an evolving process where scientists from academia, government, industry, and public interest groups can meet and resolve scientific questions on the basis of the best science currently available. Since this is a relatively new approach to toxicological questions, the "workings" of the workshop will be explained in some detail in an effort to give a clear understanding of the process and, hopefully, provide some insight into how future workshops can utilize the experiences of this one to optimize their chances for success. Background At the request of the White House Office on Science and Technology Policy (OSTP), the Environmental Protection Agency (EPA) has worked with the National * Executive Panelists (the workshop's executive panel, whose members provided direction to the Consensus Workshop on Formalde- hyde and coordinated this report): Michael Gough (Office of Technol- ogy Assessment, Congress of the United States, Washington, DC 20510); Ronald Hart (Consensus Workshop Chairman, National Center for Toxicological Research, Building 13, Room 123c, Jefferson, AR 72079); Bruce W Karrh (E.I. du Pont de Nemours & Co., 11400 Nemours Building, Wilmington, DE 19898); Adalbert Koestner (Department of Pathology, Michigan State University, A-622 East Fee Hall, East Lansing, MI 48824); Robert Neal (Chemical Industry Institute of Tbxicology, P 0. Box 12137, Research Tiangle Park, NC 27709); David Parkinson (University ofPittsburgh, School ofMedicine, Department of Occupational and Environmental Health, Pittsburgh, PA 15213); Frederica Perera (Natural Resources Defense Council, 122 East 42 St., New York, NY 10168) (present address: Columbia University School of Public Health, Division of Environmental Health Sciences, 60 Haven Ave., New York, NY 10032); Kenneth E. Powell (Centers for Disease Control, 1600 Clifton Rd., Atlanta, GA 30333); Herbert S. Rosenkranz (Center for the Environmental Health Sciences, Case Western Reserve University, School of Medicine, Cleveland, OH 44106). The names of the authors are in alphabetical order. These affiliations are for identification only and should not be in any way construed as representing the official opinion or position of any organization. Document editors were Ronald W Hart, Angelo Terturro and Lorraine Neimeth; William F. McCallum was Workshop Executive Secretary. * Executive Panelists (the workshop's executive panel, whose members provided direction to the Consensus Workshop on Formalde- hyde and coordinated this report): Michael Gough (Office of Technol- ogy Assessment, Congress of the United States, Washington, DC 20510); Ronald Hart (Consensus Workshop Chairman, National Center for Toxicological Research, Building 13, Room 123c, Jefferson, AR 72079); Bruce W Karrh (E.I. du Pont de Nemours & Co., 11400 Nemours Building, Wilmington, DE 19898); Adalbert Koestner (Department of Pathology, Michigan State University, A-622 East Fee Hall, East Lansing, MI 48824); Robert Neal (Chemical Industry Institute of Tbxicology, P 0. Box 12137, Research Tiangle Park, NC 27709); David Parkinson (University ofPittsburgh, School ofMedicine, Department of Occupational and Environmental Health, Pittsburgh, PA 15213); Frederica Perera (Natural Resources Defense Council, 122 East 42 St., New York, NY 10168) (present address: Columbia University School of Public Health, Division of Environmental Health Sciences, 60 Haven Ave., New York, NY 10032); Kenneth E. Powell (Centers for Disease Control, 1600 Clifton Rd., Atlanta, GA 30333); Herbert S. Rosenkranz (Center for the Environmental Health Sciences, Case Western Reserve University, School of Medicine, Cleveland, OH 44106). The names of the authors are in alphabetical order. These affiliations are for identification only and should not be in any way construed as representing the official opinion or position of any organization. Document editors were Ronald W Hart, Angelo Terturro and Lorraine Neimeth; William F. McCallum was Workshop Executive Secretary. Workshop Foci (2) Reaching consensus does not mean formal voting. Instead, the chairman was to guide the discussion in an effort to get agreement of all members of the panel on each topic of discussion-asking at the end whether there is a consensus. (2) Reaching consensus does not mean formal voting. Instead, the chairman was to guide the discussion in an effort to get agreement of all members of the panel on each topic of discussion-asking at the end whether there is a consensus. Recent scientific studies have heightened concern about the possible human health effects of formalde- hyde. The data relating to some of these health effects have been reviewed by governmental, independent and international panels of scientists. These reviews of the formaldehyde data have resulted in regulatory actions or proposals by several federal agencies; however, con- troversy remains over a number of scientific issues. At the request of the Regulatory Work Group on Science and Technology, White House Office of Science and Technology Policy, the National Center for Toxicological Research (NCTR) convened a scientific consensus work- shop on formaldehyde to discuss and seek general agreement on the existing scientific data and identify future research needs. (3) The alternate chairman/secretary was to take notes during the panel meetings. The chairman was to have the responsibility for submission of the panel draft reports in time for typing and prior to the Wednesday morning session. g (4) The draft report from each working group was to be reviewed by each member of that panel. Ideally, each panel's report was to be drafted and reviewed by every member before leaving the workshop. If this was not accomplished, a draft was to be sent to each panel member for comment. If a second draft were to be necessary, the same procedures would be followed. The Consensus Workshop on Formaldehyde met and discussed in an objective and nonadversarial fashion the relevant scientific evidence on formaldehyde in the general areas of epidemiology, exposure, toxicology and risk estimation. The endpoints discussed for each of the above general areas could include, but were not re- stricted to: carcinogenicity, genotoxicity, irritation, re- productive effects/teratology, behavioral effects, im- munotoxicology/sensitization, neurotoxicity, biochemi- cal effects/metabolism and histopathological effects. The emphasis of the workshop was to develop consensus on the scientific issues concerning formaldehyde; however, the results may also be used to assist in future regula- tory decision making. CONSENSUS WORKSHOP ON FORMALDEHYDE 324 form of questions, which focused the discussion; (3) selecting the experts in the various fields to comprise the section panels; and (4) choosing the section chair- man and alternate chairman of each panel. the workshop and the process followed in selecting topics and participants as well as in discussing topics. The conclusions of each working group were to speak for themselves and not require interpretation, summary or introduction. p Three months before the meeting, the chairman of the Executive Panel notified the chairmen of the various sections that a compilation of all current information on formaldehyde, both published and unpublished litera- ture, was available. The compilation was able to be searched by keywords, permitting the massive amount of material to be amenable to analysis. This data base (Appendix I) was also available at the Consensus Workshop, as were copies of whatever reprints were required by the panel members. (7) In the final document, a disclaimer was to be cited that the statements contained therein should in no way be construed as representing the official opinion or position of any agency, organization or institution to which participants in the workshop belong. (7) In the final document, a disclaimer was to be cited that the statements contained therein should in no way be construed as representing the official opinion or position of any agency, organization or institution to which participants in the workshop belong. p p p g (8) The findings of the Risk Estimation Panel were to be circulated to all panelists for evaluation and comment, with a turnaround time not to exceed three weeks. The Risk Estimation Panel was then to have the responsibil- ity for final drafting of their report based on their deliberation and the comments from the other panelists. Workshop Ground Rules p (9) During the deliberation of the Risk Assessment Panel, the chairperson or designate was to report only the findings of the panel as a whole. If a panel chairman or designate were to have a personal comment, he/she could notify the chairman of the Risk Assessment Panel in writing as was the case for any other member of the panel and the audience. p (9) During the deliberation of the Risk Assessment Panel, the chairperson or designate was to report only the findings of the panel as a whole. If a panel chairman or designate were to have a personal comment, he/she could notify the chairman of the Risk Assessment Panel in writing as was the case for any other member of the panel and the audience. Before the meeting, the chairman of the Executive Panel and the section chairmen discussed the role of the section chairmen and what was expected in terms of the report from each section. Ground rules established by the Executive Panel for conducting the workshop were as follows. (1) Each panel's report was to focus on those areas where consensus was reached on an issue by the panel, including the major considerations and factors in reach- ing agreement. Other general ideas, as well as special issues where consensus was not reached, were to be listed and the scientific basis preventing consensus stated. p (10) The Risk Assessment Panel report was to be bound by the consensus findings of the panels. Valid scientific disagreements within the panel were to be treated as stated in item (1) above. History of this Consensus Workshop Upon the determination of OSTP that it would be useful for a Consensus Workshop to convene and attempt to resolve some important controversies con- cerning the scientific status of formaldehyde,the EPA and NCTR entered into an Interagency Agreement, which determined that an Executive Panel, drawn from academia, government, industry and public interest groups, would be appointed and provide direction to the workshop. This direction consisted of: (1) defining the format of the workshop, which eventually consisted of eight separate panels; (2) determining the topics, in the CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 325 d. What is the size and composition of populations exposed to various ranges of concentrations of formaldehyde by various routes? Because of the wide variety of information available within a topic, the nature of the area addressed and difference in the scope and depth of the questions, the different panels chose to present the answers differently. Some panels gave answers directly, others decided to present the response in a narrative form. To preserve the nuances and subtleties of the different panels of experts, there has been no editing of the panel reports, other than that required for journal publication. This led to some significant differences in the style, pacing and flow of the narrative. * y y Topic 2. What data that are currently lacking would be most important in resolving controversies about exposure? TIpic 1: Sources, Modes and Levels of Exposure la. Many methods have been used for measuring formaldehyde levels in air and other media. A selection of these methods has been critically reviewed by Balmat (1) for the Formaldehyde Institute. The information in Table 1 has been extracted and condensed from this review to provide a concise summary of the 19 methods that provide the basis for the monitoring data reviewed by the panel. To facilitate comparisons, columns 4 and 5 in Table 1 provide estimates of the sensitivity of each method for short-term (15-min) and long-term (usually 8-hr) sampling (devices intended for sampling over periods of several days may achieve higher sensitivities than those listed in Table 1). These reported sensitivi- ties do not necessarily represent values measured using standard test atmospheric concentrations, but are calcu- lated from reported limits of quantitation, assuming recommended sampling rates. The last two columns list the main advantages and disadvantages of each method, including known interferences, classified as positive ( + ) or negative (-) where appropriate. Criteria for precision and accuracy of each method have been established (1). However, these criteria are valid only when the methods are used according to their written procedures, and deviations from these procedures in sample collection or analytical procedures can greatly influence the precision and accuracy of any procedure. Workshop Orientation Each panel specifically concentrated on the "science" and the scientific questions of numerous issues concern- ing formaldehyde and deliberately avoided philosophical, policy and/or regulatory aspects. Despite the organiza- tional difficulties in obtaining a "balanced input" from divergent scientific viewpoints, the workshop demon- strated that, in considering science issues with give-and- take discussion, many of the differences have been significantly narrowed and consensus reached on many important toxicological issues. It is hoped that this workshop has been instrumental in updating and synthe- sizing the scientific data on formaldehyde and strength- ening the factual basis for environmental health risk assessment of this substance. Each panel prepared and reviewed a report from its deliberations, and the chairmen of each panel presented the reports to the workshop in plenary session. The panel reports follow in their entirety. Because of the density of information, complexities, and variances of the panel reports, the Executive Panel decided not to attempt further summarization. y p Evaluation of the reliability of monitoring data re- quires critical assessment of the methodology used for sampling and analysis and of the interfering compounds likely to be present. As shown in Table 1, each method has advantages and disadvantages. Choice ofan inappro- priate method, improper conduct of the sampling or analysis, or the presence of interferences may lead to substantial positive or negative biases. Workshop Foci y p (5) Written comments or requests to present in- formation/data from the floor were to be received by the panel chairman for consideration and inclusion in the panel deliberation. No other comments were to be accepted. (6) Executive Panel members were not to revise or edit the working group reports. Their duties were: (a) to read each panel's report and to identify either areas requiring clarification or inconsistencies between vari- ous panels' findings so that the chairmen of the relevant panels could attempt to correct these problems and (b) to write a short introduction explaining the purpose of CONSENSUS WORKSHOP ON FORMALDEHYDE * Italic number/letter designations (e.g., 2a) refer to the specific topic areas; the same number is used more than once if the discussion again becomes relevant to a particular topic. i i d i * Italic number/letter designations (e.g., 2a) refer to the specific topic areas; the same number is used more than once if the discussion again becomes relevant to a particular topic. t Exposure Panel: Ian Nisbet (Chairman); Bernd Seifert (Alternate Chairman/Secretary); Jean L. Balmat; John M. Fajen; Richard Gammage; Eugene R. Kennedy; Michael Silverstein. g p p t Exposure Panel: Ian Nisbet (Chairman); Bernd Seifert (Alternate Chairman/Secretary); Jean L. Balmat; John M. Fajen; Richard Gammage; Eugene R. Kennedy; Michael Silverstein. Exposure Panel Reportt Topic 1. What are the sources, modes and levels of exposure in various segments of the population? p g a. What are the available monitoring (collection and analytical) methods and what are the reliability, accuracy, sensitivity, specificity, comparability and limitations of these methods? a. What are the available monitoring (collection and analytical) methods and what are the reliability, accuracy, sensitivity, specificity, comparability and limitations of these methods? p g In the judgment of the panel, adequate methods exist for both short-term and long-term monitoring of formal- dehyde concentrations in a variety of conditions, includ- ing occupational and residential situations. A number of these methods have been standardized and evaluated under laboratory conditions. However, panel members expressed concern about several aspects of published formaldehyde studies: (a) some reports included inade- quate or no documentation of the methods used; (b) some studies involved inappropriate choice or improper modification of methods; (c) some methods (e.g., pas- sive monitors) are coming into widespread use without complete evaluation under field conditions. For this reason, some studies probably have achieved substan- tially less precision and accuracy than claimed for the b. What is known about the levels and sources of exposure in residences, public buildings, occupa- tional areas, outdoor air, water, soil, consumer products and medical procedures? How do these exposures vary in duration, concentration and frequency? b. What is known about the levels and sources of exposure in residences, public buildings, occupa- tional areas, outdoor air, water, soil, consumer products and medical procedures? How do these exposures vary in duration, concentration and frequency? c. What factors (environmental and physiological) affect exposure in man and experimental animals? CONSENSUS WORKSHOP ON FORMALDEHYDE 326 Table 1. Exposure Panel Reportt Sampling and analytical methods for formaldehyde.a Analytical technique Sensitivity, ppm Method designation Sampling Analysis 15 min Long-term Advantages Disadvantages Chromotropic acid, Midget impinger P&CAM 125 Paraosaniline, original Midget impinger Pararosaniline, modified Pararosaniline, TGM-555 MBTH Acetylacetone, spectrophotometric Acetylacetone, fluorimetric 2,4-DNPH, aqueous ethanol 2,4-DNPH coated adsorbent P&CAM 318 NIOSH S327 OSHA, acidic hydrazine P&CAM 354 MIRAN Draeger Passive monitor 3 M DuPont Air Quality Research Envirotech Midget impinger Continuous Absorber Midget impinger Midget impinger Midget impinger Adsorbent tube Reactive adsorbent Midget impinger Midget impinger Reactive adsorbent Continuous Reactive adsorbent Reactive adsorbent Reactive adsorbent Reactive adsorbent Moist adsorbent Spectrophotometry Spectrophotometry 0.16 0.02 Spectrophotometry 0.045 Colorimetric 0.05 Spectrophotometry 0.10 Spectrophotometry 0.10 Fluorimetry HPLC 0.04 0.04 (1 hr) 0.0005 (8 hr) Sensitive and selective, simple technique High sensitivity, simple technique 0.001 High sensitivity, (8 hr) simple technique, uses no Hg compound NA High sensitivity, near real-time monitoring 0.003 (8 hr) Simple, good sample stability Mild pH, sensitive and simple technique Mild pH, sensitive and simple technique 0.00006 0.000015 (1 hr) 1.32 0.10 (3 hr) HPLC Ion chromatography 0.8 Polarography 1.62 Polarography 0. a(CA)-chromotropic acid; (PUR)-Purpald. Exposure Panel Reportt Table 3 summarizes data from six studies of formaldehyde levels in residences in different parts of the United States, Canada and the United Kingdom. Although there is some question about the randomness and representativeness of some of the samples, the panel judges that the mean levels from the large-scale studies whose results are tabulated in Table 3 provide reasonable estimates of the long-term average concentrations offormaldehyde in various broad categories of housing types. g g yp In conventional homes more than about five years old, mean concentrations of formaldehyde are usually below 0.05 parts per million (ppm), and only a small fraction exceeds 0.1 ppm (the American Society of Heating, Refrigeration and Air-Conditioning Engineers [ASHRAE] ceiling guideline for comfort and the stand- ard established in several foreign countries). In special- ized residences (mobile homes, UFFI homes, new houses, energy-efficient and perhaps weatherized homes), mean levels of formaldehyde are significantly higher and not infrequently exceed 0.1 ppm. Mean levels appear to be highest in mobile homes (Table 3). Levels of formaldehyde are highest in new residences and decline steadily as the rates of emission from insulation or building materials decline. The first half- life is 4 to 5 years for mobile homes and for new houses (39) where pressed-wood products are the primary source of formaldehyde. For UFFI homes the first half-life is usually less than 1 year (41,42). In conventional homes more than about five years old, mean concentrations of formaldehyde are usually below 0.05 parts per million (ppm), and only a small fraction exceeds 0.1 ppm (the American Society of Heating, Refrigeration and Air-Conditioning Engineers [ASHRAE] ceiling guideline for comfort and the stand- ard established in several foreign countries). In special- ized residences (mobile homes, UFFI homes, new houses, energy-efficient and perhaps weatherized homes), mean levels of formaldehyde are significantly higher and not infrequently exceed 0.1 ppm. Mean levels appear to be highest in mobile homes (Table 3). Table 2 summarizes the remaining data available to the panel. Many of these data have previously been compiled by the U.S. Environmental Protection Agency (6) and Clement Associates (7). We have added results from recent studies by the National Institute for Occupational Safety and Health (NIOSH) (8) and sev- eral additional published reports. The compilation by Hattis et al. (9) was not available to the panel. Exposure Panel Reportt industry. published data are available for only 34 job categories in 29 industries and occupations. For no job category are the data sufficient to characterize temporal or within- plant variability, or to characterize exposure in more than a small handful of plants. Hence, the panel concludes that the available data, although useful in showing the range of exposures encountered in some workplaces, are not sufficiently systematic or represen- tative to characterize the entire spectrum of exposures in U.S. industry. p g Methods for biological monitoring of exposure to formaldehyde by measuring increases in concentrations of formate in urine have been proposed (2-4). However, natural variability in formate levels is too high for these methods to be useful except in populations exposed to ambient formaldehyde levels greater than 1 ppm. y In addition to the data summarized in Table 2, the SOCMA (5) survey suggested that substantial exposure to formaldehyde also occurs in several other industries, including industrial and specialty chemicals and manu- facture of hardwood plywood, particleboard and abra- sive products. In addition, both the SOCMA survey and the National Occupational Hazard Survey (35) identified a number of other industries with potential formalde- hyde exposures, for which the panel has not encoun- tered any monitoring data. y g pp Ib. Occupational Exposure. The panel reviewed four surveys and compilations of data on occupational exposure to formaldehyde as well as a number of primary studies. The largest single source of data is a study conducted for the Synthetic Organic Chemical Manufacturers' Association (SOCMA) (5), in which 50 industries with the potential for significant worker exposure to formaldehyde were identified and 17 indus- tries were surveyed. Company monitoring data were tabulated for 10 industries and estimates were made of the frequency and range of exposures in all 17 indus- tries. However, the utility of this information is severely limited since only 89 of the 3365 companies to which questionnaires were sent provided monitoring data, and the report did not specify the methods of analysis or the circumstances in which the data were collected. For these reasons, the panel felt that these data could not be scientifically evaluated. The panel also excluded from its review several other studies for which methods of analysis or other critical pieces of information were omitted. y g lb. Indoor Exposure. Exposure Panel Reportt Gas chromatography 7.32 Infrared Visual Sensitive, specific, good collection efficiency Specific, solid adsorbent 0.025 Specific, solid (8 hr) sorbent 2 0.27 Few steps in procedure, (1.5 hr) stable sample 0 0.01 (2.5 hr) 0.5 (4 hr) Sensitive Selective, sorbent tube, stable sample 0.4 NA Real-time analysis 0.5 NA Real-time analysis Spectrophotometry 3.2 (CA) Spectrophotometry 8 (CA) Spectrophotometry 6.7 (CA) Spectrophotometry 0.72 (PUR) 0.1 (8 hr) 0.25 (8 hr) 0.21 (8 hr) 0.06 (3 hr) Ease of sampling Ease of sampling, simple analytical procedure Ease of sampling Ease of sampling Interferences: phenol (-), other organics (-) Interferences: SO2 (+), uses toxic materials, uses double impingers, poor sample stability Interference: SO2 (+), uses double impingers, poor sample stability, results are temperature dependent Interference: SO2 (+), uses toxic materials, time consuming and involved maintenance Poor specificity Interferences: other aldehydes (+), amines (-), SO2 (-), unstable chromogen Interferences: other aldehydes (+), amines (-), SO2 (+), unstable chromogen High level of analytical sophistication High level of analytical sophistication, user must prepare tubes, short shelf-life, variable blank level, poor sensitivity High level of analytical sophistication, poor sample stability, inter- ferences: formic acid (+), variable recovery High level of analytical sophistication, poor sensitivity, interference: other aldehydes (+) High level of analytical sophistication, inter- ference: acetaldehyde (+), high blanks High level of analytical sophistication, poor sensitivity Subject to multiple interferences ( + ) Questionable accuracy and precision Sensitive to humidity and face velocity Poor sensitivity, variable blanks Limited shelf life, results sensitive to humidity Results not easily interpreted, color reagent unstable, turbidity correction, interferences: other aldehydes (+) Table 1. Sampling and analytical methods for formaldehyde.a Analytical technique a(CA)-chromotropic acid; (PUR)-Purpald. CONSENSUS WORKSHOP ON FORMALDEHYDE 327 methods. In general, negative biases (due to interfer- ences and sample instability) are likely to be more frequent than positive biases. However, in the panel's judgment, these errors are probably less serious than errors introduced by the inadequacies and biases in sampling that are discussed below. published data are available for only 34 job categories in 29 industries and occupations. For no job category are the data sufficient to characterize temporal or within- plant variability, or to characterize exposure in more than a small handful of plants. Hence, the panel concludes that the available data, although useful in showing the range of exposures encountered in some workplaces, are not sufficiently systematic or represen- tative to characterize the entire spectrum of exposures in U.S. Exposure Panel Reportt p The panel regards the ambient monitoring data sum- marized in Table 2 as reasonably reliable measures of the concentrations of formaldehyde in the workplaces that were sampled, except for measurements made by the charcoal tube (CT) method, which are especially subject to negative bias. Thus, at least at the time of sampling, the data indicate substantial exposure of workers to formaldehyde in several industries. Speci- fically, sample means of 1 ppm or more have been reported in the following industries and occupations: formaldehyde production; resin and plastic materials production; apparel manufacture; plywood particle board and wood furniture manufacture; paper and paperboard manufacture; urea-formaldehyde foam insulation (UFFI) dealers and installers; mushroom farms; funeral services; and pathological and biology laboratories. High concentrations of formaldehyde have also been reported in individual samples from iron foundries and plastic molding facilities. However, the panel notes that Levels of formaldehyde are highest in new residences and decline steadily as the rates of emission from insulation or building materials decline. The first half- life is 4 to 5 years for mobile homes and for new houses (39) where pressed-wood products are the primary source of formaldehyde. For UFFI homes the first half-life is usually less than 1 year (41,42). Although mean formaldehyde levels in different classes of residences are reasonably well established, temporal patterns of fluctuation are poorly character- ized and not well understood. The cross-sectional stud- ies in Table 3 were not designed to investigate patterns of variability, and the ranges cited there reflect both between-house differences and within-house variations. Limited studies of within-house fluctuations (43,44) have revealed both diurnal variations (up to twofold) and seasonal variables (up to tenfold in some homes). Highest levels occur in the summer and in the heat of CONSENSUS WORKSHOP ON FORMALDEHYDE 328 Table 2. Formaldehyde monitoring data in occupational settings. Sampling methods Exposure levels, ppm Area or No. Duration/ Industry Job Range Mean Median pers. obs. Period Methodsa frequency Ref. Exposure Panel Reportt Comments Formaldehyde Production - 1.4 - P 4 hr TWA CT,IC (-0)b production operators (SIC 2869) Lab tech Resin and Production plastic operators materials production Resin plant (SIC 2821) Resin plant UF resin production (2 plants) 1.31 1.39 0.05-0.37 0.24 0.09-0.17 0.13 0.12-0.55 - 0.18-5.4 - 0.2-0.74 - 0.6-0.34 - UF resin 0.12-5.4 0.90 production 0.20-0.74 0.39 0.06-0.34 0.19 p p A 8 A 2 A A A - A p 18 p 5 p 5 - 4 hr TWA CT,IC 4hrTWA CT,IC 405 min BI,CT GC BI,CO Intermittent SS,IC SS,IC SS,IC SS,IC BI,CA BI,CA BI,CA (10)b (10)b (11)b (12)b Manufacture glue for plywood, samples taken over glue vat (13) Resin production Resin drumming Drum washing Foam agent blending (14)b Cooks Drum washer Foaming agent blender Textiles 0.04-0.73 0.31 warehouse 0.08-0.51 0.25 Textile < 0.1-1.3 - facilities < 0.1-1.4 - Textile 0.11-1.33 0.69 manufacture 0.15-1.2 0.53 A,P 11 A,P 11 0.8 A,P 28 0.7 A,P 15 0.64 P 6 0.45 A 13 CT,SP BI, SP - +3 hr Grab 8 hr/day Apparel Permanent (SIC 23-) press Permanent press Warehouse Sewing machine operators Clothing pressers 0.15-0.38 0.31 0-2.7 0.74 0.11-0.57 0.39 0.04-0.19 0.12 0.51-0.91 0.72 0.3-1.8 1.2 0.005-0.95 0.07 - A 9 A 32 - 0.37 P 13 30-45 min 0.15 A 9 164-341 min 0.71 P 16 7-8 hr 1.2 P 41 30 min 0.054 P 40 3-4 hr BI,I BI,I 8 hr/day 8 hr/day (18)" Early date; area samples taken in workroom (19)" Early date; area samples taken in cutting, pressing, sewing, and storage areas (15) (15) 8 hr/day (20) 1-2.5 A (16)b Wood Particle- furniture board manufacture veneering (SIC 2511, 2512, 2521, 2531, 2541) Plastic Injection molders mold (SIC 3079) operators 0.008-0.25 0.12 0.9-6.4 2.75 0.2-0.55 0.40 0.2-2.5 0.70 0.01-0.1 0.037 - A 11 A A 9 A 13 - BI,CA BI,CA BI,CA BI,CA P 9 100-350 CA min (13) Plant Veneer press Veneer core press Core, veneer, adhesives (21) Breathing zone samples Textile finishing (SIC 226-) (15)b (16)b (17) All workers Plywood particle- board (SIC 243-) Table 2. Formaldehyde monitoring data in occupational settings. CONSENSUS WORKSHOP ON FORMALDEHYDE 329 Table 2. (continued) Sampling methods Exposure levels, ppm Area or No. Duration/ Industry Job Range Mean Median pers. obs. Period Methodsa frequency Ref. Exposure Panel Reportt Comments Plastic Area 0.01-0.53 0.20 A 8 55-425 CA - (21) Area samples molders samples (sic 3079) Operators (cont'd) < 2 Near 2-4 grinder hopper Sand mold 0.1-0.7 production ND-1.1 Paper and Paper paperboard treatment (SIC 26-) resin im- pregnated) Ireated paper products Coating preparation <2 <2 p 28 min 3 3 A 3 DT DT 0.31 0.2 P 28 4 hr 0.17 0.1 A 29 4 hr 0.04-0.16 0.08 p 0.03-0.07 0.06 A 0.01-0.23 0.05 - P 0.02-0.28 0.05 P 0.14-0.99 - 0.59 A 0.14-0.90 - 0.34 P < 0.01-3 0.8-0.42 1 0.51 0.01 A 7 0.42 A 4 8 hr/day (22) 15 4 hr BI,CT 8 hr/day (11) CA 7 4 hr BI,CT 8 hr/day CA 30 4 hr BI,CT 8 hr/day CA 10 4 hr BI,CT 8 hr/day CA 64 ( 6)b 37 - Grab - ca. 30 sec (23) 20 times/day 30-200 min - (21) Breathing zone samples; limit of detection is 2 ppm (21) Wear machines processing acetal resins Filter treating of paper Filter treating of paper Series B collating Press build-up By mix and storage tanks; data indicated no exposure in work area where majority of work time spent Foundries Bronze steel, foundry, iron and core machine nonferrous operators (SIC 332-, 336-) Rubber hose production (SIC 3069) Asphalt shingle production (SIC 2952) 0.24-0.80 0.53 0.12-0.69 0.39 Iron foundry, < 0.02-18.3 cpre machine operators Iron foundry, 0.07-0.33 0.16 core machine operators Molding 0.03-0.13 0.09 0.07-0.78 0.21 ND-0.04 0.04 Producers 0.03-0.07 0.05 0.55 P 4 3-4 hr BI,CA 8 hr/day 0.39 A 11 ca. 2 hr BI,CA - 0.43 P 14 p 3 P 6 A 6 p 10 0.05 A 2 BI,CA 8 hr/day BI,CO BI,CO BI,CO BI,CO (24) (16)b (25) Complete core production cycle took 1 hr (26) Molding for products to measure temperature, PF resins (27)b Formaldehyde used as a preservative (28)b Area samples near process machinery Fiberglass Installers insulation installation (SIC 3296) 0.007-0.033 0.023 (TWA) 0.019 P 13 8 hr/day (29) Rapid worker turnover rate reported, with most employees Table 2. (continued) CONSENSUS WORKSHOP ON FORMALDEHYDE 331 Table 2. (continued) Sampling methods Exposure levels, ppm Area or No. Duration/ Industry Job Range Mean Median pers. obs. Period Methodsa frequency Ref. Comments Hospital 2.2-2.3 2.25 P 2 BI - (8) lab 1.9 P 1 CT (SIC 8071) 2.0-2.0 2.0 A 2 CT Government 2.4 P 1 CT - (8) lab 0.8 A 1 CT (SIC 8071) Dialysis ND-0.90 0.42 A 9 - CT (8) unit 0.27-0.63 0.41 P 5 CT (SIC 8081) 0.04-0.50 0.51b A CEA Animal < 0.38-1.04 - P 15 CA dissecting 0.05-0.40 0.15 A 6 BI lab 0.11-0.29 0.18b A 3 CEA (SIC 8071) Garment < 0.14-0.63 0.23-0.33 P 40 CT (8) manufactur- < 0.03-0.40 0.19-0.26 A 43 - CT ing (3 plants) 0.03-0.40 0.21 A 43 BI (SIC 2321) 0.05-1.12 0.46 A 42 CEA Chemical 0.04-1.6 0.55 P 3 BI (8) manufac- 0.03-0.43 0.17 A 5 BI turing Glass 0.42 0.42 P 1 CT (8) manufac- 0.45-0.64 0.54 A 2 CT turing (SIC 3211) Hospital 0.37-0.73 0.55 - A 2 BI (8) (SIC 8062) Paraformal- < 0.25-0.85b0.56b P 10 CA (8) dehyde 0.28-3.40 1.17"b A 8 CEA packaging (SIC 2879) Offices 0.02-0.12 0.06 - A 39 BI (8) (3 locations) < 0.04 < 0.04 A 9 CT (SIC 8111) Autopsy Resident 1.58c P 10 CA (4) rooms (SIC 8071) Pathologist 1.24c P 10 Ca Technician 0.57c p 9 CA Assistants 0.16c P 2 CA 0.13-13.57 0.72 A 23 CA aAbbreviations for analytical procedures: AA = acetylacetone procedure; BI = bisulfite impingers; CA = chromotropic acid procedure; CL = chemiluminescence procedure; CO = colorimetric analysis; CT = charcoal tubes; SS = solid sorbents; DT = Draeger tubes; FS = Fourier transform spectrometer; GC = gas chromatography; IC = ion chromatography; MB = MBTH procedure; SP = spectrophotometric procedure; CEA = CEA instruments Model 555. bAs reported by USEPA (6). cAverage. Table 2. (continued) Sampling methods aAbbreviations for analytical procedures: AA = acetylacetone procedure; BI = bisulfite impingers; CA = chromotropic acid procedure; CL = chemiluminescence procedure; CO = colorimetric analysis; CT = charcoal tubes; SS = solid sorbents; DT = Draeger tubes; FS = Fourier transform spectrometer; GC = gas chromatography; IC = ion chromatography; MB = MBTH procedure; SP = spectrophotometric procedure; CEA = CEA instruments Model 555. bAs reported by USEPA (6). cAverage. the day; doubling of formaldehyde levels has been observed with temperature increments of3-80C (39,45). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 330 Table 2. (continued) Industry Job Urea-formal- See dehyde comments foam insulation dealers and installers Suburban shopping center insulated with UF foam Fertilizer manufactur- ers (SIC 2873) Exposure levels, ppm Area or No. Range Mean Median pers. obs. Period 0.07-2.0 - - 0.8-1.6 1.05 0.3-3.1 1.44 < 0.5-3.0 1.56 0.2-1.9 0.9 A 36 A 30 A 16 -P,A 11 Sampling methods Duration/ Methodsa frequency Ref. Comments IC (13) Application of UF foam to com- mercial and residential buildings; ex- posures for applicators, laborers, trucking, and warehouse area BI,CA (16)" Area samples in CT,IC stores DT (16)" For production of time-released nitrogen fertilizers (urea forms), release formaldehyde as they release nitrogen < 0.51-10 + 2.68 ND-2.70 ND-4.93 A 12 DT Intermittent (30)b Maximum value P 3 Short-term CT,IC Intermittent was sample - A 3 CT,IC Intermittent taken at source; formaldehyde used as fumigant/ disinfectant Funeral Embalmers 0.09-5.26 0.74 homes (SIC 7261) Embalmers 0.20-3.99 1.1 1.30-3.93 2.7 A 187 0.54 A,P 8 70-190 min CA (31) Wayne State University study of 6 funeral homes; mean concen- tration increased to 1.34 ppm when ventila- tion off (32)b Area and personal samples; ven- tilation working Area and personal samples; ventilation inoperative CT 2.49 A,P 5 300 min CT Pathologists Autopsy (SIC 8071) room Autopsy room Biology Biology instructors lab (SIC 8071) 0.06-7.9 4.8 2.20-7.9 4.35 2.75-14.8 8.3 A 10 A 6 A 8 BI,CA Intermittent (33)b Intermittent (16)b BI,CA Intermittent (6) Minimum concen- trations with doors and windows open; maximum con- i i h Mushroom farm (SIC 0721) Table 2. (continued) CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE In an intensive study, over a period of 1 year, of 40 homes, more than half of which were less than 5 years old, a level of 0.1 ppm was exceeded in more than half the homes during at least one 24-hr period (39). However, the panel judged the available data inade- quate to characterize the frequency or magnitude of short-term peak (acute) exposures of various groups within the population. the day; doubling of formaldehyde levels has been observed with temperature increments of3-80C (39,45). In an intensive study, over a period of 1 year, of 40 homes, more than half of which were less than 5 years old, a level of 0.1 ppm was exceeded in more than half the homes during at least one 24-hr period (39). However, the panel judged the available data inade- quate to characterize the frequency or magnitude of short-term peak (acute) exposures of various groups within the population. pling of nonresidential indoor environments, such as offices, public buidings, schools, retail stores, and vehicles, has been sparse. , p Indoor sources of formaldehyde have been identified both by association with high ambient levels and by direct measurement of emission rates. The strongest sources are articles fabricated with urea-formaldehyde resins that are used in large amounts in the indoor environment. Examples include UFFI, hardwood ply- wood (decorative) paneling, and particleboard under- lays or decking. Medium-density fiberboard is a suf- ficiently strong emitter (46) that even smaller articles such as furniture can elevate indoor levels of formalde- hyde significantly (38). rAansient increases in formalde- p p The information provided by past surveys is limited by failure to standardize or report sampling design or details of protocols, such as location and timing of samples, ventilation rates, human activity, etc. Sam- CONSENSUS WORKSHOP ON FORMALDEHYDE 332 Table 3. Reported levels of formaldehyde in indoor air in residences. No. CONSENSUS WORKSHOP ON FORMALDEHYDE of Formaldehyde, ppm Type of residence residences Range Mean Reference House without UFFIa 41 0.01-0.1 0.03 (36) House with UFFI 636 0.01-4 0.12 (36) (complaint and non-complaint)' Houses without UFFI 378 (3% > 0.lppm) 0.035 (37) House with UFFI 1146 (10% > 0.lppm) 0.054 (37) (complaint and non-complaint)b Mobile homes 431 0.01-3 0.38 (38) Mobile homes - 0-1.77 0.38 (38) (Washington, complaint)b Mobile homes 0-3.0 0.4 (38) (Minnesota, complaint)b Mobile homes 0.02-4.2 0.9 (38) (Wisconsin, complaint)b East Tennessee homes 40 < 0.02-0.4 0.06 (39) Age 0-5 yr 18 0.08 (39) Age 5-15 yr 11 0.04 (39) Age >15 yr 11 0.03 (39) California, Colorado 64 0.02-0.11 0.05 (40) and S. Dakota homes (conventional) California homes 52 0.03-0.3 0.1 (40) (mobile, energy-efficient, weatherized) U.K. buildings without UFFI 50 < 0.02-> 0.3 0.047 (41) U.K. buildings with UFFI 128 0.01-> 1 0.093 (41) aUFFI: urea-formaldehyde foam insulation. bComplaint: homes in which residents had complained of symptoms putatively associated with exposure to formaldehyde. cNumber not stated. Table 3. Reported levels of formaldehyde in indoor air in residences. on. ad complained of symptoms putatively associated with exposure to formaldehyde. Table 4. Formaldehyde in consumer products and medical products. Type of product Household products Household disinfectants (diluted for application); U.S. labeling required at > 1% Cosmetics (used as preserv- atives and in shampoos; FDA 1981 listed 805 pro- ducts containing formaldehyde) Level recommended by CTFA Highest permitted content in EEC (except fingernail hardener at 5%); EEC labeling required at 0.05% Disinfectants for medical use Concentrates Diluted for use Formaldehydecontent, up to 1% up to 7% generally < 1% (1% of products in range 1-10%) < 0.2% 0.2% Mostly 5-10% (range: 1-30%) < 1% Reference (50) (50) (51) (51) (52) (50) (50) I Table 4. Formaldehyde in consumer products and medical products. hyde levels can also be caused by burning cigarettes or gas-fired space heaters. In extreme cases (heavy smok- ing or poorly tuned heaters), these transient increases can exceed 0.1 ppm (47). lb. Environmental Exposures. Environmental lev- els of formaldehyde have been summarized by the National Research Council (NRC) (38) and other reviewers. Concentrations reported in ambient air have generally been below 10 to 15 parts per billion (ppb), except for situations of heavy traffic and/or photochemi- cal smog, when concentrations up to 90 to 150 ppb have been reported (38,48). CONSENSUS WORKSHOP ON FORMALDEHYDE Formaldehyde production (SIC 2869) Full 271-913a Part 145-487a Resin and plastic materials and manufacture Full 4,728 (SIC 2821) Part 1,272 Industrial and specialty chemicals and Full 2,680 manufacture (SIC 284-, 286-, 287-, 289-) Part 2,220 Paints, dyes, and pigments manufacture Full 7,480 (SIC 2851, 2865) Part 29,920 Adhesive and sealants manufacture (SIC 2891) Full 2,380 Part 420 Textile finishing (SIC 226-) Full 17,800 Part 2,200 Apparel manufacture (SIC 23-) Full 771,420 Part 125,580 Hardwood plywood manufacture (SIC 2435) Full 7,500 Softwood plywood manufacture (SIC 2435) Full 7,500 Plastic board manufacture (SIC 2492) Full 2,831 Part 1,069 Mobile home manufacture (SIC 2491, 2452, 3792) Full 31,500 Wood furniture (SIC 25-) Full 47,322 Part 2,178 Paper and paperboard manufacture (SIC 26-) Full 6,000-45,000a Plastic molding (SIC 3079) Full 15,073 Part 1,527 Abrasive products manufacture (SIC 3291) Full 3,996 Part 2,004 Foundries (SIC 332-, 336-) Full 28,638 Part 14,362 Producers of rubber and misc. plastic products Full 6,128-27,584a (SIC 30-) Urea-formaldehyde foam insulation dealers and Full 2,000-15,000a installers Fertilizer producers (SIC 2873) Full 500-900a Funeral services (SIC 7261) Part 52,000-70,200a Pathologists Part 12,400a Biology instructors (college/university) Part 13,000a Biology instructors (high school) Part 22,000a Total for all industries 1,341,084 aFrom USEPA (6); all other estimates from SOCMA (5). Table 5. Extent of exposure to formaldehyde in 23 industries and occupations in the U.S. Paper and paperboard manufacture (SIC 26-) Plastic molding (SIC 3079) Abrasive products manufacture (SIC 3291) Foundries (SIC 332-, 336-) aFrom USEPA (6); all other estimates from SOCMA (5). industries and three occupations, estimates were de- rived by EPA (6) by use of data from the Bureau of the Census, from the National Occupational Hazard Survey, and from various assumptions about the number of workers per plant. Except for the Bureau of the Census data on pathologists, the panel regards the EPA data as quite tenuous. The SOCMA data are more soundly based in data from the industries they cover, but are nevertheless subject to substantial biases because ofthe low response rate (2.7%) to the questionnaire. Thus, except for the estimate for pathologists, each of the estimates in Table 5 could be in error by a factor of up to 2 or 3. than in adults. The panel did not review the uptake of formaldehyde through the skin or mucous membranes. Environmental factors (especially temperature) in- fluencing emission rates of formaldehyde have been discussed in the previous section. CONSENSUS WORKSHOP ON FORMALDEHYDE These urban concentrations appear to have decreased since the 1960s (38,48,49) and are likely to decrease further as automobile emissions are progressively reduced. Rain water in Western Europe contained 0.3 to 1.4 ,ug/L formaldehyde in 1969 and 0.1 to 0.17 ,ug/L formaldehyde in the mid-1970s (49). The panel has not reviewed these data in detail because outdoor exposures appear to be much smaller than indoor exposures. p Ib. Consumer Products andMedical Devices. Ta- ble 4 lists data on the occurrence of formaldehyde in consumer products, drugs, and disinfectants. Formalde- hyde is used quite widely in cosmetics and household disinfectants. However, the panel was not able to find any data on the extent of exposure to these products via skin contact or inhalation. Formaldehyde is also used to disinfect medical devices such as kidney dialysis units, giving rise to potential exposures of users and medical p Ib. Consumer Products andMedical Devices. Ta- ble 4 lists data on the occurrence of formaldehyde in consumer products, drugs, and disinfectants. Formalde- hyde is used quite widely in cosmetics and household disinfectants. However, the panel was not able to find any data on the extent of exposure to these products via skin contact or inhalation. Formaldehyde is also used to disinfect medical devices such as kidney dialysis units, giving rise to potential exposures of users and medical personnel. The panel was concerned about a report from Europe of high concentrations (up to 15-25 ppm) of formaldehyde in incubators used for intensive care of premature infants (53). p ( ) Ic. The principal physiological factor affecting respi- ratory intake of formaldehyde is the breathing rate (minute volume), which is dependent on physical activ- ity and is higher (per unit of body weight) in children p Ic. The principal physiological factor affecting respi- ratory intake of formaldehyde is the breathing rate (minute volume), which is dependent on physical activ- ity and is higher (per unit of body weight) in children CONSENSUS WORKSHOP ON FORMALDEHYDE 333 Table 5. Extent of exposure to formaldehyde in 23 industries and occupations in the U.S. Workers exposed Tlype Industry (SIC code) (full/part-time) No. Epidemiology Panel Report* For reasons stated above, it is not possible to break down the occupationally exposed populations according to the levels of their exposure to formaldehyde. The data in Table 5 represent a cross section of industrial populations, and only limited information is available on the duration of their exposure or on rates of job turnover. The number of workers exposed at any one time is only a fraction of the total number of workers exposed during their working lives, but this fraction cannot be estimnnted from the data available. Topic 1. What is the epidemiologic evidence concern- ing the relationship between formaldehyde exposure and human illness (neoplastic and nonneoplastic)? ( p p a. What are the limitations and/or strengths of the available epidemiologic data (e.g., power, confound- ing variables)? g b. Can any or all of the data from epidemiologic studies on the relationship between cancer and formaldehyde be combined in order to provide a greater data base for statistical evaluation? Id. Residential Exposures. EPA (6) estimated that about 2.2 million persons were living in mobile homes less than five years old and that 1.3 to 1.6 million people were living in homes insulated with UFFI during the preceding five years. The Consumer Product Safety Commission (CPSC) estimated that 1.75 million people were living in holnes insulated with UFFI during the preceding 9 years. These estimates were based on construction and installation data and -reasonable esti- mates of family size, and the panel regards them as reasonable. We assume that the remainder of the U.S. population (220 million) is exposed to formaldehyde at levels characteristic of conventional homes (Table 3). For reasons stated above, it is not possible to character- ize temporal patterns or short-term peak exposures to formaldehyde. g c. Are there any epidemiologic hypotheses that can be developed from case reports of illness following exposure to formaldehyde? p y d. What levels of exposure or what types of exposure to formaldehyde have been reliably associated with human biological responses as evidenced from epidemiologic data? e. Does the epidemiologic evidence indicate that some segments of the population are particularly sensitive to any adverse health effects of formalde- hyde? If so, which segments and at what levels of exposure? p Topic 2. After reviewing completed and ongoing studies, what attainable additional studies might clarify any exposure disease relationships? * Epidemiology Panel: E. A. Acheson (Chairman); Kenneth J. Rothman (Alternate Chairman/Secretary); Aaron Blair; John F Gamble; Peter F Infante; Keith R. Long; Edward A. Mortimer, Jr.; Marvin A. Schneiderman. Epidemiology Panel Report* y p p This report, which discusses the epidemiologic evi- dence concerning the relationship between formalde- hyde exposure and human illness, addresses three issues. First the panel considered the evidence relating to neoplasms; next, nonneoplastic illness; finally, recom- mendations are made regarding further research direc- tions. The scientific papers published, in press, and in manuscript, that were considered comprise the refer- ences. With the single exception of nasal cancer, where case reports are quoted, our discussion is limited to studies in which the material can be related to a definable population. As the Consensus Workshop in- cludes panels on carcinogenicity, biochemistry, expo- sure and risk estimation, this panel has only to a limited extent attempted an evaluation of the epidemiologic data in the light of other biological information, or in relation to estimates of exposure for different occupa- tions. Nevertheless, whenever possible, qualitative infor- mation on level of exposure by occupation was considered. CONSENSUS WORKSHOP ON FORMALDEHYDE Little appears to be known about the physical or chemical form of formalde- hyde present in the ambient air. In occupational settings, exposure can be reduced by ventilation and other work practices. The panel found no information on the effectiveness of gloves or other protective clothing. d i l i Id. Occupational Exposures. Table 5 (54) lists estimates of the numbers of workers exposed to formal- dehyde in 23 industries and occupations in the U.S. For 17 industries, estimates were derived by SOCMA (5) on the basis of industry-wide questionnaires. Companies responding to the questionnaires provided data on the number of employees in various job categories poten- tially exposed to formaldehyde, and these were scaled up to the entire industry, assuming that the proportion of exposed workers was the same in all plants. For six EPA (6) cited data from the Bureau of the Census indicating that there are about 1.4 million biology and nursing students in the U.S., most or all of whom are likely to be exposed to formaldehyde intermittently during laboratory studies. Other categories of labora- tory workers, such as laboratory and medical techni- cians, have not been enumerated. CONSENSUS WORKSHOP ON FORMALDEHYDE 334 Topic 2: Currently Lacking Data 2. The panel did not identify substantial controver- sies about exposure, although a number of gaps and deficiencies in the available data limit the accuracy and completeness of exposure assessment. The most impor- tant data needs identified by the panel were the following. g Systematic data are needed on the extent and magni- tude of occupational exposure to formaldehyde, includ- ing representative, industry-wide information about personal exposures. Priority attention should be given to industries with a large number of persons exposed (e.g., textiles and clothing, and biological laboratories), workplaces with reportedly high concentrations (e.g., foundries and funeral services), and plants in which epidemiological studies are being or have been conduct- ed. An important first step would be systematic compila- tion and review of data already collected by employers, since the SOCMA (5) survey indicated that extensive data already exist in company files. CONSENSUS WORKSHOP ON FORMALDEHYDE 335 experience of the adult male working population. This panel considered no evidence relating to persons first exposed in childhood or old age, to women or to the fetus. have been exposed, but may also differ in the pattern and intensity of exposure to formaldehyde. The panel noted that in industry many of the workers had been exposed for only short periods (i.e., less than one year) while for professional workers the possibility of expo- sure at varying intervals was present for many years. In addition, personal habits, which almost certainly influ- ence cancer mortality, differ between the professional and industrial groups. I b. As shown in Table 6, information on all sites was not available for all studies. There is a potential problem involved in simply summing observed and expected numbers and then computing an overall observed-to- expected ratio. With this direct summing one or two large studies then provide the weight of evidence. If some unrecognized distortions or biases had affected one or more of these large studies, the summary would also be biased and thus be potentially misleading. Some readers may wish to make their own separate or different summaries using the data. Several summary approaches are possible. For example, a summary could be prepared which omits one (or more) of the separate studies, based perhaps on the quality of the study, geography, etc. The panel advised caution against basing evaluation on the "total" column in Table 7 without considering the inherent differences between the professional and industrial cohorts with regard to exposure and possible compounding factors. g p Ic, Id. Selected Sites of Cancer. NASAL CANCER g p Ic, Id. Selected Sites of Cancer. NASAL CANCER Ic, Id. Selected Sites of Cancer. NASAL CANCER No case of nasal cancer has been reported in any of the epidemiological studies listed in Table 7 [O obs; 3.0 exp; 95% confidence limits (CL) for the standardized morbid- ity ratio (SMR) 0-123] (55,59,61-63). This expected figure and, therefore, the confidence interval for the SMR, take no account of induction period or degree of exposure. In other epidemiologic studies of nasal cancer, the evidence suggests that the risk does not begin to rise until 15 to 25 years after first exposure. CONSENSUS WORKSHOP ON FORMALDEHYDE In a study of British chemical workers (64), it can be seen that the study had a 46% chance of detecting a fivefold increase of risk of this rare tumor if risk commenced immediately after the beginning of exposure and was limited to persons exposed to "high levels"; there was only a 28% chance if the risk did not begin to rise until 20 years after first exposure. These data therefore suggest that it is too early to exclude an important increase in risk after a prolonged induction period. In a case-control study of 167 men with nasal cancer carried out jointly in Denmark, Finland and Sweden (65) "a scrutiny of occupations for which exposure to formalde- hyde possibly may have occurred gave no indication of any association." In a case-control study of 160 patients with nasal cancer in North Carolina and Virginia (66) (available for review only in abstract form), no mention was made of any association with formaldehyde. e posu e poss b e co pou d g acto s. la, lb. Although the summary of observed and expected numbers provides a convenient basis for evaluation, it involves a number of important assump- tions which limit its validity and usefulness. The bulk of the expected numbers has been calculated on the basis of an analysis of proportionate mortality, and these have been summed with those from studies using other types of data. Some would regard this as inappropriate. Further, it should be emphasized that a substantial proportion ofthe person-years at risk relates to individu- als within the industrial cohorts who were exposed to very low levels of formaldehyde or, as in the case of professionals, who may have been intermittently ex- posed to higher levels. Historical exposure estimates were made in only two studies (55,56). Moreover, in a proportion of men the period between exposure and completion of follow-up has been brief. The overall effect of these factors and of shortcomings in some of the studies is to make interpretation of associations between formaldehyde and cancer difficult. Where such data existed, the induction period was considered (Table 6), since the inclusion of persons with short potential induction times and very low levels of expo- sure biases the relative risk of the direction of unity. Finally, few studies had information about smoking and all studies lacked data about consumption of alcohol. Topic 1. Formaldehyde and Human Illness 1, la. Formaldehyde and Cancer. The plan has been to consider in turn each category of neoplasm to which attention has been drawn in the literature. Most of the studies reviewed use the occurrence of death as the measure of outcome and almost all refer to the y Systematic data on the variability in concentrations of formaldehyde in indoor air are required to determine the frequency and magnitude of short-term peak exposures. This will require systematic sampling de- signs, stratified among various categories of houses, with sampling designed to detect both short-term (hourly) and long-term (seasonal) variations. It will also require better standardization of protocols and valida- tion of analytical methods. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 2) Matanoski 0 0.5 0 0.6 0 0.4 0 0.2 ND ND ND ND 0 1.3 ND ND ND ND ND ND 8 7.1 8 6.1 1 6.8 1 2.1 ND ND 2 1.7 0 0 0 0 ND ND 0 ND ND ND 1.3 1.6 0.2 0.5 ND 1.2 5 6.1 0.82 7 3.1 2.3 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 9 5.8 1.6 3 1.8 1.7 9 4.7 1.9 7 2.7 2.6 10 3.7 2.7 6 1.7 3.5 3 2.6 1.2 ND ND ND 4 1.2 3.3 ND ND ND 5 4.6 1.1 ND ND ND Acheson Liebling, Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No. 2) Matanoski Acheson Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No. 2) Matanoski 5 12.5 ND ND 1 0.7 ND ND 25 20.6 19 15.5 18 14.6 8 6.5 8 3.8 2 3.0 ND ND 20 26.3 2 2.3 3 2.0 ND ND 12 8.5 12 6.9 10 6.7 4 2.5 1 1.5 1 1.1 ND ND 0.40 ND 1.4 ND 1.2 1.2 1.2 1.2 2.3 0.7 ND 0.8 0.9 1.5 ND 1.4 1.7 1.5 1.6 0.7 0.91 ND ND ND ND ND ND ND ND ND ND ND ND ND 11 9.1 1.21 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1 0.95 1.00 ND ND ND 10 4.3 2.3 7 4.1 1.7 ND ND ND ND ND ND ND ND ND ND ND ND Acheson Liebling, Marsh Tabershaw Fayerweather 9 ND ND ND 11.4 ND ND ND 0.8 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Walrath (New York) Walrath (California) Stroup Levine 72 66.8 41 42.8 12 43.0 19 20.2 1.1 1.0 0.3 0.94 35 34.7 1.0 28 30.8 0.9 6 29.3 2.0 19 18.4 1.0 CONSENSUS WORKSHOP ON FORMALDEHYDE 336 Table 6. CONSENSUS WORKSHOP ON FORMALDEHYDE O/E data by site and by study.a No induction period considered Induction period considered Site Study 0 E O/E 0 E O/E Nasal passages Professional Industrial Buccal cavity and pharynx Professional Industrial Brain Professional Walrath (New York) Walrath (California) Stroup Levine Harrington Matanoski Acheson Liebling, Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington Matanoski Acheson Liebling, Marshb Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (Study No. 2) Matanoski 0 0.5 0 0.6 0 0.4 0 0.2 ND ND ND ND 0 1.3 ND ND ND ND ND ND 8 7.1 8 6.1 1 6.8 1 2.1 ND ND 2 1.7 0 0 0 0 ND ND 0 ND ND ND 1.3 1.6 0.2 0.5 ND 1.2 5 6.1 0.82 7 3.1 2.3 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 9 5.8 1.6 3 1.8 1.7 9 4.7 1.9 7 2.7 2.6 10 3.7 2.7 6 1.7 3.5 3 2.6 1.2 ND ND ND 4 1.2 3.3 ND ND ND 5 4.6 1.1 ND ND ND Industrial All lymphatic and hemopoetic Professional Industrial Leukemia (only) Professional Acheson Liebling, Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No. 2) Matanoski Acheson Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No. CONSENSUS WORKSHOP ON FORMALDEHYDE y The panel found two case reports of nasal cancer in persons exposed to formaldehyde. One individual was employed for 25 years in a fabric finishing plant where textiles were permeated with the formaldehyde-based resin. He had previously soldered components of air- craft engines for about 2 years (67). Soldering, welding and flamecutting have been associated with an in- creased risk of nasal cancer (65). The second individual had been employed for 32 years at a plant where phenol formaldehyde and formaldehyde resins were manufac- tured (68). As usual, interpretation of these case- reports is problematic. BUCCAL CAVITY AND PHARYNX. No important excess of cancers of these sites has been observed in relation to the calculated expected numbers in any of the published studies (55,59-64,69). In a reclassification of the deaths in the cohort first studied by Marsh there were a total of seven buccal and pharyngeal cancer deaths observed as compared with an estimated 3.1 expected (68). Two of these deaths occurred subsequent to Marsh's date of conclusion of follow-up. p The occupations studied fall into two distinct catego- ries and these have been considered separately. The observed and expected cases of cancer in studies of professional people who use formaldehyde in the preser- vation of human tissues (embalmers, anatomists and pathologists) (57-63) are shown separately from those for industrial workers involved in the production and use of formaldehyde. These two groups differ not only in substances other than formaldehyde to which they may BRAIN. A substantial excess of deaths from cancer CONSENSUS WORKSHOP ON FORMALDEHYDE Table 6. O/E data by site and by study.a No induction period considered Induction period considered Study 0 E O/E 0 E O/E Walrath (New York) Walrath (California) troup Levine Harrington Matanoski Acheson iebling, Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington Matanoski Acheson Liebling, Marshb Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (Study No. CONSENSUS WORKSHOP ON FORMALDEHYDE 337 Table 6. (continued) No induction period considered Induction period considered Site Study 0 E O/E 0 E O/E Harrington (No. 1) Harrington (No.2) Matanoski 10 27.4 9 22.0 12 21.4 0.4 0.41 0.6 ND ND ND ND ND ND ND ND ND Industrial Prostate Professional Acheson Skin Professional Acheson Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No. 2) Matanoski Acheson Liebling, Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No.2) Matanoski 205 6 3 ND 15 23 20 3 ND ND ND ND ND 2 ND 215.0 7.1 5.2 ND 16.4 13.1 18.7 3.4 ND ND ND ND ND 0.55 ND 8 3.6 2 3.4 2 3.5 0 0.9 ND ND ND ND ND ND 1.0 86 72.1 1.2 0.9 3 3.7 0.8 0.6 ND ND ND ND 12 14.0 0.9 0.91 1.8 1.1 0.88 ND ND ND ND ND ND 16 9.5 1.7 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 3.6 ND ND ND ND ND ND ND 2.2 0.6 0.6 0 ND ND ND 4 1.3 3.1 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Industrial Bladder Professional Industrial Kidney Professional Industrial Acheson Liebling, Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No. 2) Matanoski Acheson Marsh Tabershaw Fayerweather Walrath (New York) Walrath (California) Stroup Levine Harrington (No. 1) Harrington (No. CONSENSUS WORKSHOP ON FORMALDEHYDE 2) Matanoski Acheson Liebling, Marsh Tabershaw Fayerweather ND ND ND ND ND ND ND ND ND ND ND ND 0 0.4 0 ND ND ND ND ND ND ND ND ND 7 7.3 1.0 8 5.8 1.4 5 7.2 0.7 ND ND ND 1 2.1 0.5 2 1.9 1.1 ND ND ND ND ND ND 1 0.3 3.3 ND ND ND ND ND ND 8 5.4 4 4.0 1 4.0 1 1.7 ND ND ND ND 7 3.6 ND ND ND ND 1 0.4 ND ND 1.5 1.0 0.25 0.6 ND ND 1.9 ND ND 2.5 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 3 0.6 5.3 2 2.4 0.83 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Digestive system Professional Walrath (New York) Walrath (California)c Stroup Levine Harrington (No. 1) Harrington (No. 2) Matanoski 68 68 38 17 12 8 ND 65.2 55.7 66.4 22.6 19.8 15.5 ND 1.0 1.2 0.6 0.8 0.6 0.5 ND ND ND ND 33 25.4 1.3 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Table 6. CONSENSUS WORKSHOP ON FORMALDEHYDE 2) Matanoski 5 12.5 ND ND 1 0.7 ND ND 25 20.6 19 15.5 18 14.6 8 6.5 8 3.8 2 3.0 ND ND 20 26.3 2 2.3 3 2.0 ND ND 12 8.5 12 6.9 10 6.7 4 2.5 1 1.5 1 1.1 ND ND 0.40 ND 1.4 ND 1.2 1.2 1.2 1.2 2.3 0.7 ND 0.8 0.9 1.5 ND 1.4 1.7 1.5 1.6 0.7 0.91 ND ND ND ND ND ND ND ND ND ND ND ND ND 11 9.1 1.21 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1 0.95 1.00 ND ND ND 10 4.3 2.3 7 4.1 1.7 ND ND ND ND ND ND ND ND ND ND ND ND Industrial Acheson Liebling, Marsh Tabershaw Fayerweather 9 ND ND ND 11.4 ND ND ND 0.8 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Lung Professional Walrath (New York) Walrath (California) Stroup Levine 72 66.8 41 42.8 12 43.0 19 20.2 1.1 1.0 0.3 0.94 35 34.7 1.0 28 30.8 0.9 6 29.3 2.0 19 18.4 1.0 Table 6. O/E data by site and by study.a CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE (continued) inued) No induction period considered Induction period considered 0 E O/E 0 E O/E 10 27.4 9 22.0 12 21.4 0.4 0.41 0.6 ND ND ND ND ND ND ND ND ND 205 6 3 ND 15 23 20 3 ND ND ND ND ND 2 ND 215.0 7.1 5.2 ND 16.4 13.1 18.7 3.4 ND ND ND ND ND 0.55 ND 8 3.6 2 3.4 2 3.5 0 0.9 ND ND ND ND ND ND 1.0 86 72.1 1.2 0.9 3 3.7 0.8 0.6 ND ND ND ND 12 14.0 0.9 0.91 1.8 1.1 0.88 ND ND ND ND ND ND 16 9.5 1.7 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 3.6 ND ND ND ND ND ND ND 2.2 0.6 0.6 0 ND ND ND 4 1.3 3.1 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0 0.4 0 ND ND ND ND ND ND ND ND ND 7 7.3 1.0 8 5.8 1.4 5 7.2 0.7 ND ND ND 1 2.1 0.5 2 1.9 1.1 ND ND ND ND ND ND 1 0.3 3.3 ND ND ND ND ND ND 8 5.4 4 4.0 1 4.0 1 1.7 ND ND ND ND 7 3.6 ND ND ND ND 1 0.4 ND ND 1.5 1.0 0.25 0.6 ND ND 1.9 ND ND 2.5 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 3 0.6 5.3 2 2.4 0.83 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 68 68 38 17 12 8 ND 65.2 55.7 66.4 22.6 19.8 15.5 ND 1.0 1.2 0.6 0.8 0.6 0.5 ND ND ND ND 33 25.4 1.3 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Site Digestive system Professional CONSENSUS WORKSHOP ON FORMALDEHYDE 338 Table 6 (continued) No induction period considered Induction period considered Site Study 0 E O/E 0 E O/E Digestive system Acheson ND ND ND ND ND ND Industrial Marshc 8 6.3 1.3 ND ND ND Tabershaw 0 4.1 0.0 ND ND ND Fayerweatherd ND ND ND 6 7.5 0.8 aData from: Acheson (55); Fayerweather (56); Harrington or Harrington (No. CONSENSUS WORKSHOP ON FORMALDEHYDE social class gradients are relatively unimportant in this tumor . . ., and there is no excess either the mortality experience of psychiatrists, or based on the records of Olmsted County, in which the Mayo Clinic is located, the excess brain cancer mortality remained. Olmsted County has a very high autopsy rate, and brain cancer mortality is not likely to have been seriously underestimated. Thus, detection bias would not seem to account for the excessive brain cancer risk among anatomists. The association between professional groups engaged in preservation of human tissues and brain cancer does not necessarily implicate formaldehyde. Aside from formaldehyde and human tissues themselves, however, it is unclear what other important occupational expo- sures these professional groups shared. The British study, which shows a deficiency of brain tumors among industrial workers (55), provides evidence against an association, although professional and industrial work- ers studied have differed in level, duration and fre- quency of exposure to both formaldehyde and other substances, chemical and biological. LYMPHATIC AND HEMOPOIETIC SYSTEM. When taken as a single group, no significant excess of deaths from these cancers was observed (105 obs, 94.6 exp; 95% CL for the SMR 91-134) (55,57-59,61-63,70). However, an excess exists that approaches significance for profes- sional workers (80 obs, 64 exp; 95% CL for the SMR i i i Table 7. Observed and expected deaths for professionals and industrial workers exposed to formaldehyde, with exact 95% confidence limits (CL). ble 7. Observed and expected deaths for professionals and industrial workers exposed to formaldehyde, with exact 95% confidence limits (CL). of the brain is noted among the professional workers exposed to formaldehyde (40 obs, 22.6 exp; 95% CL for the SMR 126-241) (58-63). Among industrial workers, on the other hand, there is a substantial deficit (6 obs, 13.2 exp; 95% CL for the SMR 17-99) (55,70). The latter result, however, is derived from only two studies of which one contributes five observed cases and 12.5 expected. An excess of an approximately similar order of magnitude is seen in each of the three professional groups, namely embalmers, anatomists and pathologists, and within each of three separate groups of embalmers; the SMR increased when induc- tion period was taken into consideration (see Table 6) (55, 70). Among anatomists 10 cases were observed and 3.7 expected; all the tumors were glioblastomas or astro- cytomas. CONSENSUS WORKSHOP ON FORMALDEHYDE 1) (57); Harrington (No. 2) (58); Levine (59); Matanoski (60); Stroup (61); Walrath (New York) (62); Walrath (California) (63); Liebling (66); March (64); Tabershaw (65). ND = no data. bIncluding two "post closing" deaths. cStomach, colon and pancreas. dC l Table 6 (continued) dColon and rectum. Table 7. Observed and expected deaths for professionals and industrial workers exposed to formaldehyde, with exact 95% confidence limits (CL). Professional Industrial Total Site of cancer Obs/Exp CL Obs/Exp CL Obs/Exp CL Nasal 0/1.7 0-2.17 0/1.3 0-2.84 0/3.0 0-1.23 Mouth 20/23.8 0.51-1.30 12/9.2 0.67-2.28 32/33.0 0.66-1.37 Brain 40/22.6 1.26-2.41 6/13.2 0.17-0.99 46/35.8 0.94-1.71 Lymphatic and hematopoietic 80/64.0 0.98-1.53 25/30.6 0.53-1.21 105/94.6 0.91-1.34 Leukemia 40/27.2 1.05-2.00 9/11.4 0.36-1.50 49/38.6 0.94-1.68 Other lymphatic and hematopoietic 40/36.8 0.78-1.48 16/19.2 0.48-1.35 56/56.0 0.76-1.30 Lung 175/243.6 0.62-0.83 214/227.3 0.82-1.08 389/470.9 0.75-0.91 Prostate 61/51.6 0.90-1.52 2/0.6 0.40-12.04 63/52.2 93-1.54 Skin 12/11.4 0.54-1.84 0/0.4 0-9.22 12/11.8 0.52-1.78 Bladder 23/24.3 0.60-1.42 1/0.3 0.18-18.6 24/24.6 0.62-1.45 Kidney 21/18.6 0.70-1.73 1/0.4 0.06-13.93 22/19.0 0.72-1.75 Digestive System 211/245.2 0.74-0.98 8/10.4 0.33-1.52 219/255.6 0.75-0.98 Other causes Cirrhosis of liver 83/59.3 1.11-1.74 10/9 0.53-2.04 93/68.3 1.10-1.67 Nonneoplastic respiratory disease 109/163.7 0.55-0.80 243/241.1 0.88-1.14 352/404.8 0.78-0.96 of the brain is noted among the professional workers exposed to formaldehyde (40 obs, 22.6 exp; 95% CL for the SMR 126-241) (58-63). Among industrial workers, on the other hand, there is a substantial deficit (6 obs, 13.2 exp; 95% CL for the SMR 17-99) (55,70). The latter result, however, is derived from only two studies of which one contributes five observed cases and 12.5 expected. An excess of an approximately similar order of magnitude is seen in each of the three professional groups, namely embalmers, anatomists and pathologists, and within each of three separate groups of embalmers; the SMR increased when induc- tion period was taken into consideration (see Table 6) (55, 70). Among anatomists 10 cases were observed and 3.7 expected; all the tumors were glioblastomas or astro- cytomas. Among pathologists, all four brain cancers were of the glioma or astrocytoma cell type. Data from some autopsy series indicate that about 30% to 50% of primary intracranial neoplasms are of these cell types. Brain cancer is difficult to diagnose and may be more effectively detected among persons with better access to sophisticated medical care, particularly among groups such as medical faculty members (71). In discussing his study of medical professionals, Harrington (58) re- marked that, a . . . * Dr. Acheson, who could not attend the second meeting of the panel in Boston, suggested, based upon follow-up data, from studies he conducted which were not available at the time of the second meeting (72,73), that this sentence be replaced with "There is evidence within this factory of a relation between degree of exposure to formaldehyde and lung cancer, but the trend disappears when account is taken of length of exposure." CONSENSUS WORKSHOP ON FORMALDEHYDE The authors adjusted for age, sex, pay class, plant site and smoking history but presented crude odds ratios having found that adjust- ment made little difference. Numbers of many cancers were insufficient for meaningful analysis. No nasal cancers were reported. Among the seven cancers of the buccal cavity and seven melanomas of the skin, only one case of each was exposed to formaldehyde. Odds ratios were not elevated substantially for cancers of the brain, kidney, lung, or lymphatic and hematopoietic system. Risk gradients were noted for cancer of the prostate and bladder by cumulative exposure index (CEI). Compared with a value of 1.0 among nonexposed, odds ratios were 3.0 for CEI - 20 and 3.6 for CEI > 20 for cancer of the prostate. For bladder cancer, the odds ratios were 2.6 for CEI - 20 and 5.2 for CEI > 20. A case-control study of lung cancer in Danish doctors could not be evaluated because of an error in the analysis (74). pp y LUNG. A deficiency of deaths from cancer of the lung is noted which is statistically significant among the professional workers (175 obs, 243.6 exp; 95% CL for the SMR 62-83) (57-63) and both groups combined (389 obs, 470.9 exp; 95% CL for the SMR 75-91), but not among the industrial workers (214 obs, 227.3 exp; 95% CL for the SMR 82-108) (55,64,70). The most likely explanation for the deficiencies is that exposed profes- sional workers have been compared with a reference population that has smoked more; data on smoking habits are not available in any of these studies. In the British study (55), a significant increase in mortality from lung cancer was found in one of the six factories studied (British Industrial Plastics) (SMR = 124, 95% CL 104-148) in comparison with an expected number calculated from national death rates. This factory con- tributed over half of the lung cancer deaths and also had the highest average exposures; a trend of increasing mortality (as measured by the SMR) with increasing exposure to formaldehyde was found in the same factory and its significance was borderline at the 5% level. No such relation was found in any of the other factories or when all six factories were considered together. The number ofmen, however, exposed to "medium" or "high" levels of formaldehyde in the other factories was small. CONSENSUS WORKSHOP ON FORMALDEHYDE 339 27.2 exp; CL for the SMR 105-200), particularly among those with an induction time of 15 years or more (17 obs, 8.4 exp; 95% CL for the SMR 118-324). In the past leukemia has been reported more frequently in the higher social classes. Among the anatomists (61), an excess of leukemia deaths, in particular, deaths due to chronic myeloid leukemia, persisted when compared with the mortality experience of psychiatrists. Thus, detection bias would not seem to account for the excessive leukemia risk among anatomists. The number of cases of leukemia reported among industrial groups exposed to formaldehyde is approximately as expected. Remarks about exposures and working practices of the various groups made in the previous paragraph also apply here. might only be detectable through study of populations who experience high exposure levels, given the steep dose response exhibited by experimental animals. 27.2 exp; CL for the SMR 105-200), particularly among those with an induction time of 15 years or more (17 obs, 8.4 exp; 95% CL for the SMR 118-324). In the past leukemia has been reported more frequently in the higher social classes. Among the anatomists (61), an excess of leukemia deaths, in particular, deaths due to chronic myeloid leukemia, persisted when compared with the mortality experience of psychiatrists. Thus, detection bias would not seem to account for the excessive leukemia risk among anatomists. The number of cases of leukemia reported among industrial groups exposed to formaldehyde is approximately as expected. Remarks about exposures and working practices of the various groups made in the previous paragraph also apply here. p y p In individual studies, attention has been drawn to small excesses of deaths from cancer of the prostate, skin (including melanoma), kidney, bladder, and of the digestive system. As can be seen from Table 7, in none of these sites, with the possible exception of prostate cancer, do the figures approach statistical significance in either professionals or industrial workers. There is at present scant evidence of an association between expo- sure and cancer of any of these sites. y Results from the DuPont case-control (56) study were evaluated and compared with findings from SMR and proportionate morbidity ratio (PMR) studies. Exposure to formaldehyde for the 481 cancer deaths and 481 living controls was estimated from work histories. Approxi- mately 20% of both the control series and the cases were exposed to formaldehyde. CONSENSUS WORKSHOP ON FORMALDEHYDE Among pathologists, all four brain cancers were of the glioma or astrocytoma cell type. Data from some autopsy series indicate that about 30% to 50% of primary intracranial neoplasms are of these cell types. Brain cancer is difficult to diagnose and may be more effectively detected among persons with better access to sophisticated medical care, particularly among groups such as medical faculty members (71). In discussing his study of medical professionals, Harrington (58) re- marked that, a . . . social class gradients are relatively unimportant in this tumor . . ., and there is no excess in medical practitioners as a group." When the expected numbers in the study of anatomists (61) were based on either the mortality experience of psychiatrists, or based on the records of Olmsted County, in which the Mayo Clinic is located, the excess brain cancer mortality remained. Olmsted County has a very high autopsy rate, and brain cancer mortality is not likely to have been seriously underestimated. Thus, detection bias would not seem to account for the excessive brain cancer risk among anatomists. g The association between professional groups engaged in preservation of human tissues and brain cancer does not necessarily implicate formaldehyde. Aside from formaldehyde and human tissues themselves, however, it is unclear what other important occupational expo- sures these professional groups shared. The British study, which shows a deficiency of brain tumors among industrial workers (55), provides evidence against an association, although professional and industrial work- ers studied have differed in level, duration and fre- quency of exposure to both formaldehyde and other substances, chemical and biological. g LYMPHATIC AND HEMOPOIETIC SYSTEM. When taken as a single group, no significant excess of deaths from these cancers was observed (105 obs, 94.6 exp; 95% CL for the SMR 91-134) (55,57-59,61-63,70). However, an excess exists that approaches significance for profes- sional workers (80 obs, 64 exp; 95% CL for the SMR 98-156). This excess is primarily attributable to an excess of deaths from leukemia in this group (40 obs, CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE No relation was found between lung cancer mortality and exposure to formaldehyde as measured by length of service. Similarly, no relation was found between lung cancer mortality and interval since first exposure to formaldehyde. Men who entered the BIP factory be- tween 1936 and 1945 (when exposure levels were probably highest) had the highest mortality from lung cancer. As the data now stand, there is some evidence of a dose-response relation between exposure to formalde- hyde and lung cancer; the evidence derives from the one British factory where the largest number of cohort members experienced the highest formaldehyde expo- sure levels.* If formaldehyde is a human carcinogen, it y The data are sparse and conflicting and do not yet provide persuasive evidence of a causal relation be- tween exposure to formaldehyde and cancer in man. As far as nasal cancer is concerned, the evidence is against a substantial (e.g., 10-fold) immediate increase in risk, but sufficient information is not yet available to exclude such an effect if risk starts to increase 20 years or more after first exposure. An increase in risk of brain cancer and leukemia is noted among each of three professional groups who preserve human tissues with solutions containing formaldehyde and other chemicals. In view of the small numbers of person-years of follow-up in subjects followed for 20 years or more and various methodological limitations of the studies, it is not possible from the available epidemiological data to exclude the possibility that formaldehyde is a human carcinogen. le. Although on general grounds there is reason to believe that there is a wide variation in the susceptibil- ity of individuals to carcinogens there is insufficient evidence to date by which to identify any particular subgroup as having greater than average susceptibility to a putative carcinogenic action of formaldehyde. CONSENSUS WORKSHOP ON FORMALDEHYDE 340 Id. Formaldehyde and Other Conditions. MORTAL- ITY. No excess mortality from disease ofthe respiratory system (other than cancer) as a whole has been noted although lack of information on smoking severely limits interpretation. No detailed examination of mortality from particular respiratory diseases has been under- taken (Table 7). Carcinogenicity/Histopathology/ Genotoxicity Panel Report* Although some symptoms were present, the changes in PFT were clinically insignificant, and there is no convincing evidence formaldehyde exposure results in restriction or obstruction at the doses studied. There is some suggestion that the symptoms are reversible and of minor import. However, because of the demonstrated irritant potential of formaldehyde, selection bias may be occurring in the exposed populations so that these studies are likely to underestimate adverse effects of formaldehyde exposure. Topic 1. What conclusions can be drawn from the available experimental data relative to the carcinogeni- city/genotoxicity of formaldehyde? Are there data from studies that permit projections to be made about potential human responses? p p a. What role does the cytotoxicity of formaldehyde play in its carcinogenicity in experimental animals? i i i i g y p b. What is the significance of benign tumors and potential preneoplastic lesions in the carcinogenic response in rats exposed to formaldehyde by inhalation? c. What do genotoxicity studies tell us about the potential of formaldehyde to be an initiator or promoter for carcinogenesis or a mutagen in somatic or germ cells? g d. What nonneoplastic changes occur when experi- mental animals and man are exposed to formalde- hyde? What is the health significance of these changes? g Topic 2. What critical questions remain to be an- swered? CONSENSUS WORKSHOP ON FORMALDEHYDE A statistically significant excess mortal- ity from cirrhosis of the liver has been observed in the professional groups (83 obs 59.3 exp; 95% CL for the SMR 111-174) (57-59,62,63).'No such excess was found in industrial workers (10 obs, 9.0 exp; 95% CL for the SMR 53-204), but these figures were derived from one study (55). The possibility of an excess consumption of alcohol should be considered as a possible explanation for the excess among embalmers, although there is no direct evidence to support this conjecture. degree of exposure to formaldehyde and smoking habits should be examined. The panel wished to draw particular attention to the consistent excesses of malignant brain tumors (and leukemia) reported in several groups of professionals who use formaldehyde in the preservation of human tissues. Studies should be undertaken to measure the patterns of exposure to formaldehyde in these occupa- tions, and to identify other chemical and biological exposures. p Longitudinal studies of the effects (if any) of exposure to formaldehyde gas and particulates on pulmonary function are needed as the data currently available are derived from cross-sectional studies. Topic 1: Conclusions from Experimental Data 1. Data on the carcinogenicity of formaldehyde vapor are available for the rat, mouse, and hamster. Carcinogenicity for the nasal epithelium has been demonstrated in two strains of rats exposed by inhala- tion for 6 hr/day, 5 days/week, for up to 2 years, to concentrations of approximately 14 ppm formaldehyde (83,84.t The same exposure regimen in one experi- ment, but at a concentration of 5.6 ppm, produced a greatly reduced incidence of nasal cancer, 2 out of a total * Carcinogenicity/Histopathology/Genotoxicity Panel: Richard Griese- mer (Chairman); Craig Boreiko; Frederick J. de Serres; Victor J. Feron; J. Justin McCormick; James A. Swenberg; Benjamin E Trump; A. Upton; J. Ward. t The number of rats with nasal carcinoma at concentrations of 14.3 ppm was an adjusted incidence of 103 out of 206. * Carcinogenicity/Histopathology/Genotoxicity Panel: Richard Griese- mer (Chairman); Craig Boreiko; Frederick J. de Serres; Victor J. Feron; J. Justin McCormick; James A. Swenberg; Benjamin E Trump; A. Upton; J. Ward. t Th b f i h l i i f t The number of rats with nasal carcinoma at concentrations of 14.3 ppm was an adjusted incidence of 103 out of 206. Carcinogenicity/Histopathology/ Genotoxicity Panel Report* direct evidence to support this conjecture. MORBIDITY The panel summarized studies measur- ing primarily symptoms and/or changes in pulmonary function (74-82). Symptoms (particularly irritation of the eyes, nose and throat) were consistently reported more frequently in the exposed populations, and were reported even when there were no reductions in pulmo- nary function. No important reductions in forced vital capacity were observed. Reductions in forced expir- atory volume in one second (FEV) and forced expir- atory volume % (FEV/FVC) when observed were small. These were not detected when exposure to formalde- hyde was solely as a vapor. There was either a weak or absent association of reduced pulmonary function test with exposure in the few studies where this factor was analyzed. Workshift (acute) changes in pulmonary func- tion test (PFT) have been assessed only when other dust was present and/or the formaldehyde itself was a particulate or incorporated in particles. Acute PFT reductions were not consistently present, were small and showed no regular association with exposure. Although some symptoms were present, the changes in PFT were clinically insignificant, and there is no convincing evidence formaldehyde exposure results in restriction or obstruction at the doses studied. There is some suggestion that the symptoms are reversible and of minor import. However, because of the demonstrated irritant potential of formaldehyde, selection bias may be occurring in the exposed populations so that these studies are likely to underestimate adverse effects of formaldehyde exposure. pp j MORBIDITY The panel summarized studies measur- ing primarily symptoms and/or changes in pulmonary function (74-82). Symptoms (particularly irritation of the eyes, nose and throat) were consistently reported more frequently in the exposed populations, and were reported even when there were no reductions in pulmo- nary function. No important reductions in forced vital capacity were observed. Reductions in forced expir- atory volume in one second (FEV) and forced expir- atory volume % (FEV/FVC) when observed were small. These were not detected when exposure to formalde- hyde was solely as a vapor. There was either a weak or absent association of reduced pulmonary function test with exposure in the few studies where this factor was analyzed. Workshift (acute) changes in pulmonary func- tion test (PFT) have been assessed only when other dust was present and/or the formaldehyde itself was a particulate or incorporated in particles. Acute PFT reductions were not consistently present, were small and showed no regular association with exposure. lopic 2. Future Studies Further case-control and cohort mortality studies are needed, both of industrial and professional workers exposed to formaldehyde and of persons exposed to formaldehyde in mobile and other homes. These studies should include estimates of the degree of exposure to formaldehyde and other chemicals; the analyses should take into account various possible induction times for a formaldehyde effect, as well as possible confounding by smoking habits and alcohol consumption. The possibil- ity that there may be an inverse relation between CONSENSUS WORKSHOP ON FORMALDEHYDE 341 of 235 rats of both sexes (83). About 80% of both sexes at this dose level survived 2 years and, therefore, were at risk to late developing lesions. No nasal tumors, other than polypoid adenomas, were found in the same experi- ment in rats exposed to concentrations of 2 ppm. greater effective target site doses. This panel considers differences in ambient air concentrations versus dose to target sites to be a likely explanation for the apparent nonlinearity in concentration response. y p la. Rodents exposed to formaldehyde by inhalation showed clear indications of cell damage in the nasal turbinates at concentrations that induced squamous cell carcinomas (90). The cytotoxic response was character- ized by cell death, restorative cell proliferation and hyperplasia, and occurred in the same regions of the nasal passages that developed squamous cell carcinomas. This resulted in alteration of the nasal epithelium from a tissue with low cell replications (i.e., 0.1-0.5% of the cells) to one with increased cell replication in the early time points sampled. Concentration has been shown to play a greater role than the concentration-time product in both acute studies and following exposure of rats for up to six months. No squamous cell carcinomas were observed at concentrations inducing less cytotoxicity. p pp For mice, only one adequate study has been reported in which 2 out of a total of 215 mice exposed to a concentration of 14 ppm developed nasal cancers after 24 months of exposure (83). Although not statistically significant in comparison with matched control mice, the finding is considered strongly suggestive in compari- son to historic control mice in which nasal cancers are rare. The difference in susceptibility of rats and mice might be attributed to greater reductions in respiratory minute volume in mice than rats during exposure to the irritant vapor (85). lopic 2. Future Studies p Eighty-eight male Syrian hamsters exposed to 10 ppm formaldehyde vapor, 5 hr/day, 5 days/week for their lifetime had no detected respiratory cancers (86). However, only two microscopic sections of the nose were examined for each animal. In another experiment by the same group of investigators, male hamsters exposed to 30 ppm formaldehyde for 5 hr a day, once a week for lifetime also had no tumors observed in the respiratory tract. When male hamsters received a 5-hr exposure to 30 ppm formaldehyde 2 days prior to each of 10 weekly injections of diethylnitrosamine (DEN), the average number of tracheal tumors per tumor-bearing animal was reported to be increased as compared with those hamsters receiving DEN alone. Inspection of the data presented in the report, however, showed a correspond- ing decrease in lung tumors in hamsters exposed to both agents, suggesting that the effect on the trachea may be within the limits of experimental variability. The reported cocarcinogenicity requires confirmation. g y y The implications of these findings are that cytotoxic- ity might influence both initiation and promotion. If more cells of the target tissue replicate, there is a greater availability of single-stranded DNA for adduct formation, decreased time for repair of DNA adducts, and a greater chance of fixation of mutagenic events. Increased cell proliferation also contributes to tumor promotion. Thus, there is a greater likelihood of tumor development with the increased cell proliferation associ- ated with cytotoxic exposures. Other chemicals classi- fied as tumor promoters (i.e., nitrilotriacetic acid, phenobarbital, saccharin and 2,3,7,8-tetrachlorodibenzo- p-dioxin) also induce tumors primarily at doses which result in cytotoxicity and chronic hyperplasia in target organs. These factors are likely to contribute to the nonlinearity of the dose response, i.e., proportionately greater response at higher doses. p g y q The available data indicate that hamsters are much less susceptible to the carcinogenic effects of formalde- hyde than rats and that mice are intermediate in response. It should be emphasized, however, that the data available for species comparisons are limited to very few experiments. It may be relevant that acetal- dehyde, a structurally related compound, is carcino- genic for the nose and larynx of Syrian hamsters (87) and for the nasal passages of rats (88). g lb. Benign Tumors. Carcinogens induce both be- nign and malignant tumors. Carcinogens which induce benign tumors almost always induce malignant tumors in the same experiment or other experiments. lopic 2. Future Studies However, in a more recent dominant lethal assay using higher doses and a different mouse strain, margin- ally positive results were obtained, but only in the first and third week of the seven weeks studied (113). Negative results were obtained when the induction of micronuclei (114 or chromosomal aberrations (97,113) were used as an endpoint. pp I b. Potential Preneoplastic Lesions. Preneoplastic lesions may be induced by carcinogens. The morpho- logic, histochemical and biologic properties of these lesions are best studied by serial sacrifice experiments and reversibility studies. In these types of studies, specific lesions may be characterized and a sequence of the histopathogenesis of cancer postulated. Some infor- mation of this type is available for formaldehyde. Based on incidence in serial sacrifice studies, epithelial dyspla- sia and squamous metaplasia in rats and mice exposed to formaldehyde appear to regress after stopping the carcinogen exposure. It is uncertain if these reversible lesions are potentially preneoplastic, but their reversibil- ity suggests that they are not committed to progress. More severe and typical lesions occurred only in the highest dose groups where tumors occurred. Adequate data are not available to draw any conclusions on regression or progression of the latter potentially preneoplastic lesions in the formaldehyde bioassay. p A small increase in sister chromatid exchanges has been reported in the bone marrow of mice exposed to high (> 25 ppm) formaldehyde concentrations. Unfor- tunately, technical problems were encountered during the formaldehyde exposures and the actual concentra- tions required to elicit this effect are not known (95,115). ( , ) The observation that formaldehyde is both genotoxic and carcinogenic suggests that in vivo exposures cause irreversible genetic alterations causally related to the acquisition of malignancy. In vitro studies suggest, however, that the genotoxic properties of formaldehyde are relatively weak. One can thus question whether promotional influences play a major role in the etiology of formaldehyde-induced neoplasms. Studies conducted to date suggest that formaldehyde possesses only weak activity for the promotion of transformation in cultures of C3H/1OT1/2 cells (116) and similar weak promoting activity on mouse skin (117; Ward, personal communi- cation). A second study on mouse skin failed to detect promotion following the application of nonirritating doses of formaldehyde (118). In addition, the exposure of DEN-treated hamsters to 30 ppm formaldehyde (86) was reported to increase the incidence of DEN-induced tracheal adenomas. lopic 2. Future Studies Promoters exhibit marked tissue and species specificity and the possibility that formalde- hyde might possess strong tumor-promoting properties in the rat nasal cavity cannot be excluded. TIeatment with carcinogenic concentrations of formaldehyde in- duces extensive cell proliferation, a phenomenon re- ported to be sufficient for the promotion of tumorigene- sis on mouse skin (119,120). p p y y Ic. The 1977 review of Auerbach et al. of the literature on the biological effects of formaldehyde (91) indicated that the ability of formaldehyde to cause genetic alterations in Drosophila larvae, fungi, and bacteria was known as early as the 1940s. Since 1977, new studies have demonstrated that formaldehyde can induce single-strand breaks in DNA (92-94), DNA- protein crosslinks (92-96), sister chromatid exchanges (97,98), and chromosome aberrations (99). It has also been shown to induce mutations in bacteria (100-102), yeast (103-105), Drosophila (106), mammalian cells (97,107) and human cells (108). In vitro evidence of formaldehyde's carcinogenic potential is provided by its ability to transform BALB/c 3T3 mouse cells (97), BHK 21 hamster cells (107) and C2H-1OT1/2 mouse cells (100,109) to enhance the transformation of Syrian hamster embryo cells by SA7 adenovirus (110), and to inhibit DNA repair (111). , Most importantly, perhaps, the mouse spot test is reported to be negative in two separate studies (115,120), although the full data were not available to the panel. In one of these studies (115), difficulties were encountered in regulating the levels of formaldehyde exposure and levels in excess of 25 ppm probably occurred. Further- more, while genotoxic effects were not observed in this study, there was some suggestion of embryotoxicity as evidenced by the production of white midventral spots. Still, since this assay is considered a good assessment of the ability of an agent to cause systemic effects, these negative results suggest that germ-cell assays would also be negative. Thus, there may not be a need for additional studies such as the specific locus test and the heritable translocation test. In reviewing the above literature, we have found that the recent work is more likely to find formaldehyde a mutagen than earlier studies and is also more likely to show a dose-response relationship. These results are most probably attributable to the greater sophistication in the way the later assays were carried out. lopic 2. Future Studies Benign tumors in rodents may progress to malignant tumors. It is impossible to predict whether or not a specific benign tumor will progress to a malignant tumor. In the case of formaldehyde (83), a high incidence of malignant tumors (squamous cell carcinomas) was in- duced in rats exposed to 14 ppm formaldehyde gas. Papillomas, which the panel believes represent the benign counterpart of the squamous cell carcinoma, were not observed. A second type of benign lesion, polypoid adenoma, occurred in all exposure groups and one control animal. Six of the 20 lesions originally diagnosed as polypoid adenomas in the study conducted at the Chemical Industry Institute of Toxicology (CIIT) (83) were reviewed by pathologists on the panel and in the audience. They considered the lesions to represent a mixture of polypoid adenomas, nonneoplastic polyps and hyperplasias. (Subsequent independent review of the lesions originally diagnosed as polypoid adenomas in the CIIT study essentially confirmed the presence of No compound-related skin cancers were found in any of the inhalation bioassays. y Several studies on the pathogenesis of formaldehyde- induced nasal cancer have been completed (89). Data from these are included in this panel's report as well as the report of the Structure Activity/Biochemistry/ Metabolism Panel. Collectively, these suggest that the toxic effects offormaldehyde are proportionately greater at high concentrations (i.e., 6-15 ppm) than at lower concentrations (i.e. < 2 ppm). For example, increasing the exposure concentration of formaldehyde from 6 to 14 ppm, less than a 3-fold increase, resulted in a 50-fold increase in squamous cell carcinomas in rats. Possible mechanisms for this include impairment of mucociliary clearance, detoxification, and DNA repair leading to CONSENSUS WORKSHOP ON FORMALDEHYDE 342 polypoid adenomas in the study, although a few lesions were considered to be borderline between adenoma and hyperplasia.) The panel recommends that this group of lesions not be combined with squamous cell carcinomas for risk estimation because of the differences in the cell type of origin. The polypoid adenomas can be evaluated separately and in combination with the nonsquamous carcinomas that were observed in the 14 ppm rats. Various studies have been undertaken to determine whether formaldehyde has a genotoxic effect in vivo. In mice, the dominant lethal test was found to be negative (112). * Immunology/Sensitization/Irritation Panel: Jack Pepys (Chair- man); Paul Nettesheim (Alternate Chairman/Secretary); Yves Alaire; James R. Beall; Jack H. Dean; K. C. Gupta; Michael D. Lebowitz; Howard Maibach; Roy Patterson. Topic 2. Future Studies Formaldehyde-DNA adducts have recently been par- tially characterized. Also, formaldehyde-induced DNA- protein crosslinks are reported to exist (Structure Activity/Biochemistry/Metabolism Panel). However, the biological relevance ofthese formaldehyde-induced DNA lesions has not been ascertained. Future studies should determine whether such adducts preferentially form on single stranded DNA within cells, what repair mecha- nisms remove such lesions, and whether the DNA lesions are associated with the cytotoxic, mutagenic, and transforming activity of formaldehyde. y g There is no convincing experimental evidence that formaldehyde has primary toxic effects at body sites distant from the site of exposure. lopic 2. Future Studies It should be noted that in the above studies, the relationship between cytotoxicity induced by formaldehyde and mutagenicity (108) or transformation (100,107,109) in- duced by this agent is typical of most mutagens/carcino- gens that are positive in these assays. The data we have reviewed are consistent with formaldehyde acting as a weak mutagen (i.e., less than a 10-fold increase over background). Id. There is no convincing evidence in experimental animals that inhalation exposure causes significant CONSENSUS WORKSHOP ON FORMALDEHYDE 343 primary toxicologic effects in organs other than the upper respiratory tract. either separately (from squamous carcinomas), or in combination with nonsquamous carcinomas. q Formaldehyde is genotoxic in a number of assays and is weakly mutagenic in human cells in culture as well as in other mammalian cells, Drosophila, fungi and bac- teria. Formaldehyde is a weak promoter in cell culture and on mouse skin. In vivo studies in mice are, in the main, negative, but marginally positive results have sometimes been seen at very high doses. Immunology/Sensitization/ Irritation Panel Report* p Topic 1. What is the significance of reports that formaldehyde causes irritation and/or sensitization fol- lowing topical or inhalation exposure? g y y Comparative molecular dosimetry studies following formaldehyde inhalation exposure are urgently needed to evaluate the relationship between exposure and dose to target site. g p p a. Is formaldehyde a primary sensitizing agent or does it elicit a response only in presensitized populations? g Additional carcinogenicity studies in rats at doses above, below, and between 2 and 14 ppm would be beneficial in better defining the dose-response curve. p p b. If irritation, primary or secondary sensitization occurs, who are the susceptible populations? g p Since rodents are obligate nose breathers, compara- tive toxicity/carcinogenicity studies in primates which have oronasal breathing patterns similar to man would be useful to further validate dosimetry studies as a means of carcinogenic risk assessment. , p p p c. What are the possible mechanisms for formalde- hyde-induced irritation/sensitization? / y d. Do threshold levels exist for irritation/sensiti- zation? If so, what are the levels and the concentra- tion ranges? Do thresholds differ for different populations (sensitized)? g An animal model for initiation/promotion in the nasal epithelium should be developed. p Since formaldehyde and acetaldehyde are nasal carcinogens, information should be collected on struc- ture-activity relationships of other aldehydes. p p e. Is there evidence for effects of formaldehyde on the immune system? i 2 i i y Topic 2. What experiments would be most important to resolve any controversies in this area? Topic 2. What experiments would be most important to resolve any controversies in this area? ToDpic 1. Significance of Sensitization Reports Asthma is defined here as bronchial hyperactivity to specific immunological or nonspecific, nonimmunological factors. These can have effects one upon the other in clinical circumstances. Clinical obser- vations and controlled bronchial provocation tests have shown a heterogeneity of patterns of asthmatic reac- tions. These can be immediate or nonimmediate. The immediate reactions have features of Type I, IgE and IgG (STS) reactions. py lb. Irritation and allergic contact dermatitis to formaldehyde occur. Although there is extensive litera- ture on the effects of age, sex, race, and other factors on the ability of chemicals to induce allergic contact sensitization and irritation, there is no specific informa- tion on these matters for formaldehyde. y With respect to inhalant sensitization to formalde- hyde, it is not known whether there are susceptible groups in the population. Conceivably, asthmatics might be more sensitive to the irritant action of formaldehyde, triggering bronchial reactions (38,75,121,127,136,153, 161,163). Nonimmediate asthmatic reactions have three forms. One comes on after 1 hr, is maximal at about 2 to 3 hr, and resolves in 5 hr. The next, and most common, comes on after several hours, is maximal at about 5 to 8 hr and resolves by 16 hr. Then there is the nocturnal reaction, coming on in the early hours of the morning and capable of recurrence at the same time each night for many nights, following a single challenge exposure as re- ported by Hendrick and Lane (123) in nurses using formalin for disinfection in a dialysis unit. The different patterns of asthmatic reaction may be present together. There is no evidence, however, that these patterns are necessarily due to the types of allergy they may resemble. In chronic dialysis patients sensitization to formalde- hyde has been reported resulting from its release into the bloodstream from equipment and tubing sterilized with formaldehyde (171-175). y Ic. Cutaneous Exposure. Formaldehyde skin irrita- tion is nonimmunologic; studies as to how its mecha- nism differs from other forms of cutaneous irritation are not available. Formaldehyde allergic contact dermatitis presumably requires conjugation with protein and is taken to be a classical type of delayed hypersensitivity. Formaldehyde-induced contact urticaria has been docu- mented and is presumably a Type I allergy. Nonim- munologic contact urticaria which requires multiple applications on the same site has been documented (144). Information on mechanisms is lacking. 1. ToDpic 1. Significance of Sensitization Reports Formaldehyde gas is carcinogenic for rats and proba- bly for mice, producing nasal tumors after inhalation. Limited experiments in Syrian hamsters have not demonstrated carcinogenicity. In rats, the carcinogenic response appears nonlinear, being disproportionately higher at the higher concentrations (14 ppm). Any assessment of reports on irritation by and/or sensitization to formaldehyde requires some definition of terms. Allergy can be defined as the acquired, specific, altered capacity to react. The general criteria suggestive ofthis are: a period ofexposure to nonirritant or irritant concentrations, i.e., the period of sensitiz- ation; the elicitation of reactions by small amounts, far below those capable of inducing sensitivity or irritant effects. This is a distinguishing feature between irrita- tion and sensitization. Last, only a'few exposed subjects are likely to be sensitized. This is in contrast to irritant effects which are likely to occur in most subjects who are more heavily exposed to irritant concentrations. Demonstration of relevant, appropriate, humoral anti- bodies or sensitized lymphocytes provides strong evi- dence that the reaction is allergic. Cell killing and the reparative process that occurred in the inhalation bioassays may contribute to carcino- genicity, but the specific role of cytotoxicity in formalde- hyde carcinogenicity is unknown. Cytotoxicity is not unique to formaldehyde bioassays and may play a role in the carcinogenicity of other chemicals as well. g y A sample of the benign lesions observed in the Chemical Industry Institute of Toxicology (CIIT) study, originally diagnosed as polypoid adenomas, was re- viewed by the panel and was considered to be a mixture of polypoid adenomas, nonneoplastic polyps and hyper- plasias. Uncertainties about the nature of the remain- ing lesions in the study made the lesion unsuitable for use in risk assessment. Subsequent to the Consensus Workshop, an independent review essentially confirmed the original diagnoses in the CIIT study, indicating that they were then suitable for use in risk assessment, g Four different types of allergy can be induced. CONSENSUS WORKSHOP ON FORMALDEHYDE 344 Type I. Immediate, anaphylactic reactions, coming on in minutes, are maximal in about 10 to 15 min and last about 1 to 1.5 hr. These reactions are mediated by IgE antibody, mainly in atopics, but also by IgG (short-term sensitizing) antibody. "Atopy" is defined here as the production of specific IgE to one or more common allergens with or without symptoms, a feature found in many studies in about 40% of individuals tested. ToDpic 1. Significance of Sensitization Reports gas or formaldehyde products, or both, and in which allergic reactions were elicited (123,152,160-165). The interpretation of the findings is uncertain since ques- tions about the nature of the materials, the methods of testing and the statistical analysis of the data remain unanswered. la. Formaldehyde is definitely a primary skin sensi- tizing agent inducing allergic contact dermatitis (Type IV allergy) and probably immunologic contact urticaria (probably Type I allergy) (38,75,121,135,136,145,149, 154,155,166-170). Type II. The antigens are altered body constituents, and where chemical haptens are involved, the antibod- ies are directed against the hapten-carrier complex. Type II. The antigens are altered body constituents, and where chemical haptens are involved, the antibod- ies are directed against the hapten-carrier complex. There is extensive experience suggesting that human beings with allergic contact dermatitis to one chemical are, in some situations, more likely to develop allergic contact dermatitis to another. On the other hand, other situations exist in which sensitization to one chemical iterferes with sensitization to a second. This panel is not aware of any controlled study that documents the ability of any other chemical to influence the sensitization rate to formaldehyde. g p p Type III. Immune-complex complement-dependent allergy is mediated by precipitating antibodies. The reactions come on after several hours, are maximal at about 5 to 8 hr and resolve in about 24 hr. They are preceded in skin tests by an immediate, Type I introduc- tory reaction. y Type IV Delayed-type allergy is mediated by sensi- tized T-lymphocytes. It is the allergy of contact dermati- tis and develops after about 24 hr, being maximal at about 48 to 96 hr and resolves slowly. The different types of allergy may be present together. y Type IV Delayed-type allergy is mediated by sensi- tized T-lymphocytes. It is the allergy of contact dermati- tis and develops after about 24 hr, being maximal at about 48 to 96 hr and resolves slowly. The different types of allergy may be present together. y It is not known whether populations already sensi- tized to chemicals other than formaldehyde are at greater risk for inhalant sensitization to formaldehyde or its products. Assessment of a role for presensitization in people exposed to formaldehyde needs study in terms of atopy. Studies with reactive low molecular weight chemicals have shown the participation of Types I and III allergy in allergic respiratory diseases such as rhinitis, asthma, etc. (121,122). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 345 develop short-term tolerance (31,125,130,180,181). There is evidence in experimental animals that with repeated exposure increased responses are obtained (130). develop short-term tolerance (31,125,130,180,181). There is evidence in experimental animals that with repeated exposure increased responses are obtained (130). induction of allergic contact dermatitis in man has only been imprecisely determined as less than 5% formalin in water. The approximate thresholds for elicitation of allergic contact dermatitis in sensitized subjects to formaldehyde range from 30 ppm for patch testing to 60 ppm for actual use concentrations of formalin. Because of the small data base these values must be regarded with caution until further studies become available (144). p p ( ) It requires higher concentrations of formaldehyde in the air to stimulate bronchial receptors, since the water-soluble formaldehyde reacts with and is retained in the upper respiratory tract (175). pp p y At even higher concentrations, receptors in the peripheral lung tissues (175), in the conducting airways and the alveoli, have been stimulated in animals (131,177,182,183). To achieve this, nasal catheters were used to bypass the upper airways. Stimulation of receptors in the peripheral lung might occur if small particulate material from urea, phenol or other formal- dehyde products and pressed wood dusts were depos- ited in these regions of the lung followed by hydrolytic release of formaldehyde (182,184). Other chemical com- ponents would have to be excluded as possible causes before the effects could be attributed to released formaldehyde. ( ) The threshold for induction of immunologic contact urticaria has not been determined. For the induction of repetitive nonimmunologic contact urticaria is under 1% formalin in water or less (144). Id. Inhalation Exposure. Precise thresholds have not been established for the irritant effects of inhaled formaldehyde. However, within the range of 0.1 to 3 ppm, most people experience irritation of the eyes, nose, and throat (78,79,124,125,127,130,153,179,181, 202-205). Between 10 and 20 ppm, symptoms are severe, and it becomes difficult to take a normal breath (206). Lower airways and pulmonary effects are likely to occur between 5 and 30 ppm. Exposures between 50 and 100 ppm cause serious injury to the respiratory tract, such as pulmonary edema, inflammation, pneumonitis and pneumonia (130,136). Asthmalike symptoms have been elicited by irritant concentrations. y At least three mechanisms exist whereby low molecu- lar weight chemicals may cause sensory irritation (185), i.e., nucleophilic addition, disulfide bond cleavage and physical interaction. CONSENSUS WORKSHOP ON FORMALDEHYDE In the case of formaldehyde, nucleophilic addition is probably the most important mechanism. The reactivity is directed for example toward SH or NH2 groups in proteins. Formaldehyde reacts with SH and NH2 groups in a reversible way. The interaction with SH is less reversible than that with NH2 (186). y There is no information regarding threshold values for sensitization to formaldehyde as an inhalant allergen. y g le. Only two animal studies on the potential toxic effects of inhaled formaldehyde on the immune system have come to our attention (207,208). No impairment of immune functions was found except in one of the studies where lethal concentrations were used (208). Inflammation (with Cellular and Tissue Damage). a at o ( t g ) Experiments in animals show that cellular damage and inflammation is induced with increasing severity at concentrations of formaldehyde of 1 to 15 ppm. Expo- sure for several hours is required and is accompanied by impaired ciliary activity (176,187). ToDpic 1. Significance of Sensitization Reports There are numerous reports that formaldehyde vapor exposure causes direct irritation of both the skin and respiratory tract (36,38,124-144). By comparison the evidence for allergic airway responses to formnalde- hyde is less extensive (75,121,126,131,136,138,145-159). Sufficiently well-controlled scientific studies are not available to definitively establish the development of respiratory tract allergy to formaldehyde gas per se. There are a few clinical reports in which tests were made for respiratory sensitivity to either formaldehyde g Ic. Inhalation Exposure. Irritation and potential predisposition to infections are seen. Two types of irritation can be caused by formaldehyde: sensory; and inflammation (175,176). Sensory. Receptors of eyes, nose and throat may be affected (36,38,124,127,177-179). Man and animals can Topic 2. Future Studies. Cutaneous. Future studies should include defining the operational criteria for scientific documentation of the diagnosis of allergic contact dermatitis in man; determining the amount of formaldehyde required to induce and elicit the following reactions in man: irritant dermatitis, allergic contact dermatitis, nonimmunologic contact urticaria and contact urticaria; ascertaining the cross reaction pattern in allergic contact dermatitis in animal and man to formaldehyde-related chemicals; utilizing the above information, ascertaining popula- tions susceptible to irritation, allergic contact dermati- tis and immunologic and nonimmunologic contact urti- caria; determining the relationship in allergic contact dermatitis between the degree of patch test reactivity and intolerance to actual product use. i p y Earlier studies implicate formaldehyde as a potential factor predisposing particularly children to respiratory tract infections (181,188-190). Appropriate epidemiologi- cal and animal studies need to be designed using specific criteria for infection and better protocols of the type used to investigate other irritant gases, such as O3, SO2, and NO2 (177,191-193). ic. Systemic Sensitization. Sensitization arising from release of formaldehyde into the circulation during dialysis has been reported. This is associated with frequent eosinophilia and some severe hypersensitivity and asthmalike reactions (194,195). The presence of auto-anti-N-like antibodies reacting with formaldehyde conjugated red blood cells is evidence of Type II auto-allergy (171-174,196-201). The anaphylactic reac- tions are suggestive of lype I allergy. ic. Systemic Sensitization. Sensitization arising from release of formaldehyde into the circulation during dialysis has been reported. This is associated with frequent eosinophilia and some severe hypersensitivity and asthmalike reactions (194,195). The presence of auto-anti-N-like antibodies reacting with formaldehyde conjugated red blood cells is evidence of Type II auto-allergy (171-174,196-201). The anaphylactic reac- tions are suggestive of lype I allergy. Inhalation. Controlled cohort epidemiological stud- ies of exposed and unexposed persons with particular emphasis on the formulation and application of proto- cols should be conducted. The application of standard- ized procedures is recommended. Clinical studies should use basic protocols for pulmo- nary function studies and for testing for specific and nonspecific sensitivity. Comparisons of gaseous and Inhalation. Controlled cohort epidemiological stud- ies of exposed and unexposed persons with particular emphasis on the formulation and application of proto- cols should be conducted. The application of standard- ized procedures is recommended. Inhalation. Controlled cohort epidemiological stud- ies of exposed and unexposed persons with particular emphasis on the formulation and application of proto- cols should be conducted. The application of standard- ized procedures is recommended. * Structure Activity/Biochemistry/Metabolism Panel: Sidney Wein- house (Chairman); James E. Gibson (Alternate Chairman/Secretary); Joseph Arcos; Ruth E. Billings; Henry d'A. Heck; Thomas R. Tephly; Andrew G. Ulsamer; Phillip G. Watanabe. Background for the information in this report may be found in the data base in Appendix I to the Consensus Workshop document and in the references (38,49, 141,143,186,209-211). Topic 1. Metabolism of Formaldehyde It is mutagenic in three strains of Salmonella typhimurium and initiates neoplastic transformations in C3H/1OT1/2 mouse fibroblasts when treated together with the tumor promoter, 12-0-tetradecanoyl phorbol 13-acetate (TPA). Formaldehyde at concentrations of 0.017 to 0.083 mM also caused transformation of BALB/c3T3 and baby hamster kidney cells and caused mutations in cultured human lymphoblasts at concentrations greater than 0.13 mM and chromosomal aberrations in diploid human fibroblasts at 2 to 8 mM. Topic 1. Metabolism of Formaldehyde la. There is now a large and growing body of data which infers that formaldehyde, like many other chemi- cal carcinogens, acts as an electrophile with macromole- cules such as DNA, RNA and protein to form reversibly bound adducts or irreversible crosslinks and that the modifications in DNA caused thereby are relevant to the neoplastic transformation. p y A larger data base should be established on sensory and irritant thresholds in normal subjects in all age groups including individuals with respiratory tract symptoms. y p Laboratory studies need to be conducted to search for serological and cellular antibody activity in groups who are or appear to be sensitive to formaldehyde and in particular in the hemodialysis patient population in whom allergic sensitivity to formaldehyde has been reported. This may provide a basis for immunological studies of respiratory tract responses to formaldehyde. Animal models need to be developed for observations of biological effects of differing formaldehyde exposures and for studies of the mechanisms of formaldehyde irritation and sensitization. Laboratory studies need to be conducted to search for serological and cellular antibody activity in groups who are or appear to be sensitive to formaldehyde and in particular in the hemodialysis patient population in whom allergic sensitivity to formaldehyde has been reported. This may provide a basis for immunological studies of respiratory tract responses to formaldehyde. p lb. Formaldehyde is not unique as a carcinogenic aldehyde, since other aldehydes, such as acetaldehyde, acrolein, malondialdehyde and glycidaldehyde are also carcinogenic. It is likely, though not certain, that all of these aldehydes have a common mode of action: through modification of DNA by formation of adducts or cross- links. Differences in the degree and tissue targets of carcinogenesis are readily attributable to differences in activities and localizations of activating and/or compet- ing enzyme systems, and to differences in physical properties, solubilities and partition between cell membranes. p y p y Animal models need to be developed for observations of biological effects of differing formaldehyde exposures and for studies of the mechanisms of formaldehyde irritation and sensitization. Chemical characterization of formaldehyde products is needed. The behavior of such products in the atmo- sphere and their biological effects and interrelation- ships need to be investigated. la. Supporting a role ofDNA modification in formal- dehyde toxicity is its clear-cut mutagenic activity. CONSENSUS WORKSHOP ON FORMALDEHYDE 346 particulate materials for provocation test purposes are needed. Prospective comprehensive surveillance stud- ies correlated with formaldehyde exposure measure- ments should be made, with detailed criteria to docu- ment the health hazards. bolism recognizes as a basic challenge the question of how a vitally important intermediate in the normal metabolism of cells, a compound which serves as a building-block for the synthesis of purines, pyrimidines, many amino acids and lipids, a key molecule in one- carbon metabolism which is present in normal tissues, can induce cancer in animals on inhalation. Sulfur dioxide and 03 exposure of animals has been shown to promote airway infection to bacterial and viral agents. Similar studies should be conducted with formaldehyde. Appropriate epidemiological studies with acceptable criteria for respiratory infection should be conducted to determine the effects of formaldehyde on respiratory infections in humans. Topic 2. Future Studies. Clinical studies should use basic protocols for pulmo- nary function studies and for testing for specific and nonspecific sensitivity. Comparisons of gaseous and gg yp gy Id. CutaneousExposure. Threshold levels have been reported for cutaneous irritation and allergic contact dermatitis in the guinea pig and man. For human skin, a single application of 1% formalin in water with occlusion will produce an irritant response in approximately 5% of the population (144). The threshold for open application has not been determined. The threshold level for the Id. CutaneousExposure. Threshold levels have been reported for cutaneous irritation and allergic contact dermatitis in the guinea pig and man. For human skin, a single application of 1% formalin in water with occlusion will produce an irritant response in approximately 5% of the population (144). The threshold for open application has not been determined. The threshold level for the Clinical studies should use basic protocols for pulmo- nary function studies and for testing for specific and nonspecific sensitivity. Comparisons of gaseous and CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 347 to dosage, in contrast with the linear response of DNA-protein crosslinks at 6 ppm and above, suggests a role for nongenetic factors in formaldehyde carcino- genesis. However, it should be recognized that only certain specific sites on DNA might lead to carcino- genesis. Moreover, the rapid and nondiscriminate attack of formaldehyde on tissue components could involve membrane function, transport and repair processes, and it is known that mucociliary removal of formalde- hyde is inhibited at high concentrations. In view of this complex network ofreinforcing and competing reactions, it is not surprising that the carcinogenic response is not linear or that it does not parallel the degree of cross- links. Although the cause is still uncertain, this appar- ent nonlinearity cannot be construed to indicate the existence of a threshold for exogenous formaldehyde carcinogenicity. Although nongenetic factors may play a role, more sensitive methods will be required to deter- mine whether a linear relationship between dose and response exists at low exposure levels. The mutagenic- ity of formaldehyde in a variety of experimental sys- tems and the formation of DNA adducts and cross links both in vivo and in vitro point to modification of DNA or in the repair of damaged DNA as critical steps in the induction of cancer by formaldehyde. nine and other methyl-containing substances, and all of these can be formed by the many reversible reactions involving tetrahydrofolate derivatives. Metabolic reac- tions involving tetrahydrofolic acid (THFA) derivatives allow formaldehyde carbon to enter virtually every metabolite in the cell. The total concentration of formaldehyde, free and bound, in freshly collected livers of F-344 rats is 0.1 to 0.2 pumole/g fresh weight, very little of which is in the form of N ,N10-methylene THFA. , y Oxidation. The major pathway of formaldehyde oxidation is to formic acid, for which three enzymatic pathways are known. By far the most important is catalyzed by formaldehyde dehydrogenase, which oxi- dizes the formaldehyde-glutathione adduct with NAD + to yield S-formylglutathione and NADH. The former is hydrolyzed to formate and glutathione by a hydrolase present in excess. The affinity of formaldehyde for formaldehyde dehydrogenase in respiratory and olfac- tory mucosa is 2.5 ,uM, which is in the range of normal formaldehyde concentration, whereas the nonspecific aldehyde dehydrogenase, has an affinity 200-fold lower; completely outside the range for physiological sig- nificance. CONSENSUS WORKSHOP ON FORMALDEHYDE Formaldehyde is also oxidized to formate by hydrogen peroxide, catalyzed by catalase, but the quantitative significance and sites of action are not known. The exclusive formation of nasal tumors in animals exposed to formaldehyde does not signify any special susceptibility and can only be attributed to its location at the site of entry of gaseous formaldehyde. ic. Formaldehyde disappears from the plasma with a half-time of about 1 to 1.5 min. Removal is so rapid that an increase cannot be detected in the plasma of animals or humans immediately following inhalation exposure to high concentrations. In intact animals, a high percentage of the formaldehyde administered is converted to CO through formate with smaller amounts excreted in the urine as formate and several other minor metabolites. y g y The reason why endogenous formaldehyde, formed constantly during normal metabolism and occurring at low concentrations in body fluids, is apparently not harmful still remains a mystery. The mystery is com- pounded by the fact that exposure to formaldehyde does not lead to any appreciable rise in body fluid levels. More information is required on the metabolism and pharmacokinetics of exogenous formaldehyde. Although there are differences in formaldehyde carcinogenicity among different species, at present there is no reason to assume that humans would be more or less susceptible than the rat. As with many other carcinogens, there are no interspecies differences in metabolism that would provide information on possible differences in the direc- tion or magnitude of carcinogenic responses. Despite its rapid removal, the possibility exists that transient increases in formaldehyde may occur in the intact animal. The panel could not exclude the possibil- ity that formaldehyde may be transported to, and exert toxic effects at, distant sites following inhalation, but definitive evidence for effects of formaldehyde per se at distant sites is lacking. g Id. Formaldehyde from Xenobiotics. Besides its formation from normal metabolites, formaldehyde is produced in the oxidative demethylation of drugs and other foreign substances by the microsomal cytochrome P-450 monooxygenases. Such endogenous formaldehyde may exert toxic effects; for example, hexamethylphos- phoramide is a potent nasal carcinogen and a mutagen acting, presumably, by conversion of its methyl groups intracellularly to formaldehyde. Other substances that may be toxic by virtue of conversion to formaldehyde would be methanol and methyl chloride, but existing evidence for a toxic role of formaldehyde is to the contrary. Structure Activity/Biochemistry/ Metabolism Panel Report* Topic 1. What is known about the metabolism and fate of exogenous and endogenous formaldehyde in experi- mental animals and man? a. What are the data concerning binding of formalde- hyde or its metabolic products to cellular macro- molecules? Reactivity. Formaldehyde reacts virtually instanta- neously with primary and secondary amines, thiols, hydroxyls, and amides to form methylol derivatives, which in some instances can lose water to form the corresponding Schiff bases. The thiols have a somewhat higher affinity for formaldehyde than amines and ad- ducts with glutathione or beta-mercaptoethanol are quite stable. b. Are there common biological effects induced by short-chain aldehydes that can be predicted on the basis of structure activity relationships? y p c. What are the quantitative relationships between exposure levels and concentrations of formalde- hyde in particular tissues and organs? y p g d. Are there compounds that exert an effect by forming formaldehyde during metabolism? q Formaldehyde forms unstable adducts with DNA, RNA and proteins, but when crosslinked, these are far more stable. DNA-protein adducts have been demon- strated in several eukaryotic and prokaryotic cell types, and are formed at the same concentrations of formalde- hyde that cause mutation and cell transformation. Topic 2. What future studies would resolve uncertain- ties in this area? / / The Panel on Structure Activity/Biochemistry/Meta- y Metabolism. Formaldehyde is normally formed endo- genously, as the N5,N10-methylenetetrahydrofolic acid, which equilibrates rapidly with other tetrahydrofolate derivatives. The major sources are serine and glycine; however, other sources are N-methylamino acids, methio- CONSENSUS WORKSHOP ON FORMALDEHYDE * Reproduction/Teratology Panel: Godfrey P Oakley, Jr. (Chairman); Rochelle Wolkowski-Tyl (Alternate Chairman/Secretary); Sarah H. Broman; Thomas F Collins; Carole A. Kimmel; Thomas A. Marks. CONSENSUS WORKSHOP ON FORMALDEHYDE 348 (212) intubated pregnant mice on days 6 through 15 of gestation with 0, 74, 148 or 185 mg/kg/day. At the highest dose, 22 of 34 pregnant mice died. At that dose, there was an increased incidence of resorptions, but that increase was not statistically significant. At no dose did the incidence of malformations differ between the treated and control groups. There were also no treatment-related differences in the mean number of implantations, stunted fetuses, live fetuses per litter, or average fetal body weight per litter. At a dose which killed more than 50% of the dams, no adverse reproduc- tive outcomes were observed except for the increase in the incidence of resorptions that was not statistically significant. There is a need for human studies and for a model of the human upper respiratory airway system to deter- mine rate constants for absorption, transport, and metabolism. This should include studies of formalde- hyde bound to airborne particulates. y p Information is needed on degranulation of rough endoplasmic reticulum caused by formaldehyde and its effect on the membrane thiol-disulfide exchange enzymes. g y Other questions concern whether chromosome breaks are related to formaldehyde dose; whether formalde- hyde induces DNA repair enzymes and mixed function oxidases in nasal epithelium; whether the nasal carcino- gen, hexamethylphosphoramide is carcinogenic systemi- cally and whether abnormal dietary fatty acids can so alter membrane structure as to affect cross-linking in glutathione-depleted animals. g Ic. Although the Marks study (212) exposed ani- mals to high enough doses to kill the dams, it is not likely that embryos were exposed to formaldehyde itself due to the very short biological half-life (142). Another approach used to investigate whether formaldehyde is teratogenic has been to give hexamethylenetetramine (HMT), which is metabolized to formaldehyde in vivo. Hurni and Ohder (213) exposed pregnant beagle dogs to 1250, 600 or 0 ppm HMT in the feed during days 4 through 56 of gestation. At 1250 ppm, 10 of 56 pups were stillborn, the growth rate between birth and weaning was decreased, and there was an increase in early postnatal mortality. There were no malformed pups. In a study in rats by Della Porta et al. (214), exposure to HMT at 1% in drinking water from 2 weeks before pregnancy through weaning produced no mal- formed animals. Pups drinking the same solution after weaning had a temporary decrease in weight gain. Reproduction/Teratology Panel Report* Topic 1. Is there evidence that formaldehyde pro- duces reproductive toxicity or is teratogenic in experi- mental animals or man? a. Is there evidence that formaldehyde causes germ cell mutations in experimental animals which are clinically significant? a. Is there evidence that formaldehyde causes germ cell mutations in experimental animals which are clinically significant? y g b. Ifthere is evidence for reproductive or teratogenic effects, are there biological models to explain the activity? y c. Is there evidence for reproductive or teratogenic effects from substances related to formaldehyde or known to be metabolized to formaldehyde? Topic 2. What experimental or epidemiologic studies would be important to resolve any controversies in this area? CONSENSUS WORKSHOP ON FORMALDEHYDE In a two-generation study by Natvig et al. (215), rats fed a diet of 1600 ppm HMT (100 mg/kg/day) showed no difference in weight gain or fertility in the first or second generation. Staples (216) reviewed studies involv- ing hexamethylphosphoramide. This compound did not cause reproductive problems in rats gavaged with up to 200 mg/kg/day or by inhalation of 0.334 ppm. Staples (216) also indicated that glycerol formal (a condensation product of glycerin and formaldehyde) has been re- ported to be teratogenic in rats after administration subcutaneously or intramuscularly at all dose levels tested from days 6 through 15 of gestation. The panel is not aware of any human exposure or if this compound is metabolized to formaldehyde in vivo. g p It would be desirable to learn whether modification of glutathione concentration affects toxicity of formalde- hyde in vivo and in vitro. The following questions emerge: Does exposure to airborne formaldehyde lead to excretion of mutagens in the urine? What are the structures of the modified bases in DNA-formaldehyde adducts? What are the nature and degree of inhibition of DNA repair after modification by formaldehyde? Topic 2. Future Studies Information is needed on relationships between ambi- ent formaldehyde levels in air and its concentration at target cells. g Effects of airborne formic acid should be studied to see whether it may be responsible for formaldehyde effects. i Information is required on penetration, transport and tissue distribution of inhaled gaseous formaldehyde and the time course of formaldehyde concentration in blood and tissues after administration by various routes. Significance of Nonlinear Carcinogenic Response to Formaldehyde Exposure. At 15 ppm, formalde- hyde is an effective nasal carcinogen in the rat, but there are few tumors at 6 ppm. This non linear response y Comparative studies on related aldehydes would be desirable to reveal their toxic effects, metabolism and reactions with macromolecules. CONSENSUS WORKSHOP ON FORMALDEHYDE Topic 2. Future Studies Animal Studies. There was consensus that no ade- quate reproductive studies exist using inhalation expo- sure which is the primary route ofhuman exposure. The panel has no toxicokinetic information to suggest that inhalation studies would actually result in exposures as high as in the intubation study which was negative (212). However, classic teratology studies using inhala- tion exposure would resolve the question of teratogenic- ity with such exposure. We understand that such a teratology study is being initiated soon in Canada (Dr. W J. Martin, Chairman, Formaldehyde Council of Canada, communication to the panel). In addition, the panel believes it would be reasonable to do the following studies: combine or modify several existing protocols to evaluate reproductive effects; sperm morphology and vaginal cytology (226); fertility assessment by continu- ous breeding (226); evaluate animals at the end of the continuous breeding experiment to seek signs of func- tional impairment in offspring. p Hemminki et al. (221,222) studied spontaneous abor- tions among hospital staff engaged in sterilizing instru- ments with chemical agents. They reported no increase in spontaneous abortions associated with use of for- maldehyde. There is indirect evidence that would argue against formaldehyde being a major human teratogen. Over the last 30 years, the annual production and domestic use of formaldehyde in the United States has gone up fivefold from one billion pounds to about five billion pounds. Birth defects have been reasonably stable over the last 30 years, although in the last decade, there have been some exceptions to this rule. The reported incidence of ventricular septal defects and patent ductus arteriosus has increased and that of anencephaly and spina bifida has declined (223). If data from the above experiments indicate reproduc- tive and/or developmental risk, further studies should be performed. Studies should be done to investigate possible differences in the kinetics and metabolism of formaldehyde with differing routes of exposure in experimental animals. These studies should provide data for comparison between animal studies and human exposure data for risk assessment. la. Panel members did not find evidence of germ- cell mutations of clinical significance in mammals. One study by Fontignie-Houbrechts (113) indicated an in- creased pre- and post-implantation loss in the first week of mating, following exposure of males to 50 mg/kg by injection, and increased preimplantation loss in the third week. However, Epstein et al. CONSENSUS WORKSHOP ON FORMALDEHYDE 349 urea-formaldehyde resins. Concentrations of 1.2 to 3.6 ppm formaldehyde were observed in the exposed groups. The methods of data collection were not very well explained in the translated article, so that the panel had many questions about methodology and the interpreta- tion of these data. The author concluded that exposed women had a three-fold increase in menstrual disorders and produced more babies with birth weights between 2500 and 3000 g than did controls. However, the panel noted that only a few neonates weighed less than 2500 g and the rates did not differ for exposed and nonexposed women. The panel members did not view this evidence as very strong since many factors that could not be evaluated from the report could have explained the finding. In a better designed survey, Ols0n and D0ssing (77) found that 30% of women working in an environ- ment of 0.43 mg/m3 formaldehyde had a history of menstrual irregularities, whereas a reasonably matched control group had no history of menstrual irregularity. The exposed women reported eye irritation, headache and use of analgesics more frequently than did controls. The panel felt that these two papers, rather than showing a cause-and-effect relationship between formal- dehyde exposure and menstrual disorders, point to a reproductive effect that needs further evaluation. exposure, suggests that exposure to formaldehyde is not causing nondisjunction in humans (225). g j ( ) In summary, the panel could find no evidence clearly demonstrating that formaldehyde caused adverse repro- ductive outcomes. What it found was a paucity of information from which to make inferences and data that suggested hypotheses to be tested in future studies. This panel feels that formaldehyde poses little, if any, risk as a potential human teratogen. This judgment is based on the irritation potential of formaldehyde at extremely low ambient concentrations (0.05 ppm), exist- ing data from in vivo mammalian studies, and toxico- kinetic and metabolism data indicating an extremely short half-life (not detected to 1.5 min) of the parent compound, and relatively short half-life (80 to 90 min) of the only known metabolite (formate) in the blood, regardless of the route of exposure (142). Ibpic 1. Reproductive Toxicity There is a paucity of data addressing possible repro- ductive and developmental hazards of formaldehyde in experimental animals or man. Our answers to the questions posed about adverse reproductive effects should be viewed as far from conclusive regarding either the safety or lack thereof due to exposure to formaldehyde. y 1. Other adverse reproductive effects were reported by Hagino (217), who found prolonged diestrus, but no impairment of reproductive function in female rats stressed with strong formalin vapor (concentration not specified). Ovarian involution and endometrial atrophy were found in female mice exposed to 40 ppm formalde- hyde by inhalation -an exposure level that killed 80% of the animals (211,218). The panel members reviewed several other studies in experimental animals (216,219) and agreed that there were sufficient methodologic problems to render them of little value. h ili y There has been one adequate study of possible teratogenic effects in mammals. Marks and colleagues p The human data are extremely meager. Shumilina (220) studied workers exposed and not exposed to CONSENSUS WORKSHOP ON FORMALDEHYDE Topic 2. Future Studies (112) found no evidence of increased dominant lethal effects in mice exposed at doses up to 40 mg/kg, IP. Cassidy (224) reported increased sperm abnormalities in rats following expo- sure to 200 mg/kg, but not to 100 mg/kg, given orally. Thus, the data are not consistent and do not adequately test the possibility that formaldehyde causes germ-cell mutations. Epidemiology Studies. There are several possible epidemiologic approaches to studying the relationship between formaldehyde and adverse reproductive out- comes. In one approach, a group of exposed women and a group of unexposed women would be located and adverse reproductive effects sought. Women who might have or had industrial exposures and women who live in house trailers would be reasonable subjects for such a study. If such a study were to be done, every effort should be made to determine whether menstrual irregu- larity resulted from exposures. The panel realizes that a great deal of care will be needed in the design and Human germ-cell mutations causing nondisjunction could result in an increase in Down's syndrome. The constancy of maternal age-specific rates of Down's syndrome over the last 30 years, in face of increased CONSENSUS WORKSHOP ON FORMALDEHYDE 350 execution of this study if a valid answer is to be found. Other reproductive outcomes should be studied. The panel realizes that a very large number of subjects will be needed to get a reasonable evaluation on whether exposure causes birth defects or developmental delays. Evaluation of the rate of low birth weights should be feasible. 1. Neurochemical Studies. Formaldehyde is mainly metabolized to formate. Formate anion is an inhibitor of cytochrome oxidase (231-233) and the inhibition of cytochrome c oxidase by formate increases with decreas- ing pH. Because of man's evolutionary loss of the enzymes uricase and formyltetrahydrofolate reductase, man cannot utilize formate in many syntheses via the folate pathway (234). This enhances the human toxicity of chemicals that are metabolized to formate. The central nervous system is especially vulnerable to hypoxia which may be a relevant feature quite apart from acidosis in possible nervous system toxicity of formaldehyde (235). Paternal exposure could be causing adverse reproduc- tive outcomes. Studies of male workers and controls would evaluate this possibility. A more practical and cheaper way to seek to identify human reproductive outcomes associated with exposure is to seek histories of exposures where there have been adverse reproductive outcomes. Topic 2. Future Studies The panel suggests that existing case-control studies of birth defects be reviewed for evidence of exposure. The panel believes it would be reasonable to do a case-control study of birth defects specifically seeking relationships with exposure. Exposure to formic acid vapor (20 ppm for 3 and 8 days, 6 hr daily) caused increased cerebral acid protein- ase activity in rats (235). Cerebral glutathione in- creased initially, probably as a reactive phenomenon, but decreased at 8 days with increased acid proteinase activity, both reflecting increased lipid peroxidation (in cerebral hypoxia). Acid proteinase activity in cell frac- tions increased above the control range during the third week of exposure to rats to 20 ppm of formic acid vapor 5 days/week, 6 hr daily (236). Hypoxic labilization of the lysosomal complex was suggested as an explanation for the finding. * Behavior/Neurotoxicity/Psychological Effects Panel: Leon Golberg (Chairman); Marc Schenker (Alternate Chairman/Secretary); Jean Chen Shih; Michael J. Colligan; Anna Seppalainen. Behavioral/Neurotoxicity/ Psychological Effects Panel Report* Topic 1. What is known about the effects of formalde- hyde on the biochemistry and/or morphology of the nervous system? g Dimethylformamide, a general solvent in industry, is demethylated in vivo to produce monomethylformamide and formaldehyde (237). When rats were exposed through drinking water to 13.7 mM dimethylformamide, acid proteinase activity increased in the glial cell fraction after 2 and 7 weeks while under the same conditions cerebral glutathione concentration was be- low the control range (238). y Topic 2. Does formaldehyde induce behavioral or psychological changes? If so, what is the evidence and what methods can be used to measure the changes? g Topic 3. What critical experimental questions remain? What epidemiological studies might be important in this area? g Topic 3. What critical experimental questions remain? What epidemiological studies might be important in this area? 1. Effects of Formaldehyde on the Biochemistry of the Nervous System. Not much work has been done on the effects of formaldehyde on the biochemistry of the nervous system. Heck et al. (227) have shown that there was radioactivity in the brain after the animals were exposed to '4C-formaldehyde for 6 hr. The concen- tration of radioactivity in brain after exposure of F-344 rats to airborne 14C-formaldehyde for 6 hr (formaldehyde concentration range 5-24 ppm) was 0.37 (dpm/g tissue/dpm/g plasma), compared to 4.95 in the esopha- gus and 2.05 in lung. The chemical nature of the radioactive labeled compound remained to be studied. It is unlikely to be formaldehyde itself because it is a very reactive compound. Since formaldehyde readily forms condensation products with a number of biogenic amines, it is possible that these condensation products may be found in the brain. This aspect has been reviewed by Golberg (228). Another possibility is that formaldehyde may be converted rapidly to formic acid. The latter may join one-carbon metabolism by the tetrahydrofolate pathway, and in turn be incorporated into amino acids such as methionine and serine (229). Formation of methionine could account for the labeled choline observed by DuVigne et al. (230). High concentrations of formaldehyde (196 + 32 ppm), moderate levels of formic acid (11 + 3 ppm) and low concentrations ofacrolein (0.07 ± 0.03 pm) were detected in the air containing oxidative thermodegradation prod- ucts of polyacetal plastic (Deldrin, DuPont) (239). Rats were exposed to this mixture for 6 hr, once or three times. CONSENSUS WORKSHOP ON FORMALDEHYDE 351 and reaction to such changes, which, in turn, feeds back into the central nervous system; and (3) as a result of the individual's psychological reaction and concomitant CNS response to the olfactory properties of the substance. In practice, these processes are interdependent, yet this simple analysis of a complex series of responses under- lines the need to control for "expectancy" effects in formaldehyde research to permit a differentiation of the direct effects of formaldehyde on psychological func- tions from its secondary effects. The authors assert that even 1.0 ppm formaldehyde elicits "a significant depression in the rat trigeminal nerve response to a standard odorant" However, it is important to realize that the rats were under urethane anesthesia and had been treated with atropine before surgery, with further supplements as required to limit the production of mucus. Similar exposure to 5 ppm ozone for 1 hr actually increased neural responsiveness to amyl alcohol. y There is a considerable body of information on the acute irritant effects of formaldehyde on healthy human subjects. In one such study, Weber-Tschopp et al. (202) reported that irritant effects such as eye blinking rate as well as subjective irritation and annoyance increased as a function offormaldehyde concentration. The thresh- old for such effects was judged to lie between 1 and 2 ppm. EPIDEMIOLOGIC CONSIDERATIONS. Several epidemio- logic studies have evaluated neuropsychological symp- toms potentially due to occupational or environmental exposure to formaldehyde (243-245). While a high prevalence of symptoms has frequently been observed and ascribed to formaldehyde, serious deficiencies in study design, assessment of exposure and outcome measurement limit the interpretation of these studies. pp IC. MORPHOLOGICAL CHANGES IN RESPONSE TO FOR- MALDEHYDE EXPOSURE. Potts et al. (240) injected for- maldehyde intravenously over several hours to achieve a total dose of 0.9 g/kg in monkeys. No histologically detectable effect was observed in the central nervous system. On the other hand, Bonashevskaya (241) re- ported lesions in the cerebral amygdaloid complex after exposure to formaldehyde. Structural and cytochemical shifts occurred in the amvTgdaloid complex of rats after 3 months at 1 to 3 mg/mi formaldehyde in hermetically sealed chambers. The shifts were described as "dis- turbed uniformity" in the content of Nissl bodies and RNA in the limits of the same amygdaloid nucleus. Some neurons showed extensive accumulation, and others reduction of these constituents. CONSENSUS WORKSHOP ON FORMALDEHYDE Measures taken to monitor the concentrations of formaldehyde in the exposure chambers were not reported. p Dally and co-workers reported symptoms prevalences among residents of homes in Wisconsin that had health- related complaints, possibly due to formaldehyde expo- sure (243). The median formaldehyde concentration was 0.47 ppm (range < 0. 1-3.68 ppm) and a positive (inverse) association was present between age of the structure and formaldehyde concentration. The great- est prevalence of symptoms was for irritation of mucous membranes, but of the 256 subjects, 53% reported headaches and 38% reported difficulty in sleeping. p g The major weakness in this study is selection bias of the population, i.e., only subjects from homes where complaints were registered were studied. An appropri- ate control population was not included. Responses may also have been biased by other considerations such as reports in the press and ongoing litigation. No attempt was made to evaluate respondent bias nor were responses related to measured levels of formaldehyde or other poten- tial covariates collected during the investigation. p p 2. A number of reports exist which link chronic formaldehyde exposure to a range of psychological/ behavioral problems including depression, irritability, memory loss and decreased attentional capacity, and sleep disturbances. Unfortunately, these studies, re- viewed by Sauer (242), for the most part have involved field surveys using subjective self-report symptom inventories. Control data, describing the incidence of such symptoms from unexposed cohort samples, are often inadequate or completely absent. This is an extremely thorny problem when dealing with formal- dehyde, which in addition to any direct toxic effects possibly associated with it, produces distinct olfactory cues which when detected by the individual may stimu- late a spectrum of secondary psychological reactions (e.g., expectancies, irritations, anxieties, fears, etc.). These reactions may in turn exacerbate, mask, or interfere with the more direct neurologic, biochemical, and physiological responses to the substance. g g Similar criticisms exist for the studies of Sardinas et al. (244) and Garry and co-workers (245). Both of these investigations, in Connecticut and Minnesota, respec- tively, found a high prevalence of headache but failed to include control populations or account for selection bias. Behavioral/Neurotoxicity/ Psychological Effects Panel Report* They gasped for air for hours after the exposure. The fumes decreased the cerebral RNA concentration, together with decreases in the succinate dehydrogenase and acid proteinase activities. p Irritant Action of Formaldehyde on Sensory Nerves. 1. The effect of exposure to formaldehyde was studied in anesthetized rats by Kulle and Cooper (239), who judged the response by an increase in action potential firing rate of nasopalatine and ethmoidal nerves of the trigeminal nasal sensory system. Brief (2 min) exposure over a range of 0.5-2.5 ppm formalde- hyde delivered through a nose cone yielded responses like those elicited by ozone and amyl alcohol. Exposure to 0.5, 1.0, 1.5 or 2.0 ppm formaldehyde for 1 hr, repeated up to four times, revealed a progressive depression in the response to amyl alcohol with increas- ing formaldehyde concentration. The effects of a 2.0 ppm exposure were similar whether presented sepa- rately or as the final exposure of a series. Return to air inhalation for 1 hr brought about a partial recovery of the neural response to amyl alcohol. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 352 self-assessment method to 66 subjects who worked in a mobile day care center and 26 controls working in permanent centers. Mean concentrations of formalde- hyde were 0.43 and 0.008 mg/m3, respectively, with no difference in temperature, humidity or ventilation rates. A significantly greater prevalence and greater symptom intensity of nose and throat irritation, unnatural tiredness, and headaches was present in the exposed subjects. No difference was noted in disturbed memory or concentration or in the control questions on the questionnaire. This study overcomes some of the pre- viously mentioned problems, but responses still may have been influenced by awareness of the subjects of the study goals and hypotheses. Nevertheless, this is a controlled study using a standardized questionnaire and the findings need further investigation. self-assessment method to 66 subjects who worked in a mobile day care center and 26 controls working in permanent centers. Mean concentrations of formalde- hyde were 0.43 and 0.008 mg/m3, respectively, with no difference in temperature, humidity or ventilation rates. A significantly greater prevalence and greater symptom intensity of nose and throat irritation, unnatural tiredness, and headaches was present in the exposed subjects. No difference was noted in disturbed memory or concentration or in the control questions on the questionnaire. This study overcomes some of the pre- viously mentioned problems, but responses still may have been influenced by awareness of the subjects of the study goals and hypotheses. Nevertheless, this is a controlled study using a standardized questionnaire and the findings need further investigation. phological changes in the CNS has been observed in one study and not in another. y The difficulties inherent in any study of psychological/ behavioral effects of formaldehyde have not yet been overcome in the course of conducting field surveys. g y Epidemiologic studies evaluating neuropsychological symptoms potentially due to occupational or environ- mental exposure to formaldehyde have failed to over- come the problems commonly associated with such studies. However, some studies merit further investigation. CONSENSUS WORKSHOP ON FORMALDEHYDE A report of symptoms in 20 infants under 12 months of age by Woodbury and Zenz (246) also found frequently reported problems with sleeping (14/20), but no associa- tion was present with measured concentrations of formaldehyde and this study suffers from the same biases as the adult studies. Thun and Altman (78) have pointed out some of the difficulties in prevalence surveys of symptoms in resi- dents from homes with urea-formaldehyde insulation (UFFI) foam, including the presence of odor affecting response, respondent and recall biases, and the objec- tive outcomes measured. In this case-control study, no significant difference was found in the occurrence of headaches or insomnia in residents of homes with UFFI, compared to neighborhood controls (247). ll d l i f For purposes of exposition, this suggests that formal- dehyde may affect the psychological functioning of the individual in three ways: (1) directly, as a result of the immediate toxic properties of the substance on the peripheral and central nervous systems; (2) indirectly, as a result of the individual's monitoring and awareness of the aforementioned changes and his/her interpretation g A controlled evaluation of symptom prevalence was undertaken by Ols0n and D0ssing (79). They adminis- tered a questionnaire based on the linear analogue CONSENSUS WORKSHOP ON FORMALDEHYDE Topic 3. Future Studies Biochemical. While the neurotoxicity of formalde- hyde is still a controversial issue, the study on the biochemical effect of formaldehyde on the central ner- vous system will provide fundamental knowledge for understanding its possible neurotoxicity. g g Reported symptoms need to be evaluated by formal tests of neuropsychologic function. Schenker and co- workers (248) found in a pilot study that residents of homes with UFFI who complained of memory impair- ment did not have abnormalities on formal tests of memory function, although many showed deficits in tests of attention span or the ability to sustain attention. This result needs to be evaluated in a controlled, population-based study. Effort should be made to identify and to characterize the structure of the radioactive compound found in rat brain after the animals were exposed to 14C-formalde- hyde. Radioactive formate, radioactive choline and radioactive condensation products between biogenic amines and formaldehyde are the possible compounds. Among these compounds the condensation products are of particular interest. Tetrahydrobetacarboline should be found if indoleamines and formaldehyde formed a condensation product. Tetrahydroisoquinolines should be found if catecholamines and formaldehyde form condensation products. One study has briefly considered neurologic function- ing in a controlled laboratory exposure. Anderson (124) reported on 16 healthy young subjects to 0.3, 0.5, 1.0 and 2.0 mg/m3 formaldehyde for 5 hr. He found subjec- tive and physiologic responses with exposure, but no effect on performance tests (speed and accuracy of multiplication and addition, and of card punching) at any of the exposure concentrations. Details of the performance tests were not presented, and the power of the study was not reported. p Once the structures of the radioactive compounds have been determined, the distribution of these com- pounds in brain, the effects of these compounds on neurotransmitter receptors, uptake, biosynthetic and degradative enzymes could be systematically studied. Furthermore, the effects of those compounds on animal behavior could be examined. y p A preliminary report by Kilburn and co-workers (249) described a study using a standardized battery of neuropsychological tests on histology technicians. Sub- jects were also exposed to xylene and toluene at work, and there was no measurement of individual exposures. Response rates and characteristics of the exposed and control populations are not reported, and no consider- ation was given to potential respondent bias in symp- toms or exposures. No data are presented on the tests of neuropsychologic function. CONSENSUS WORKSHOP ON FORMALDEHYDE Such a battery of tests could be coupled with traditional paper and pencil tests (e.g., Wechsler Intelligence Scales) for which there are standardized population norms, but it is felt that the critical comparison is against a cohort of base-line exposed controls comparable to the target group along relevant socioeconomic, educational, and occupational dimensions. wrist in the arm nerves. Measurements of the ampli- tude of the elicited sensory action potential might increase the sensitivity of the method. Such a battery of tests could be coupled with traditional paper and pencil tests (e.g., Wechsler Intelligence Scales) for which there are standardized population norms, but it is felt that the critical comparison is against a cohort of base-line exposed controls comparable to the target group along relevant socioeconomic, educational, and occupational dimensions. p Tiaditional psychodiagnostic inventories of psychopa- thology (e.g., MMPI, Beck Depression Scale) or mood state (e.g., Profile of Mood States) would also be appropriate. Cross-sectional designs should attempt to identify not only "exposed" and background or baseline- exposed cohorts, but should attempt to quantify the level and duration of exposure for all groups to permit maximum statistical analysis. Prospective studies, if feasible, should involve repeated testing of both the target and control cohorts to control for aging and historical effects. Finally, attempts should be made to gather "real-life data" (e.g., academic performance, sleep diaries, behav- ioral sampling) to complement the psychological testing. Occupationally and environmentally exposed popula- tions should be studied because they provide a unique opportunity to investigate the potential neurotoxicity of formaldehyde. Such studies allow investigation of sensi- tive populations (e.g., children), long-term exposures, and acute exposures at formaldehyde concentrations that cannot be used in a controlled laboratory setting. It is important that appropriate control populations be included in these studies and adjustment be made for potential confounders such as socioeconomic status. The studies should include a spectrum of neuropyschological tests, including standardized and validated tests when possible. Attempts are currently underway to develop such a test battery, and similar studies have been done on working populations with exposure to solvents or heavy metals. A difficult and expensive component of such studies is the measurement of formaldehyde exposure, but such measurements are an important component ofepidemiologic investigations. Several occu- pational populations exist that represent possible study groups. CONSENSUS WORKSHOP ON FORMALDEHYDE 353 describing the impact of formaldehyde on psychological/ behavioral functioning is currently nonexistent, it was agreed that considerable research is needed along these lines. Furthermore, it was felt that the primary empha- sis should be placed on long-term, or prolonged expo- sures to disentangle the potential toxic effects of formaldehyde from the transient, irritative ones, and to simulate the exposure conditions of relevant popula- tions at risk in the environment, such as inhabitants of mobile homes having urea-formaldehyde insulation and individuals experiencing working-life occupational expo- sures. The emphasis on chronic exposure studies sug- gests two particular research paradigms: (1) animal studies in which controlled, measured doses offormalde- hyde are administered over variable periods of time to assess the effects on select, well-defined behavioral and performance parameters; and (2) field/epidemiological studies in which groups of people undergoing long- range, measurable formaldehyde exposures as part of their daily routine (e.g., medical students, anatomists, plywood/fiberboard workers, mobile home dwellers) are assessed in terms of relevant psychological/behavioral parameters relative to appropriate baseline-exposed cohort samples. describing the impact of formaldehyde on psychological/ behavioral functioning is currently nonexistent, it was agreed that considerable research is needed along these lines. Furthermore, it was felt that the primary empha- sis should be placed on long-term, or prolonged expo- sures to disentangle the potential toxic effects of formaldehyde from the transient, irritative ones, and to simulate the exposure conditions of relevant popula- tions at risk in the environment, such as inhabitants of mobile homes having urea-formaldehyde insulation and individuals experiencing working-life occupational expo- sures. The emphasis on chronic exposure studies sug- gests two particular research paradigms: (1) animal studies in which controlled, measured doses offormalde- hyde are administered over variable periods of time to assess the effects on select, well-defined behavioral and performance parameters; and (2) field/epidemiological studies in which groups of people undergoing long- range, measurable formaldehyde exposures as part of their daily routine (e.g., medical students, anatomists, plywood/fiberboard workers, mobile home dwellers) are assessed in terms of relevant psychological/behavioral parameters relative to appropriate baseline-exposed cohort samples. wrist in the arm nerves. Measurements of the ampli- tude of the elicited sensory action potential might increase the sensitivity of the method. Topic 3. Future Studies This study utilizes stan- dardized tests of neuropsychologic function, but the results cannot be considered indicative of effects due to formaldehyde exposure. Electrophysiological. Acute effects of formalde- hyde have been studied on the trigeminal nasal sensory system (239). Several authors (250,251) have suggested that electrophysiological methods of studying the cen- tral nervous system are helpful in evaluating toxic effects of, for example, formaldehyde. Long-term experimental exposure to various concentrations of formaldehyde could be carried out on rats and/or rabbits and electroencephalography (EEG) with indwell- ing electrodes in hippocampal and cortical (occipital) areas could be performed. Spontaneous EEG activity could be analyzed at various time intervals during the exposure and evoked potential studies using flash light stimulus (visual evoked potentials) as well as sound stimulus (brainstem auditory evoked potentials) could and should be applied at various intervals of continous exposure. y p Conclusions. The effects of formaldehyde and/or its metabolites on the biochemistry of the nervous system have not been clearly defined. Various possibilities exist whereby such effects might be mediated. y Some evidence exists that exposure to formic acid (the principal metabolite of formaldehyde) in vapor form at high concentrations exercises nervous system toxic- ity in intact rats. No studies have demonstrated peripheral nervous system effects of formaldehyde. Possible PNS effects could be checked by applying nerve conduction measure- ments in chronically exposed rats. The irritant effects of formaldehyde may be reflected in altered function of sensory nerves such as the trigeminal nasal sensory system. The presence of mor- Given the fact that a sound, systematic data base CONSENSUS WORKSHOP ON FORMALDEHYDE Risk Estimation Panel Report* Topic 1. How can all the available data be integrated to make reasonable risk estimates (neoplastic and nonneoplastic) for humans exposed to formaldehyde at various levels and through different routes? g a. In making estimates, what data and assumptions lead the Panel to choose one method over another? y g g 1. The first task ofthe Risk Estimation Panel was to review the reports from the other panels to establish what potential risks to human health exist due to exposure to formaldehyde. The second task was to identify time-dose-response data sets that could be used for quantitative risk estimation of formaldehyde. The third task of the panel was an attempt to utilize whatever biological and mechanistic actions offormalde- hyde were identified by the other panels to assist in the choice of time-dose-response models for low dose risk estimates. b. In making risk estimates, how can data be used from: 1. metabolism studies 1. metabolism studies i i 2. biological endpoints (importance of benign tumors) 3. individual variabilities id i l 4. epidemiology 5 hi h l d 5. high or low dose extrapolation models 6 i i i i 6. interspecies variation i i Topic 2. Are any practically achievable data likely to resolve any of the uncertainties? Topic 2. Are any practically achievable data likely to resolve any of the uncertainties? Ib.2. ImmunologylSensitizationlIrritation. For- maldehyde produces irritation of the nose, eyes and throat when inhaled, and of the skin when applied topically in solution. In most individuals, this is simple irritation and not due to an allergic reaction. However, the Immunology/Sensitization/Irritation Panel found that allergic contact dermatitis and other forms of skin sensitization did occur in some individuals. There was not sufficient evidence to confirm that respiratory tract allergy occurred, although there were some suggestive findings. Risk assessment is generally regarded to consist of three main functions: hazard identification, exposure assessment, and risk estimation. Hazard identification is the qualitative determination of an actual or potential toxic effect in humans or in animals that is also presumed to pose a threat to humans. This includes identifying toxic biological endpoints, investigating mechanisms that may alter the response in humans, and identifying data sources that may be useful in perform- ing quantitative risk assessments. Hazard identification also involves the determination of the degree of the hazard, i.e., is it life threatening or not; reversible or not. * Risk Estimation Panel: David Gaylor (Chairmnan); Mary F Lyon (Alternate Chairman/Secretary); Roy Albert; Charles C. Brown; Murray S. Cohn; Robert Sielken, Jr.; Mike Wright. CONSENSUS WORKSHOP ON FORMALDEHYDE Medical students or other users of anatomy and pathology laboratories could be studied to evaluate effects of short term exposures (3-6 hr) on neuro- psychologic test performance. The fabric industry uses formaldehyde in coating certain products and appropri- ate unexposed comparison groups may exist within the industry to allow controlled evaluation of workplace exposure. Numerous other industries have workplace exposure to formaldehyde and should be evaluated for the nature of the formaldehyde exposure and the feasibility of epidemiologic investigation. Home expo- sures to formaldehyde may occur from insulation prod- ucts or from building materials themselves. Some schools may have elevated formaldehyde exposures because of new construction or from the use of trailers p With respect to the animal studies, it was felt that the test battery should encompass a range of measures involving relatively simple and direct sensory responses (e.g., absolute and difference sensory thresholds), mo- tor behaviors (e.g., balance and tremor assessments), sensory-motor activities (reaction times, avoidance learning, etc.) and cognitive processes (short- and long-term memory, discrimination learning, etc.). Infor- mation stemming from this research will identify system-specific as well as general responses to formal- dehyde, and help to identify parameters for follow-up research in the human field studies. With respect to the latter, given the limited knowledge regarding the impact of formaldehyde on human psychological/be- havioral functioning, it was felt that a broadbrush approach was most appropriate. The human perfor- mance test battery should assess an array of psychologi- cal functions ranging from relatively simple sensory responses (e.g., threshold determination, attention, vigilance) through perceptual motor operations (e.g., simple and choice reaction times, tracking, short-term memory) to complex cognitive operations (e.g., concept formation, dual-task strategies, logical reasoning). Vi- sual evoked potential (VEP) recordings could be accom- modated to study acute effects in the visual sensory system. Workers handling formaldehyde containing material might be locally exposed through the skin. This could induce local neurotoxic effects in peripheral nerves. Inhalation exposure could in the long-term also affect the peripheral nervous system (PNS). As the distal ends of long nerves are usually the first portions to suffer in chemical neurotoxicity the PNS should be checked upon. A feasible approach could be to measure the distal sensory conduction velocity from fingers to CONSENSUS WORKSHOP ON FORMALDEHYDE 354 for classrooms. These situations allow the design of controlled epidemiologic studies to evaluate behavioral/neurotoxicologic/developmental effects among large populations including young children. CONSENSUS WORKSHOP ON FORMALDEHYDE Similar studies have been performed to evaluate neuropsycho- logic effects, in children, of lead toxicity or dietary alterations. Risk estimation is the quantitative aspect of risk assessment which attempts to mathematically relate risk to exposure. In some instances, this can be done with human data. Generally, only animal data are available. Since it is necessary to determine if there are toxic effects using relatively small numbers of animals, doses well above human exposure levels are generally employed in animal bioassays. The process of quantita- tive risk estimation usually involves two main functions: (1) extrapolation of risks from high to low doses and (2) extrapolation of risks from animal exposures (e.g., laboratory animals exposed to a constant level continu- ously for life) to human populations that are genetically and environmentally heterogeneous and exposed to varying levels at various times during their life. CONSENSUS WORKSHOP ON FORMALDEHYDE The Risk Estimation Panel also agrees with the Carcinogenicity/Histopathology/Genotoxicity Panel that nonneoplastic polyps and hyperplasias do not at present represent endpoints suitable for the assess- ment oftumor risk. The Carcinogenicity/Histopathology/ Genotoxicity Panel noted the difficulty of differentiating between polypoid adenomas, nonneoplastic polyps, and hyperplasia, in the CIIT rat inhalation study. A recently completed re-examination by a Pathology Work Group resulted in diagnoses that were in close agreement with the original diagnoses ofpolypoid adenomas. Papillomas, which are generally considered to be the benign counter- part of the squamous cell carcinoma, were not seen in the CIIT rat inhalation study. Nasal adenocarcinomas were seen in the NYU formaldehyde study. The data now available lead the Risk Estimation Panel to believe that the target sites of formaldehyde are not primarily distant from the site of exposure. p gy y The Reproduction/Teratology Panel found no clear evidence of any adverse effects of formaldehyde on either reproduction or development of the fetus. However, there were grounds for suggesting that fur- ther work was needed to elucidate certain points. Concerning mutation in mammalian germ cells, both the Reproduction/Teratology Panel and the Carcino- genicity/Histopathology/Genotoxicity Panel found no good evidence of positive effects. The Carcinogenicity/ Histopathology/Genotoxicity Panel recognized formalde- hyde as a weak mutagen, and both panels noted one study in which marginally positive results were ob- tained in a dominant lethal test. However, the mouse spot test was negative. This led the Carcinogenicity/ Histopathology/Genotoxicity Panel to conclude that more extensive tests of mutation in mammalian germ cells were not at present warranted. On the basis of these findings, there are no grounds at present for attempting any quantitative estimates of risk to humans for adverse effects on reproduction, fetal development, or hereditary defects resulting from expo- sure to formaldehyde. y lb.2, lb.3, lb.4. Epidemiology. In comparing the results of experimental animal studies to human epide- miological observations, there is evidence that the affected site in animals is not necessarily predictive of the affected site in humans. The Epidemiology Panel stated that there is some evidence ofa dose-response relationship between formal- dehyde exposure and lung cancer based upon the one of six British factories where the largest number of cohort workers experienced the highest formaldehyde expo- sure levels. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 355 cancer and leukemia. The Epidemiology Panel sug- gested further investigation of the questions raised. No dose-response information is currently available for quantitative risk estimation. the home or at work. However, there was great difficulty in interpretating these data. In some cases, there were no adequate controls. In other cases, the possibility of subjective effects due to the smell of formaldehyde could not be excluded. Commonly, the exposure level was not known. q The Risk Estimation Panel recommends that no quantitative risk estimate be based on the current epidemiological information. However, we also recom- mend that future estimates might be based on addi- tional data provided by further analyses of these human populations. In view of the difficulties and uncertainties in these studies, the Risk Estimation Panel feels that there are no adequate data for attempting to assess quantitative risks of behavioral or psychological effects resulting from formaldehyde exposure in man at the present time. p p In addition, we recommend that the results of risk assessments based on animal studies should be com- pared to the information from each of these human observational studies in order to evaluate the extent to which there is any statistical consistency or inconsis- tency of this extrapolation to human data. lb.2. Reproduction and Teratology. The data con- cerning adverse effects of formaldehyde on reproduc- tion and on fetal development were reviewed by the Reproduction/Teratology Panel. Data on possible induc- tion of germ cell mutations were considered by both the Reproduction/Teratology Panel and the Carcinogenicity/ Histopathology Genotoxicity Panel. y p la, lb.2. Endpoints forRisk Estimation. The Risk Estimation Panel found that data from the CIIT rat inhalation chronic bioassay are suitable for modeling the dose-response relationship. The data from the formalde- hyde studies in Syrian hamsters and the CIIT mouse inhalation study provide information on risk, but do not provide sufficient dose-response information for quanti- tative model fitting. The panel agrees with the conclu- sion ofthe Carcinogenicity/Histopathology/Genotoxicity Panel that the malignant tumors represent an endpoint which should be used in any quantitative risk assess- ment based on the CIIT rat inhalation data. The Risk Estimation Panel follows the Carcinogenicity/Histo- pathology/Genotoxicity Panel recommendation that, be- cause of different cell types of origin, any evaluation of polypoid adenoma be done separately from squamous cell carcinomas. Risk Estimation Panel Report* These topics were addressed by the other panels at the workshop. Exposure assessment is the activity of identifying the routes and extent of exposure of the human population to toxic substances. This function involves identifying sources of toxic substances, expo- sure levels, and length of exposure, which may be occupational, environmental, or from consumer pro- ducts, which may be intermittent or continuous. These levels of exposure may be used in time-dose-response functions to estimate risks (the animal or human probability of a toxic health effect by a specific age or the average time without a toxic response). The ex- pected number of individuals with a disease in a population requires estimates of the numbers of individ- uals in various exposure groups multiplied by the probability of disease for those groups. There were difficulties in assessing the proportion of individuals who were unduly sensitive to skin contact. Data were also inadequate to establish clear threshold doses for the various effects. For normal individuals, it was possible to give a range of doses over which various harmful effects were seen, but there generally were not adequate dose-response data. In view of the frequent lack of data on dose-response and on the proportion of individuals who are unduly sensitive, the Risk Estimation Panel did not attempt at present to perform a quantitative risk estimation for the irritant or sensitization effects of formaldehyde, al- though some risks clearly exist at some current levels of exposure. / p 1b.2. Behavior/NeurotoxicitylPsychological Effects. Th B h i /N t i it /P h l i l P l 1b.2. Behavior/NeurotoxicitylPsychological Effects. The Behavior/Neurotoxicity/Psychological Panel re- ported that exposure to formaldehyde produced neuro- chemical changes in the brain of rats, and also possible effects in peripheral nerves but further studies of these phenomena were needed. i p In humans, there have been reports of various symptoms in subjects exposed to formaldehyde either in CONSENSUS WORKSHOP ON FORMALDEHYDE The Risk Estimation Panel noted that sufficient epidemiological data are not now available for a quantitative risk estimate which relates the level and duration of formaldehyde exposure to the risk of either lung or nasal cancer. p Ia, Ib.l, Ib.6, Ic. Species-to-Species Extrapolation. Ri k ti ti h b d i l d t Ia, Ib.l, Ib.6, Ic. Species-to-Species Extrapolation. Risk estimation, when based on animal data, must consider the relationship between risk observed in the test species and risk projected in the species of concern (252,253). In the case of formaldehyde, humans are the species of concern and the test species used in the quantitative modeling techniques are the rats in the CIIT chronic inhalation study. Species differences have been observed with respect to factors which may influ- Studies of three professional groups who preserve human tissues with solutions containing formaldehyde and other chemicals have shown an excess of brain CONSENSUS WORKSHOP ON FORMALDEHYDE 356 ence carcinogenic risk. An example comes from the CIIT study itself, where at high concentrations of formalde- hyde, mice modify their respiration more than rats. tumor produced in experimental animals, the mecha- nism by which tumors appear to be induced, interspe- cies comparisons, the actual dose delivered to the target tissues, and other factors. y , y p There are, however, no indications that the response by humans would be different than that exhibited by rats, mainly due to the lack of experimental data pertaining to this issue. Qualitatively, the metabolic pathways of formaldehyde in rats and humans are similar. The sites of greatest exposure may differ, since rats are obliged to breathe solely through the nose and humans may also breathe orally. Again, no information exists demonstrating that the response would be quanti- tatively different as a result of differences in distribu- tion of the inhaled dose. At the present time there is general agreement that, because of the complexity of the carcinogenic process and the fact that we understand so little of the pathogenesis of cancer, there is uncertainty in the nature of the dose-response relationships for cancer production at low levels of exposure, specifically at those levels which do not produce observable effects in animal experiments or in epidemiological studies. CONSENSUS WORKSHOP ON FORMALDEHYDE p p g The use of data obtained at high doses in long term animal bioassays to predict carcinogenic risk at low dose is one of the most controversial issues in risk assess- ment methodology. This is also true in the case of formaldehyde, where the CIIT rat dose-response curve is highly nonlinear between the concentrations of 2 and 15 ppm. The observed experimental data in the CIIT inhalation study on rats can be fit reasonably well by using a sufficiently flexible family of models (e.g. Weibull, gamma-multihit, generalized multistage). The corresponding estimated models provide estimates of risk at any selected dose level. These estimates are those which are most consistent with the presumed family of dose-response models and their associated assumptions about background. If no explicit low-dose linear term is included in these models, these models assume no dosewise additivity with other carcinogens present in the environment or with background carcino- genic processes. The panel recommends that the suggestion of the Structure Activity/Biochemistry/Metabolism Panel be utilized in risk estimation: "Although there are differ- ences in formaldehyde carcinogenicity among different species, at present there is no reason to assume that humans would be more or less susceptible than the rat." The panel can only assume that rats and humans exposed to the same concentration of formaldehyde for the same proportion of lifetime will exhibit a similar carcinogenic response, all other influences being equal. The panel. recognizes that this assumption is based on the lack of information and may change as further data become available. The Structure Activity/Biochemistry/Metabolism Panel noted that the nonlinear carcinogenic response to dose observed in the CIIT Study was in contrast to the linear response of DNA-protein crosslinks at 6 ppm and above. The Carcinogenicity/Histopathology/Geno- toxicity Panel considered impairment of mucociliary clearance, detoxification, and DNA repair as leading to relatively greater effective target site doses at higher doses, resulting in a likely explanation for the nonlinear carcinogenic dose-response. Following initial exposure, high formaldehyde concentrations impair mucociliary clearance, stimulate cell proliferation, and increase the replication rate in respiratory epithelium (89). This, at least initially, increases the single-strand fraction of respiratory mucosal DNA, which is susceptible to covalent binding with formaldehyde. The latter data, however, do not indicate the concentration of formalde- hyde at a target site relative to the concentration in air. Therefore, the panel recommends the use of airborne concentration for dose-response modeling at this time. CONSENSUS WORKSHOP ON FORMALDEHYDE The actual target dose is not known at this time but more information is being collected which bears on this issue (254). Alternative expressions for the target dose may be utilized. g p Upper and lower confidence limits on the risks being modeled may be computed to reflect the variability in the data under the presumed family of models. These confidence limits do not reflect the uncertainty in the choice of the family of models. Not all points in a confidence interval or all points below an upper confidence limit for the above presumed family of models are equally likely to be the true value of the quantity being bounded. These estimates and confidence limits are helpful as long as the underlying dose-response relationship is reasonably well described by the pre- sumed family of models. The major difficulty is that, because of the complexity of carcinogenic processes and our limited understanding, there is usually considerable uncertainty about the shape of the dose-response rela- tionship for doses near zero and hence the appropriate- ness of the low-dose behavior of the presumed family of models. The panel realizes that the very low-dose behavior of different families of models can be dramati- cally different. y la., Ib.1, Ib.5, Ic. Low Dose Extrapolation. Mathe- matical risk estimation models are only mathematical constructs which can assist in evaluating dose-response relationships. Risk estimation models are no better than the data and the biological and mathematical assumptions on which they are based. Predictions of human risk derived from animal data must be consid- ered in conjunction with human (epidemiological) data and qualitative biological data, such as the type of la., Ib.1, Ib.5, Ic. Low Dose Extrapolation. Mathe- matical risk estimation models are only mathematical constructs which can assist in evaluating dose-response relationships. Risk estimation models are no better than the data and the biological and mathematical assumptions on which they are based. Predictions of human risk derived from animal data must be consid- ered in conjunction with human (epidemiological) data and qualitative biological data, such as the type of y The Risk Estimation Panel is aware of several possi- ble reasons for the nonlinear carcinogenic dose-response observed with formaldehyde in the CIIT study. The Car- cinogenicity/Histopathology/Genotoxicity Panel states: "Cytotoxicity might influence both initiation and promo- tion. CONSENSUS WORKSHOP ON FORMALDEHYDE 357 ing uncertainty over the appropriateness of the low-dose behavior for the family of models used to model the dose-response relationship in the experimental dose region has encouraged many members of the panel to suggest that a linear low dose nonthreshold extrapola- tion be used in risk assessment. A straight line connect- ing the risks at the endpoints of a low dose region provides an upper bound on low dose risks for dose- response relationships which curve upward in the low dose region (260-263). Low-dose linear extrapolation is not equivalent to fitting a straight line or a one-hit model to the experimental data. A highly nonlinear model is required, in the case of formaldehyde, to fit the experimental data. Linear extrapolation is only in- tended to be used at the low doses where adequate experimental information is not directly available due to the large numbers of animals required to measure small levels of risk. A nonlinear model which adequately fits the experimental data for the CIIT chronic rat inhala- tion bioassay could be used in conjunction with a low dose linear extrapolation procedure. The Risk Estima- tion Panel is in general agreement that a linear low-dose nonthreshold extrapolation provides an upper limit on cancer risk in rats exposed to formaldehyde by inhala- tion in the sense that the true risk in rats is not likely to be greater than this limit. The Risk Estimation Panel has not reached a consensus as to either the practical application of these upper limits for human exposures or the exact range of the low-dose region over which a linear extrapolation might be reasonably performed. Increased cell proliferation also contributes to tumor promotion. Thus, there is a greater likelihood of tumor development with the increased cell proliferation associ- ated with cytotoxic exposures. These factors are likely to contribute to the nonlinearity of the dose-response, i.e., proportionately greater response at higher doses" The Structure Activity/Biochemistry/Metabolism Panel states: "Only certain specific sites on DNA might led to carcinogenesis; moreover, the rapid and nondiscriminate attack of formaldehyde on tissue components could involve membrane function, transport and repair pro- cesses, and it is known that mucociliary removal of formaldehyde is inhibited at high concentrations. In view of this complex network of reinforcing and compet- ing reactions, it is not surprising that the carcinogenic response is not linear or that it does not parallel the degree of crosslinks. CONSENSUS WORKSHOP ON FORMALDEHYDE Although the cause is still un- certain, this apparent nonlinearity cannot be construed to indicate the existence of a threshold for exogenous formaldehyde carcinogenicity. Although nongenetic fac- tors may play a role, more sensitive methods will be required to determine whether a linear relationship between dose and response exists at low exposure levels." ing uncertainty over the appropriateness of the low-dose behavior for the family of models used to model the dose-response relationship in the experimental dose region has encouraged many members of the panel to suggest that a linear low dose nonthreshold extrapola- tion be used in risk assessment. A straight line connect- ing the risks at the endpoints of a low dose region provides an upper bound on low dose risks for dose- response relationships which curve upward in the low dose region (260-263). Low-dose linear extrapolation is not equivalent to fitting a straight line or a one-hit model to the experimental data. A highly nonlinear model is required, in the case of formaldehyde, to fit the experimental data. Linear extrapolation is only in- tended to be used at the low doses where adequate experimental information is not directly available due to the large numbers of animals required to measure small levels of risk. A nonlinear model which adequately fits the experimental data for the CIIT chronic rat inhala- tion bioassay could be used in conjunction with a low dose linear extrapolation procedure. The Risk Estima- tion Panel is in general agreement that a linear low-dose nonthreshold extrapolation provides an upper limit on cancer risk in rats exposed to formaldehyde by inhala- tion in the sense that the true risk in rats is not likely to be greater than this limit. The Risk Estimation Panel has not reached a consensus as to either the practical application of these upper limits for human exposures or the exact range of the low-dose region over which a linear extrapolation might be reasonably performed. The Risk Estimation Panel could not reach a consen- sus on the way in which the uncertainty in choosing a family of nonlinear models should be incorporated into the risk assessment process. the risk assessment process. Regardless of the model chosen, the panel is aware of several general arguments often made in support of low-dose linearity. Ehrenberg et al. CONSENSUS WORKSHOP ON FORMALDEHYDE (255) have pointed out that the kinetics of the various chemical processes involved in the uptake and metabolism of chemicals, and their reactions with target molecules, become first order at low concentrations, leading to low-dose linear- ity on the delivered dose scale. It has been suggested that when the action of a given carcinogen adds to those of other causes of cancer in a given target tissue, the incremental effect of small delivered doses of the given carcinogen is virtually linear regardless of the observed shape of the dose-response relationship at the tested doses (256-259). The rationale is that the carcinogen is augmenting some background component in causing a carcinogenic event. Formaldehyde shares with other chemical carcinogens the properties of genotoxicity and an ability to react directly with DNA (as concluded by the Structure Activity/Biochemistry/Metabolism and Carcinogenicity/Histopathology/Genotoxicity Panels). Further, the latter panel found that formaldehyde can transform, as well as mutate, various cultured cell lines and enhance the transformation of Syrian hamster embryo cells harboring adenovirus. Research at the CIIT has shown, additionally, that formaldehyde can initiate and promote the actions of other promoters and initiators in in vitro mammalian cultured cell transfor- mation assays (109,116). These data suggest that formal- dehyde can interact with several carcinogenic agents or processes. i p g y p With regard to the possibility of a threshold dose for a tumor response, in the absence of any clear evidence at this time for a threshold, the Risk Estimation Panel believes that a threshold model for formaldehyde is not indicated. It is important to note that the quantal dose-response models use only one biological endpoint, tumor inci- dence, at any selected point in time and only consider the dose-response relationship in terms of this endpoint. More information may be obtained by incorporating time-to-tumor onset information (263,264). The impact of the dose level on the length of time until a tumor occurs may be part of the risk characterization. It should be noted that dose includes the components of concentration of the agent and of the duration of exposure (255). The CIIT data show that the tumor rate is strongly dependent on formaldehyde concentration, but time-to-tumor onset may also be investigated. In addition, the length of time and the number of animals that were at risk in the CIIT study is complicated because animals were scheduled for interim sacrifices. CONSENSUS WORKSHOP ON FORMALDEHYDE If more cells of the target tissue replicate, there is greater availability of single-stranded DNA for adduct formation, decreased time for repair of DNA adducts, and a greater chance of fixation of mutagenic events. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE Thus, the numbers of animals at risk need to be adjusted to take these sacrifices into account. Conclusions. Available animal and human studies provide information to qualitatively establish various toxic effects of formaldehyde exposure, but quantitative assessment is not indicated at this time for biological effects other than carcinogenicity. The uncertainty over the shape of the dose-response relationship in the low dose region and the correspond- Although some epidemiological studies noted that there may be an association between formaldehyde CONSENSUS WORKSHOP ON FORMALDEHYDE 358 exposure and some forms of cancer, the data from these studies are not sufficient, at this time, for quantitative risk modeling. 6. USEPA. Technical Document: Formaldehyde. U.S. Environmen- tal Protection Agency, Washington, DC, 1981. g y, g , , 7. Clement Associates. Formaldehyde risk assessment for occupa- tionally exposed workers. Report to Occupational Safety and Health Administration, Washington, DC (1982). s modeling. The CIIT and NYU animal studies (84) indicate that cancer results in rodents from exposure to formalde- hyde. Formaldehyde has also been shown to be geno- toxic. The CIIT chronic inhalation bioassay in rats provides a set of data suitable for quantitative risk assessment. This data set, the use of a nonthreshold low-dose linear extrapolation,' and the use of various plausible assumptions based on the available biological information regarding factors such as species to species extrapolation, metabolism and pharmacokinetics, pro- vide an upper bound for low-dose risk estimates in the sense that the true risk in humans exposed to formalde- hyde by inhalation is not likely to be greater than this limit. Nonlinear models are needed to fit the CIIT rat inhalation bioassay data over the experimental dose range, but estimates of risk from these models may vary at low doses. The panel could not reach a consen- sus on the way in which the uncertainty in choosing a family of nonlinear models should be incorporated into the risk assessment process. , g , ( ) 8. Blade, L. M. Occupational exposure to formaldehyde-recent NIOSH involvement. In: Formaldehyde: Tbxicology-Epidemi- ology-Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker, New York, 1983, pp. 1-23. gy ( y, Eds.), Marcel Dekker, New York, 1983, pp. 1-23 ), , , , pp 9. Hattis, D., Mitchell, C., McCleary-Jones, J., and Gorelick, N. Topic 2. Future Studies Required are long-term follow-up on epidemiological studies along with measurements of formaldehyde dos- age levels; improved measures of target dose; animal bioassays to study tumor rates resulting from short- term or intermittent formaldehyde exposures. 15. NIOSH. Health Hazard Evaluation Determination Report No. HE-78-116-557. Commonwealth Trading Co. NIOSH, Washing- ton, DC, 1979. , , 16. NIOSH. Industry selection for determination of extent of exposure. Memo from Larry Elliott, NIOSH Center for Disease Control, Cincinnati, OH, to NIOSH, Washington, DC, 1979. , , , , g , , 17. NIOSH. Health Hazard Evaluation Determination Report No. 80-192-828. Rock Hill Printing and Finishing Co., Rock Hill, NC, 1981. The Panelists wish to thank the following staff members of the National Center for Toxicological Research for their valuable assis- tance in planning and conducting the Consensus Workshop on Formaldehyde: Ronald W Hart, Angelo Turturro and Lorraine Neimeth (Document Editors); William F McCallum (Workshop Executive Secretary); Judy Emde (Midwest Research Institute), Ruth Bryant, Dabra Huneycutt, Robert Loe, Elaine McGarity, Alice McKenna, Arthur Norris, Faye Riggs, Linda Vetsch, Ken Weis and Ruth York. The Panelists wish to thank the following staff members of the National Center for Toxicological Research for their valuable assis- tance in planning and conducting the Consensus Workshop on Formaldehyde: Ronald W Hart, Angelo Turturro and Lorraine Neimeth (Document Editors); William F McCallum (Workshop Executive Secretary); Judy Emde (Midwest Research Institute), Ruth Bryant, Dabra Huneycutt, Robert Loe, Elaine McGarity, Alice McKenna, Arthur Norris, Faye Riggs, Linda Vetsch, Ken Weis and Ruth York. , 18. USDHEW Report on a study of formaldehyde exposures, Perfection Garment Manufacturing, Martinsburg, WV, U.S. Department of Health, Education and Welfare, Washington, DC, 1966. 19. USDHEW Formaldehyde emissions in the permanent-press fabrics industry. TR-52, Consumer Protection and Environmen- tal Health Service, Cincinnati, OH, 1968. 20. NIOSH. Health Hazard Evaluation Determination Report No. 75-143-333, Unitog Co., Warrensburg, MO. NIOSH, Washington, DC, 1976. / This report does not necessarily reflect the official views of any private or public organization but rather the personal scientific opinions of the participants. 21. NIOSH. Health Hazard Evaluation/Ibxicity Determination Re- port No. 73-1-66, NIOSH TR-066-74, Robin Products Co., Warren, MI. NIOSH, Washington, DC, 1973. i 22. NIOSH. Health Hazard Evaluation Determination Report No. 75-160-285, Corhart Refractories, Louisville, KY NIOSH, Washington, DC, 1976. CONSENSUS WORKSHOP ON FORMALDEHYDE Cause of occupational exposures to formaldehyde: a case study of methodology for assessing the health and economic impacts of OSHA health standards. Center for Policy Alternatives, Mass. Inst. Technology, Cambridge, MA, 1981. gy g 10. NIOSH. Walk-through survey report of Georgia-Pacific Chemi- cal and Resin Division, Crossett, AR. Report prepared by Larry Elliott, National Institute for Occupational Safety and Health, Center for Disease Control, Cincinnati, OH. NIOSH, Washing- ton, DC, 1980. , , 11. NIOSH. Health Hazard Evaluation Report No. 75-145-327, Formica Corp., Cincinnati, OH. NIOSH, Washington DC, 1976. 12. NIOSH. Health Hazard Evaluation Report Nos. 76-79, 80-543. Weyerhaeuser Co., Longview, WA. NIOSH, Washington, DC, 1978. 13. Herrick, H. E, Alcarese, A. A., Reisdorf, R. R, and Rumsey, D. L. Industrial hygiene characterization of urea-formaldehyde and polyurethane foam insulation. NIOSH Technical Report 83-108, Washington, DC, 1983. g , , 14. NIOSH. An industrial hygiene survey of urea-formaldehyde foam insulation manufacturing at Rapco Foam, Inc. Enviro Control, Inc., Rockville, MD, NIOSH Contract No. 210-78-0081, 1980. CONSENSUS WORKSHOP ON FORMALDEHYDE Case-control study of cancer deaths in DuPont workers with potential exposure to formaldehyde. In: Formaldehyde: Toxicology-Epi- demiology-Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker Inc., New York, 1983, pp. 47-113. g , , 36. Gupta, K. C., Ulsamer, A. G., and Preuss, P W Formaldehyde in indoor air: sources and toxicity. Environ. Int. 8: 349-358 (1982). 37. Urea-Formaldehyde Foam Insulation Information and Coordina- tion Center (UFFI/ICC). Report on the National Testing Survey to the Board of Review. Ottawa, Ontario, 1981. , , , , , pp 57. Harrington, J. M., and Shannon, H. S. Mortality study of pathologists and medical laboratory technicians. Brit. Med. J. 2: 329-334 (1974). , , 38. National Research Council. Formaldehyde and Other Aldehydes. National Academy Press, Washington, DC, 1981. 58. Harrington, J. M., and Oakes, D. Mortality study of British pathologists. In press. p g p 59. Levine, R. J., Andjelkovich, D. A., and Shaw, L. K. Mortality of Ontario undertakers: a first report. In: Formaldehyde: Toxi- cology-Epidemiology-Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker Inc., New York, 1983, pp. 127-140. 39. Hawthorne, A. R., Gammage, R. B., Dudney, C. S., Wormack, D. R., Morris, S. A., and Wesley, R. R. Preliminary results of a forty-home indoor air pollutant monitoring study. Proceedings, Conference on Measurement and Monitoring of Non-Criteria (Toxic) Contaminants in Air, Air Pollution Control Association, 1983, pp. 514. 60. Matanoski, G. M. Preliminary studies. Letter to John Martonik, OSHA, 1982. pp 40. Nero, A. V, Jr., and Grimsrud, D. T. Dependence of indoor pollutant concentrations on sources, ventilation rates, and other removal factors. Lawrence Berkeley Laboratory Rep. LBL- 16525. Berkeley, CA, 1983. 61. Stroup, N. Personal communication, 1983. 62. Walrath, J., and Fraumeni, J. F. Mortality patterns among embalmers. Int. J. Cancer 31: 407-411 (1983). y 41. Everett, L. H. Urea-formaldehyde foam and formaldehyde emission: U.K. experience with cavity wall insulation. Contribu- tion to the Formaldehyde Consensus Workshop, 1983. 63. Walrath, J. Mortality among embalmers. Am. J. Epidemiol. 118: 432 (1983) (tables provided separately). p y 64. Marsh, G. M. Proportional mortality study among chemical workers exposed to formaldehyde. In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Corp., New York, 1983. y p 42. Cohn, M. S. Revised carcinogenic risk assessment of UFFI: estimates of cancer risk due to inhalation of formaldehyde released by UFFI. CONSENSUS WORKSHOP ON FORMALDEHYDE 359 28. NIOSH. Health Hazard Determination Report No. 77-57-460, Johns-Manville Products Corp., Vernon, CA. NIOSH, Washing- ton, DC, 1978. 48. Grosjean, D. Formaldehyde and other carbonyls in Los Angeles ambient air. Environ. Sci. Technol. 16: 254-262 (1982). ( ) 49. International Agency for Research on Cancer (IARC). Mono- graphs on the Evaluation of Carcinogenic Risk of Chemicals to Humans. Vol. 29: Some Industrial Chemicals and Dyestuffs, IARC, Lyon, France, 1982, pp. 345-389. , , 29. NIOSH. Health Hazard Evaluation Determination Report No. HE 79-72-680, Energyloc Inc., Portland, OR. NIOSH, Washing- ton, DC, 1980. , , 30. NIOSH. Walk-through survey report: Report no. 125.10. Butler County Mushroom Farm, Inc., Worthington, PA. NIOSH, Washington, DC, 1980. , y , , , pp 50. Bundesgesundheitsamt (Federal Health Office of the Federal Republic of Germany) (BGA). Report on Formaldehyde (in preparation). Berlin, FRG, 1983. g , , 31. Kerfoot, E. J., and Mooney, T. F., Jr. Formaldehyde and paraformaldehyde study in funeral homes. Am. Ind. Hyg. Assoc. J. 36: 533-537 (1975). p p ) , , 51. Cosmetics, Toiletry, and Fragrance Association, Inc. (CTFA). Tentative Report of the Safety of Formaldehyde. Washington, DC, 1983. ( ) 32. NIOSH. Health Hazard Evaluation Determination Report HE- 79-146-670, Cincinnati College of Mortuary Science, Cincinnati, OH. NIOSH, Washington, DC, 1980. , 52. European Economic Community (EEC). Directive of 17 May 1982. 53. Meyer, H. D., Schluter, G., and Wagner, J. Formaldehyd Untersuchungen in Inkubatoren nach der Desinfektion. Bun- desgesundheitsblatt 25: 386-392 (1982). , g , , 33. Covino, S. J. Evaluation and control of formaldehyde exposure in a hospital autopsy room. Thesis, submitted to the Johns Hopkins University, Baltimore, MD, as partial fulfillment of the requirements for an MHS degree, 1979. g ( ) 54. Siegel, D. M., Frankos, V H., and Schneiderman, M. A. Formaldehyde risk assessment for occupationally exposed workers. Regulatory Tbxicol. Pharmacol. 3: 355-371 (1983). q g , 34. Coldiron, V R., Ward, J. B., Jr., Trieff, N. M., Jansson, H. E., Jr., and Smith, J. H. Occupational exposure to formaldehyde in a medical center autopsy service. J. Occup. Med. 25: 544-548 (1983). g y ( ) 55. Acheson, E. D., Barnes, H. R., and Gardner, M. J. Mortality study of workers exposed to formaldehyde. Personal communi- cation, 1983. ( ) 35. NIOSH. National Occupational Hazard Survey. NIOSH, Wash- ington, DC, 1974. , 56. Fayerweather, W E., Pell, S., and Bender, J. R. REFERENCES 1. Balmat, J. L. Formaldehyde Methods Manual. Formaldehyde Institute, Scarsdale, NY, 1983. g , , 23. NIOSH. Health Hazard Evaluation Determination Report No. HE 80-126-777, St. Regis Paper Co., Buckport, ME. NIOSH, Washington, DC, 1980. l i / b i i 2. Moore, B. P, and Brown, W V Chromatographic detection of small amounts of formic acid as dimethylformamide. J. Chroma- togr. 128: 178-179 (1976). g , , 24. NIOSH. Health Hazard Evaluation/Tbxicity Determination Re- port Nos. 75-177-343, NIOSH TR -HHE-75-177-343. Hersey Products Co., Inc., Dedham, MA. NIOSH, Washington, DC, 1976. i / i i g 3. Destler, W Truenung von Milch-, Glyorxyl, Ameisen-, Essig- und Propionsaure mit Hilfe der Umkehrphasen-Hochdruckflus- sigkeitschromatographie. J. Chromatogr. 152: 250-252 (1978). 4 M k A i l M d hl T 25. NIOSH. Health Hazard Evaluation/Tbxicity Determination Re- port Nos. HHE 73-116-85, NIOSH TR-085-74, Western Foundry Co., Tigard, OR. NIOSH, Washington, DC, 1973. i g g p g 4. Makar, A. B., McMartin, K. E., Palese, M. and Tephly, T. R. Formate assays in body fluids: application in methanol poisoning. Biochem. Med. 13: 117-126 (1975). i g g 26. NIOSH. Health Hazard Evaluation Determination Report No. 77-15-421, Leeds and Northrup Corp., Ellswood, PA. NIOSH, Washington, DC, 1977. 5. Synthetic Organic Chemical Manufacturers Association (SOCMA). Preliminary study ofthe costs of increased regulation of formaldehyde exposure in the U.S. workplace. Prepared by Booz, Allen & Hamilton, Inc. for SOCMA, Hamilton, NJ, 1979. g 27. NIOSH. Report No. 77-37-413, Gates Rubber Co., Denver, CO. NIOSH, Washington, DC, 1977. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE ( ) 101. Connor, T. H., Barrie, M. D., Theiss, J. C., Matney, T. S., and Ward, J. B., Jr. Mutagenicity of formalin in the Ames assay. Mutat. Res. 119: 145-149 (1983). y yg ( ) 80. Sparks, M. J., and Peters, J. M. Respiratory morbidity in workers exposed to dust cntaining phenolic resin. Int. Arch. Occup. Environ. Health 45: 221-229 (1980). 102. Temcharoen, R, and Thilly, W G. Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium. Mutat. Res. 119: 89-93 (1983). p ( ) 81. Goldmann, R, Flach, H.-D., Hey, W, Hochadel, H., Petri, N., Straussberger, K. J., and Thiess, A. M. Formaldehyde morbid- ity study. Zentralbl. Arbeitsmed. Arbeitsschutz. 32: 250-252, 254-258 (1982). 103. Magana-Schwencke, N., Ekert, B., and Moustacchi, E. Biochemi- cal analysis of damage induced in yeast by formaldehyde. I. Induction of single-stranded breaks in DNA and their repair. Mutat. Res. 50: 181-193 (1978). 82. Gamble, J. F, McMichael, A. J., Williams, T., and Battigelli, M. Respiratory function and symptoms: an environmental-epidem- iological study of rubber workers exposed to phenol-formalde- hyde type resin. Am. Ind. Hyg. Assoc. J. 37: 499-513 (1976). 104. Magana-Schwencke, N., and Ekert, B. Biochemical analysis of damage induced in yeast by formaldehyde. II. Induction of cross- links between DNA and protein. Mutat. Res. 51: 11-19 (1978). y yp yg 83. Battelle, Columbus Laboratories. A chronic inhalation toxicity study in rats and mice exposed to formaldehyde. Battelle, Columbus Laboratories, Columbus, OH (1981). 105. Chanet, R., and von Borstel, R. C. Genetic effects of formalde- hyde in yeast. III. Nuclear and cytoplasmic mutagenic effects. Mutat. Res. 62: 239-253 (1979). 84. Albert, R. E., Sellakumar, A. R., Laskin, S., Kischner, M., Nelson, N., and Snyder, C. A. Gaseous formaldehyde and hydrogen chloride induction of nasal cancer in the rat. J. Natl. Cancer Inst. 68: 597-603 (1982). 106. Szabad, J., Soos, I., Polgar, G., and Hejja, G. Testing the mutagenicity of malondialdehyde and formaldehyde by the Drosophila mosaic and the sex-linked recessive lethal tests. Mutat. Res. 113: 117-133 (1983). 85. Chang, J. C. F, Steinhagen, W H., and Barrow, C. S. Effects of single or repeated formaldehyde exposure on minute volume of B6C3F1 mice and F-344 rats. Toxicol. Appl. Pharmacol. 61: 451-459 (1981). 107. Ashby, J., and Lefevre, P Genetic toxicology studies with formaldehyde and closely related chemicals including hexa- methylphosphoramide (HMPA). In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Corp., New York, 1983, pp. CONSENSUS WORKSHOP ON FORMALDEHYDE 360 its relevance to cytotoxicity. Chem.-Biol. Interact. 38: 119-126 (1981). 72. Acheson, E. D., Barnes, H. R., Gardner, M. J., Osmond, C., Pannett, B., and Taylor, C. P Formaldehyde in the British chemical industry-an ocupational cohort study. Lancet i: 611-616 (1984). ( ) 94. Fornace, A. J., Jr. Detection of DNA single-strand breaks produced during the repair of damage by DNA-protein cross- linking agents. Cancer Res. 42: 145-149 (1982). ( ) 73. Acheson, E. D., Barnes, H. R., Gardner, M. J., Osmond, C., Pannett, B., and Taylor, C. P Formaldehyde process workers and lung cancer. Lancet, in press. g g ( ) 95. Ross, W E., McMillan, D. R., and Ross, C. F Comparison of DNA damage by methylmelamines and formaldehyde. J. Natl. Cancer Inst. 67: 217-221 (1981). g , p 74. Jensen, O., and Andersen, S. Correspondence: lung cancer risk from formaldehyde. Lancet i:913 (1982). ( ) 96. Fornace, A. J., Jr., Lechner, J. F, Grafstrom, R. C., and Harris, C. C. DNA repair in human bronchial epithelial cells. Carcino- genesis 3: 1373-1377 (1982). y 75. Kratochvil, I. Effects of formaldehyde on the health of workers in the crease-resistant clothing industry. Pracovni Lekarstvi 23: 374-375 (1971). g ( ) 97. Brusick, D. Genetic and transforming activity of formaldehyde. In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Corp., New York, 1983, pp. 73-84. ( ) 76. Schoenberg, J. B., and Mitchell, C. A. Airway disease caused by phenolic (phenol-formaldehyde) resin exposure. Arch. Environ. Health 30: 574-577 (1975). g p , , , pp 98. Obe, G., and Beek, B. Mutagenic activity of aldehydes. Drug. Alc. Depend. 4: 91-94 (1979). ( ) 77. Alexandersson, R., Kolmodin-Hedman, B., and Hedenstierna, G. Exposure to formaldehyde: effects on pulmonary function. Arch. Environ. Health 37: 279-283 (1982). p 99. Levy, S., Nocentini, S., and Billardon, C. Induction of cytoge- netic effects in human fibroblast cultures after exposure to formaldehyde or x-rays. Mutat. Res. 119: 309-317 (1983). ( ) 78. Thun, M. J., Lakat, M. F, and Altman, R. Symptom survey of residents of homes insulated with urea-formaldehyde foam. Environ. Res. 29: 320-334 (1982). y y ( ) 100. Boreiko, C. J., Couch, D. B., and Swenberg, J. A. Mutagenic and carcinogenic effects of formaldehyde. Environ. Sci. Res. 25: 353-367 (1982). ( ) 79. Ols0n, J. H., and D0ssing, M. Formaldehyde induced symptoms in day care centers. Am. Ind. Hyg. Assoc. J. 43:366-370 (1982). CONSENSUS WORKSHOP ON FORMALDEHYDE 85-97. ( ) 86. Dalbey, W E. Formaldehyde and tumors in hamster respiratory tract. Toxicology 24: 9-14 (1982). pp 108. Goldmacher, V S., and Thilly, W G. Formaldehyde is mutagenic for cultured human cells. Mutat. Res. 116: 417-422 (1983). / / 87. Feron, V J., Kruysse, A., and Woutersen, R. A. Respiratory tract tumours in hamsters exposed to acetaldehyde vapour alone or simultaneously to benzo(a)pyrene or diethylnitrosamine. Eur. J. Cancer Clin. Oncol. 18: 13-31 (1982). 109. Ragan, D. L., and Boreiko, C. J. Initiation of C3H/lOT1/2 cell transformation by formaldehyde. Cancer Letters 13: 325-331 (1981). 88, Woutersen, R. A., Appelman, L. M., Feron, V J., and Van der Heijden, C. A. Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study: interim results after 15 months. Toxi- cology 31: 123-133 (1984). 110. Hatch, G. G., Conklin, P M., Christensen, C. C., Casto, B. C., and Nesnow, S. Synergism in the transformation of hamster embryo cells treated with formaldehyde and adenovirus. En- viron. Mutagensis 5: 49-57 (1983). 89. Swenberg, J. A., Barrow, C. S., Boreiko, C. J., Heck, D. d'A., Levine, R. J., Morgan, K. T., and Starr, T. B. Non-linear biological responses to formaldehyde and their implications for carcinogenic risk assessment. Carcinogenesis 4: 945-952 (1983). 90 S b J A G d ll W d g 111. Grafstrom, R. C., Fornace, A. J., Jr., Autrup, H., Lechner, J. F, and Harris, C. C. Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells. Science 220: 216-218 (1983). g 90. Swenberg, J. A., Gross, E. A., Randall, H. W, and Barrow, C. S. The effect of formaldehyde exposure on cytotoxicity and cell proliferation. In: Formaldehyde: Toxicology-Epidemiology- Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker, New York, 1983. 112. Epstein, S. S., Arnold, E., Andrea, J., Bass, W, and Bishop, Y Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol. 23: 288-325 (1972). 113 i i b h i ff f f ld h d i h 113. Fontignie-Houbrechts, N. Genetic effects of formaldehyde in the mouse. Mutat. Res. 88: 109-114 (1981). 11 k i i i 91. Auerbach, C., Moutschen-Dahman, M., and Moutschen, J. Genetic and cytogenetical effects of formaldehyde and related compounds. Mutat. Res. 39: 317-362 (1977). 92 i i i 114. Gocke, E., King, M. T., Eckhardt, K., and Wild, D. CONSENSUS WORKSHOP ON FORMALDEHYDE Consumer Product Safety Commission, Washington, DC, 1981. 65. Hernberg, S., Collan, Y., Degerth, R., Englund, A., Enggell, U., Kuosma, E., Mutanen, RB, Nordlinder, H., Hansen, H. S., and Schultz-Larsen, K. Nasal cancer and occupational exposures. Scand. J. Work Environ. Health 9: 208-213 (1983). g , 43. U.S. EPA. Indoor Air Pollution in the Residential Environment. Vol. 1. Data Collection, Analysis and Interpretation. U.S. Environmental Protection Agency Report EPA-600/7-78-2990, Cincinnati, OH, 1979. 66. Brinton, L., Becker, J., Blot, W, Hoover, R., and Fraumeni, J. A case-control study of cancer of the nasal cavity and sinuses. Am. J. Epidemiol. 118: 436 (1983). i 44. Gammage, R. B., Hingerty, B. E., Wormack, D. R., Westley, R. R., Mathews, P G., Hawthorne, A. R., and Gupta, K. C. Temporal fluctuations of formaldehyde levels inside residences. Proceedings, Conference on Measurement and Monitoring of Non-Criteria (Toxic) Contaminants in Air, Air Pollution Control Assoc., 1983, p. 459. 67. Halperin, W E., Goodman, M., §tayner, L., Elliott, L., Keenlyside, R. A., and Landrigan, P J. Nasal cancer in a worker exposed to formaldehyde. J. Am. Med. Assoc. 249: 510-512 (1983). ( ) 68. Infante, R, and Kang, H. Nasal sinus, pharyngeal, and buccal cavity cancer and formaldehyde exposed workers. OSHA inter- nal document. i i 45. Consumer Product Safety Commission. Part IV Bar of UFFI, withdrawal of proposed information labeling rule and denial of petition to issue a standard. Fed. Reg. 47(64): 14366-14419 (1982). 69. Liebling, J., Rosenman, K. D., and Pastidies, H., and Lemeshaw, S. Cancer mortality among workers exposed to formaldehyde. Manuscript in preparation, 1983. i 46. Matthews, T. G., Hawthorne, A. R., Daffron, C. R., Reed, T. J., and Corey, M. D. Formaldehyde releases from pressed-wood products. Proceedings 17th International Washington State Univ. Particleboard/Composite Materials Symposium, Seattle, WA, 1983. i 70. Tabershaw Associates, Inc. Historical perspective mortality study of past and present employees of the Celanese chemical and plastics plant located in Bishop, Texas. Manuscript, 1982. 71 ld i dl d 47. Girman, J. R., Geisling, K. L., and Hodgson, A. T. Sources and concentrations of formaldehyde in indoor environments. Law- rence Berkeley Laboratory Report LBL-14574. Berkeley, CA, 1983. p 71. Greenwald, R, Friedlander, B. R., Lawrence, C. E., Hearne, T., and Earle, K. Diagnostic sensitivity bias-an epidemiologic explanation for an apparent brain tumor excess. J. Occup. Med. 23: 690-694 (1981). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 361 ments with formaldehyde. Cancer Res. 43: 3236-3239 (1983). 108: 537-540 (1973). 108: 537-540 (1973). y 117. Spangler, F, and Ward, J. M. Skin initiation-promotion study with formaldehyde in Sencar mice. In: Formaldehyde: Toxicology- Epidemiology-Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker, New York, 1983. 138. Stokinger, H. E., and Coffin, D. L. A formaldehyde and its homologs. In: Air Pollution, Vol. 1 (A. C. Stern, Ed.), Academic Press, New York, 1977, pp. 483-484. , , , pp 139. Health and Safety Executive. Toxicity Review 2: Formaldehyde. Her Majesty's Stationery Office, London, 1981. , , , , 118. Krivanek, N. D., Chromey, N. C., and McAlack, J. W Skin initiation promotion study with formaldehyde in CD-1 mice. In: Formaldehyde: Toxicology-Epidemiology-Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker, New York, 1983. j y y , 140. ECETOC. Technical Report: No. 1. Assessment of Data on the Effects of Formaldehyde on Humans. European Chemical Indus- try Ecology and Toxicology Centre, Belgium, 1981. y gy gy , g , 141. Gibson, J. E. Formaldehyde Toxicity. Hemisphere Publishing Co., Washington, DC, 1983. 119. Argyris, T. S. and Slaga, T. J. Promotion of carcinomas by repeated abrasion in initiated skin of mice. Cancer Res. 41: 5193-5195 (1981). , g , , 142. Federal Panel on Formaldehyde. Report of the Federal Panel on Formaldehyde. Environ. Health Perspect. 43: 139-168 (1982). 120. Argyris, T. S. Epidermal tumor promotion by regeneration. In: Carcinogenesis, a Comprehensive Survey, Vol. 7 (E. Heckler, N. E. Fusenig, W Kunz, F Marks and H. W Thielmann, Eds.), Raven Press, New York, 1982, pp. 43-48. y p ( ) 143. Clary, J. J., Gibson, J. E, and Waritz, R. S. (Eds.). Formalde- hyde: Toxicology-Epidemiology-Mechanisms. Marcel Dekker Inc., New York, 1983. y hyde: Toxicology-Epidemiology-Mechanisms. Marcel Dekker Inc., New York, 1983. , 144. Maibach, H. Formaldehyde: effects on animal and human skin. In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Corp., Washington, DC, 1983, Chapter 14, pp. 163-174. , , , pp 121. Popa, V, Teculescu, C., Stanescu, D., and Gavrilescu, N. Bronchial asthma and asthmatic bronchitis determined by sim- ple chemicals. Dis. Chest 56(5): 395-404 (1969). p ( ) ( ) 122. Pepys, J., and Hutcheroft, B. J. Bronchial provocation tests in etiologic diagnosis and analysis of asthma. Am. Rev. Respir. Dis. 112(6): 829-859 (1975). 145. Milby, T. H., Key, M. CONSENSUS WORKSHOP ON FORMALDEHYDE Schoenberg, J. B., and Mitchell, C. A. Airway disease caused by phenolic (phenol-formaldehyde) resin exposure. Arch. Environ. Health 30: 574-577 (1975). 129. Gafafer, W M. Occupational diseases: a guide to their recognition. U.S. Department of Health, Education, and Welfare, Publication No. 1097, U.S. Government Printing Office, 1964. g 130. Kane, L. E., and Alarie, Y. Sensory irritation to formaldehyde and acrolein during single and repeated exposures in mice. Am. Ind. Hyg. Assoc. J. 38: 509-522 (1977). 154. Kuzmenko, M. N., Buslenko, A. I., Kataeva, S. E., Kravchenko, T. I., and Asatryan, R. S. Study of the sensitizing action of formaldehyde under the conditions of plastic manufacture. Vrach. Delo 6: 131-134 (1975). ll i yg ( ) 131. Loomis, T. A. Formaldehyde toxicity. Arch. Pathol. Lab. Med. 103: 321-324 (1979). 155. Wallenstein, V G., and Rebohle, E. Sensibilisierungen durch Formaldehyd bei berufficher inhalativer Exposition. Allergie Ummunologie 22: 287-295 (1976). 156 ki l h h d i i 132. Morse, S. S., Stark, J. W C., and Moschadreas, D. J. Benzo(a)pyrene and aldehydes in the residential environment. An annotated bibliography for the U.S. Environmental Protec- tion Agency, GEOMET Report Number E-719, July 18, 1979, pp. 1-33. g 156. Spassovski, M. Health hazards in the production and processing of some fibers, resins, and plastics in Bulgaria. Environ. Health Perspect. 17: 199-202 (1976). pp 133. Moschandreas, D. J., Moyer, R. H., Ward, J. R., Rector, H. E., and Schakenbach, J. T. An evaluation of formaldehyde problems in residential mobile homes. Final Task Report I, GEOMET Report No. ESF-797, for the Department of Housing and Urban Development, Washington, DC, April 1980, pp. 1-146. p 157. Cockcroft, D. W, Hoeppner, V H., and Dolovich, J. Occupa- tional asthma caused by cedar urea-formaldehyde particle board. Chest 82(1): 49-53 (1982). 158. Baur, X., and Fruhmann, G. Bronchial asthma of allergic or irritative origin as an occupational disease. Prax. Klin. Pneumol. 33 (Suppl. 1): 317-322 (1979). 134. NIOSH. A Recommended Standard for Occupational Exposure to Formaldehyde. DHEW (NIOSH) Publication No. 757-009/46, Washington, DC, 1977. 13 ( i l il) i pp 159. Burge, P S., and Pepys, J. Berufsbezogener brochialer Provo- kationstest. Allergologie 2: 7-12 (1979). i i g g 160. Frigas, E., Filley, W V, and Reed, C. E. Asthma induced by dust from urea-foam insulating material. Chest 79: 706-707 (1981). 135. NRC (National Research Council). Vapor-Phase Organic Pollu- tants. National Academy Press, Washington, DC, 1976, pp. CONSENSUS WORKSHOP ON FORMALDEHYDE M., and Gibson, R. L. Chemical hazards. In: Occupational Diseases (W M. Gafafer, Ed.), U.S. Depart- ment of Health Education, and Welfare, Publ. No. 1097, U.S. Government Printing Office, 1964, Section 6, pp. 63-242. ( ) ( ) 123. Hendrick, D. J., and Lane, D. J. Occupational formalin asthma. Brit. J. Ind. Med. 34: 11-18 (1977). g , , , pp 146. Hendrick, D. J. The formaldehyde problem: a clinical appraisal. Immunol. All. Prac. 5(4): 21-32 (1983). 1 i i i 124. Anderson, I. Formaldehyde in the indoor environment-health implications and the setting of standards. In: Indoor Climate: Effects on Human Comfort, Performance and Health in Resi- dential, Commercial, and Light-Industry Buildings (P 0. Fanger and 0. Valbjorn, Eds.), Proceedings of the First International Indoor Climate Symposium, Copenhagen, August 30-September 1'/8, 1978, Copenhagen: Danish Building Research Institute, 1979, pp. 65-87. 147. Granati, A., Calsini'e, P, and Lenzi, R. Criteri di valutazione di pericolosita di agenti chimici richerche nell'industria dell'ab- bigliamento. Med. Lavoro. 1: 22-32 (1981). g ( ) 148. Skerfving, S., Akesson, B., and Simonsson, B. G. Meatwrappers' asthma caused by thermal degradation products of polyethylene. Lancet i: 211 (1980). 149. Ostapovich, I. K. Conditions of respiratory route exposure to sulfur dioxide and formaldehyde and subsequent sensitization. Gig. Sanitar. No. 2: 9-13 (1975). , pp 125. Andersen, I., and Molhave, L. Controlled human studies with formaldehyde. In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Corp., Washington, DC, 1983, Chapter 14, pp. 154-165. g 150. Zaeva, G. N., Ulanova, I. P, and Dueva, L. A. Materials for revision of the maximal permissible concentrations of formalde- hyde in the inside atmosphere of industrial premises. Gig. Truda Prof. Zab. 12(7): 16-20 (1968). , pp 126. Bardana, E. J. Formaldehyde: hypersensitivity and irritant reactions at work and in the home. Immunol. All. Prac. 2(3): 22-23 (1980). ( ) ( ) 151. Hendrick, D. J., and Lane, D. J. Formalin asthma in hospital staff. Brit. Med. J. 1: 607-608 (1975). i 127. Department of Health and Human Services (DHHS). Report of the Federal Panel on Formaldehyde, November 1980. 152. Hendrick, D. J., Rando, R. J., Lane, D. J., and Morris, M. J. Formaldehyde asthma: challenge exposure levels and fate after five years. J. Occup. Med. 24: 893-897 (1982). y , 128. Electric Power Research Institute (EPRI). Indoor-outdoor pollu- tion levels: a bibliography. EA-1025, March 1979. i i i i i i y p 153. CONSENSUS WORKSHOP ON FORMALDEHYDE Mutageni- city of cosmetic ingredients licensed by European communities. Mutat. Res. 90: 91-109 (1981). i i p 92. Ross, W E. and Shipley, N. Relationship between DNA damage and survival in formaldehyde-treated mouse cells. Mutat. Res. 79: 277-283 (1980). 115. Litton Bionetics. In vivo mutagenicity studies of formaldehyde vapors. Final Report, 1982. b h 93. Bedford, R, and Fox, B. W The role of formaldehyde in methylene dimethanesulphonate-induced DNA cross-links and 116. Frazelle, J. H., Abernethy, D. J., and Boreiko, C. J. Weak promotion of C3H/1OT1/2 cell transformation by repeated treat- CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 35: 41-48 (1978). pp 176. Chang, J. C. F, Gross, E. A., Swenberg, J. A., and Barrow, C. S. Nasal cavity deposition histopathology, and cell proliferation after single or repeated formaldehyde exposures in B6C3F1 mice and F-344 rats. Toxicol. Appl. Pharmacol. 68: 161-176 (1983). g 199. Harrison, P B., Jansson, K., Kronenberg, H., Mahony, J. F, and Tiller, D. Cold agglutinin formation in patients undergoing haemodialysis. A possible relationship to dialyser re-use. Austral. N. Z. J. Med. 5: 195-197 (1975). i 177. Coffin, D. L., and Stokinger, H. E. Biological effects of air pollutants. In: Air Pollution, Vol. II. The effects of air pollution (A. C. Stern, Ed.), Academic Press, New York, 1977. i i 200. Howell, E. D., and Perkins, H. A. Anti-N-like antibodies in the sera of patients undergoing chronic hemodialysis. Vox Sang. 23: 291-299 (1972). i 178. Patterson, R. M., Bornstein, M. I., and Garshick, E. Assess- ment of formaldehyde as a potential air pollution problem, Volume III. GCA-TR-75-32-G(8). GCA Technology Division, GCA Corp., Bedford, Mass. for EPA Contract No. 68-02-1337 (1976). 201. McLeish, W A., Brathwaite, A. F, and Peterson, P M. Anti-N antibodies in hemodialysis patients. Iransfusion 15: 43 (1975). 202 b h i h T d G dj E I it ti 202. Weber-Tschopp, A., Fischer, T., and Grandjean, E. Irritating effects of formaldehyde on men. Int. Arch. Occup. Environ. Health 39: 207-218 (1977). 179. Weber, A. Acute effects of environmental smoke on constituents. Eur. J. Respir. Dis., in press. 203. Yefremov, G. G. State of the upper respiratory tract in formalde- hyde production workers (by the data of a special investigation). Zhur. Ushn. Nosov. Gorlov. Bolezn. 30: 11-15 (1970). 180. Blejer, H. P, and Miller, B. H. Occupational health report of formaldehyde concentrations and effects on workers at the Bayly Manufacturing Company, Visalia. Study Report No. S-1806. Los Angeles: State of California Health and Welfare Agency, Depart- ment of Public Health, Bureau of Occupational Health, 1966, p. 6. 204. Hanrahan, L. P, Anderson, H. A., Dally, K. A., Eckamnn, A. D., and Kanarek, M. S. An investigation of the offgassing decay function in aging mobile homes with climate corrected formalde- hyde readings. International Symposium on Indoor Air Pollution, Health and Energy Conservation, October 13-16 1981. 205 D ll E k A D H h L P A d H 181. USDHEW Formaldehyde Vapor Emissions in the Permanent- Press Fabrics Industry. Report No. CONSENSUS WORKSHOP ON FORMALDEHYDE 362 162. Burge, P S., Harris, M. G., Lam, W K., O'Brien, I. M., and Patchett, P A. Occupational asthma due to formaldehyde. Thorax, in press. mental chronic obstructive lung disease. I. Bronchopulmonary changes induced in rabbits by prolonged exposure to formalde- hyde. Morphol. Embryol. (Bucharest) 24: 233 (1978). , p 163. Noceto, J. B., and Laffont, H. Un cas d'asthme par sensibili- sation au formol. Arch. Mal. Prof. 23: 314-316 (1962). y p y 184. Amdur, M. 0. The response of guinea pigs to inhalation of formaldehyde and formic acid alone and with a sodium chloride aerosol. Int. J. Air Poll. 3(4): 201-220 (1960). 164. Gernez-Rieux, C. A., Marchand, M., Voisin, C., and Wallaest, C. Les bronchopneumopathies professionelles en dehors des pneumoconioses. Arch. Mal. Prof. 27: 183-214 (1966). ( ) ( ) 185. Nielsen, G. D., and Alarie, Y Sensory irritation, pulmonary irritation and respiratory stimulation by airborne benzene and alkylbenzenes. Prediction of safe industrial exposure levels and correlation with their thermodynamic properties. Toxicol. Appl. Pharmacol. 65: 459-477 (1982). p ( ) 165. Alanko, K., Keskinen, H., and Saarinen, L. Occupational asthma. Duodecim 93: 306-318 (1977). 166. Odom. R. B., and Maibach, H. I. Contact urticaria: a different contact dermatitis. In: Advances in Modern Toxicology, Vol. 4. Dermatotoxicology and Pharmacology (F N. Marzull and H. I. Maibach, Eds.), Hemisphere Publishing Corp., Washington, D.C., 1977, Chapter 15, pp. 441-453. ( ) 186. Heck, H., and Casanova-Schmitz, M. Biochemical toxicology of formaldehyde. Biochem. Toxicol., in press. y p 187. Hoffmann, D., and Wynder, E. L. Organic particulate pollutants- chemical analysis and bioassays for carcinogenicity. In: Air Pollution, Vol. II (A. C. Stern, Ed.), Academic Press, New York, 1977. , , p , pp 167. Hosfall, F L., Jr. Formaldehyde hypersensitiveness -an experi- mental study J. Immunol. 27: 569-581 (1934). 188. Burdach, S. T., and Wechselberg, K. Damage to health at school complaints due to the use of materials which emit formaldehyde in school buildings. Fortsch. Med. 98(11): 379-384 (1980). y 168. Epstein, E., and Maibach, H. I. Formaldehyde allergy: inci- dence and patch tests problems. Arch. Dermatol. 94: 186-190 (1966) ( ) 169. Uehara, M. Follicular contact dermatitis due to formaldehyde. Dermatology 156: 48-54 (1978). g 189. Helwig, H. Wie ungefahrlich ist formaldehyd? Deut. Med. Wochenschr. 102: 1612-1613 (1977). 190. Ishchenko, U. N. and Pushkina, I. K. Evaluation of working conditions in a plant engaged in processing phenol formaldehyde resins. Gig. CONSENSUS WORKSHOP ON FORMALDEHYDE Sanitar. 11: 98-100 (1978). gy 170. Marzulli, F N., and Maibach, H. I. The use of graded concentra- tions in studying skin sensitizers: experimental contact sensitiza- tion in man. Food Cosmet. Toxicol. 12: 219-227 (1974). g 191. Goldsmith, J. R., and Friburg, T. Effects of air pollution on human health. In: Air Pollution, Vol. II (A. C. Stern, Ed.), Academic Press, New York, 1977. 171. Boettcher, B., Nanra, R. S., Roberts, T. K., Mallan, M., and Watterson, C. A. Specificity and possible origin of anti-N antibodies developed by patients undergoing chronic haemodialy- sis. Vox Sang. 31: 408 (1976). , , 192. Shy, C., Goldsmith, J., Hackney, J., Lebowitz, M. D., and Menzel, D. Health Effects of Air Pollution, ATS News, New York 4: 22-62 (1978). g 172. Gorst, D., Riches, R., and Renton, P Formaldehyde induced anti-N: a possible cause of renal graft failure. J. Clin. Pathol. 30: 956-959 (1977). 193. U. S. EPA. Air Quality Criteria for Particulate Matter and Sulfur Oxides, Vol. III, Research Triangle Park, 1982, Chapters 12, 14. US EPA EPA-600/8-82-029c. 173. Lynen, R., Rothe, M., and Gallasch, E. Characterization of formaldehyde-related antibodies encountered in hemodialysis patients at different stages of immunization. Vox Sang. 44: 81-89 (1983). 194. Hoy, W E., and Cestero, R. V M. Eosinophilia in maintenance hemodialysis patients. J. Dialysis 3: 73-87 (1979). ( ) 174. Fassbinder, WV, Pilar, J., Scheuermann, E., and Koch, M. Formaldehyde and the occurrence of anti-N-like cold agglutinins in RDT patients. Proc. Eur. Dialysis Transplantation Assoc. 13: 333-338 (1976). y p 195. Goh, K. O., and Cestero, R. V M. Health hazards of formalde- hyde. J. Am. Med. Assoc. 247: 2778 (1982). y 196. Sandler, S. G., Sharon, R., Bush, M., Stroup, M., and Sabo, B. Formaldehyde-related antibodies in hemodialysis patients. Trans- fusion 19(6): 682-687 (1979). 175. Alarie, Y. Toxicological evaluation of airborne chemical irritants and allergens using respiratory reflex reactions. Proceedings of a Symposium on Inhalation Toxicology and Technology (B. K. J. Leong, Ed.), Ann Arbor Science Publishers, Ann Arbor, MI, 1981, pp. 207-231. 197. Crosson, J. T., Moulds, J., Comty, C. M., and Polesky, H. F A clinical study of anti-NDP in the sera of patients in a large repetitive hemodialysis program. Kidney Int. 10: 463 (1976). p y p g y 198. Fassbinder, W, Seidl, S., and Koch, K. M. The role of formalde- hyde in the formation of haemolydialysis-associated anti-N-like antibodies. Vox Sang. CONSENSUS WORKSHOP ON FORMALDEHYDE 190-191, 215, 222-226, 41-42. 136. NRC. Indoor Pollutants. National Academy Press, Washington, DC, 1981, pp. 6, 23, 31, 37, 40-41, 49-50, 326-327. 13 h i i i id i l f 161. Frigas, E., Filley, W V, and Reed, C. E. Bronchiolar challenge with formaldehyde gas: lack of bronchial construction in 13 patients suspected offormaldehyde-induced asthma. Mayo Clinic Proc., in press. 137. North American Contact Dermatitis Group. Epidemiology of contact dermatitis in North American: 1972. Arch. Dermatol. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE Toxicol. 47: 239-240 (1980). , , , , pp 212. Marks, T. A., Worthy, W C., and Staples, R. E. Influence of formaldehyde and SonacideR (potentiated acid glutaraldehyde) on embryo and fetal development in mice. Teratology 22(1): 51-58 (1980). ( ) 236. Zitting, A., and Savolainen, H. Biochemical effects of subacute formic acid vapor exposure. Res. Commun. Chem. Pathol. Pharmacol. 27: 157-162 (1980). ( ) 213. Hurni, H., and Ohder, H. Reproduction study with formalde- hyde and hexamethylenetetramine in beagle dogs. Food Cosmet. Toxicol. 11: 459-462 (1973). 237. Savolainen, H. Dose-dependent effects ofperoral dimethylforma- mide administration on rat brain. Acta Neuropathol. 53: 249-252 (1981). ( ) 214. Della Porta, G., Cabral, J. R., and Parmiani, G. Transplacental toxicity and carcinogenesis studies in rats with hexamethylene- tetramine. Tumori 56: 325-334 (1970). 238. Zitting, A., Savolainen, H., and Nickels, J. Biochemical and toxicological effects of single and repeated exposures to polyacetal thermodegradation products. Environ. Res. 29: 287-296 (1982). ( ) 215. Natvig, H., Andersen, J., and Rasmussen, E. W A contribution to the toxicological evaluation of hexamethylenetetramine. Food Cosmet. Toxicol. 9: 491-500 (1971). ( ) 239. Kulle, T. J., and Cooper, G. P Effects of formaldehyde and ozone of the trigenimal nasal sensory system. Arch. Environ. Health 30: 237-243 (1975). ( ) 216. Staples, R. E. Teratogenicity of formaldehyde. In: Formalde- hyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Co., New York, 1983, Chapt. 6, pp. 51-62. ( ) 240. Potts, A. M., Praglin, J., Farkas, I., Orbison, L., and Chickering, D. Studies on the visual toxicity of methanol. VIII. Additional observations on methanol poisoning in the primate test object. Am. J. Ophthalmol. 40: 76-83 (1955). p pp 217. Hagino, N. Ovulation and mating behavior in female rats under various environmental stresses or androgen treatment. Japan. J. Physiol. 18: 350-355 (1968). y 218. LaBelle, C. W, Long, J. E., and Christofano, E. E. Arch. Synergistic effects of aerosols; particulates as carriers of toxic vapors. Ind. Health 11: 297-304 (1955). j p 241. Bonashevskaya, T. I. Amygdaloid lesions after exposure to formaldehyde. Arkh. Anat. Gistol.-Embriol. 65: 56-59 (1973). 242. Sauter, D. L. The development of a battery for the exploratory screening for neuropsychological deficits in children exposed to formaldehyde. Ph.D. Dissertation, University of Wisconsin, Madison, WI, 1981. Available from University Microfilms, University of Michigan. p 219. Barlow, S. M., and Sullivan, F M. Formaldehyde. In: Reproduc- tive Hazards of Industrial Chemicals (S. M. CONSENSUS WORKSHOP ON FORMALDEHYDE Barlow and F M. Sullivan, Eds.), Academic Press, New York, 1982, Chapter 24, pp. 334-345. pp 220. Shumilina, A. V Menstrual and reproductive functions in workers with occupational exposure to formaldehyde. Gig. Truda Prof. Zab. 12: 18-21 (1975). y g 243. Dally, K. A., Hanrahan, L. P, Woodbury, M. A., and Kavarek, M. S. Formaldehyde exposure in non-occupational environments. Arch. Environ. Health 36: 277-284 (1981). 221. Hemminki, K., Mutanen, R, Saloniemi, I., Niemi, M. L., and Vainio, H. Spontaneous abortions in hospital staff engaged in sterilizing instruments with chemical agents. Brit. Med. J. 285: 1461-1463 (1982). 244. Sardinas, A. V, Most, R. S., Guiletti, M. A., and Honchar, P Health effects associated with urea-formaldehyde foam insula- tion in Connecticut. J. Environ. Health 41: 270-272 (1979). 245. Garry, V E, Oatman, L., Preus, R., and Gray, D. Formaldehyde in the home; some environmental disease perspectives. Minn. Med. 107-111 (1980). ( ) 222. Hemminki, K., Mutanen, P, and Niemi, M. L. Letter: Spontane- ous abortions in hospital sterilizing staff. Brit. Med. J. 286: 1976-1977 (1983). 246. Woodbury, M. A., and Zenz, C. Formaldehyde in the home environment: prenatal and infant exposures. In: Formaldehyde Tbxicity (J. E. Gibson, Ed.), Hemisphere Publishing Co., Washington, DC, 1983, pp. 203-211. 223. Centers for Disease Control. Temporal trends in the incidence of birth defects, United States (1979). Morbidity and Mortality Weekly Reports, 28 August 1979. y p g 224. Cassidy, S. L., Dix, K. M., and Jenkins, T. Evaluation of a testicular sperm head counting technique using rats exposed to dimethoxyethyl phthalate (DMEP), glycerol A-monochlorohydrin (GMCH), epichlorohydrin (ECH), formaldehyde (FA) or methyl methanesulfphonate (MMS). Arch. Ibxicol. 53: 71-78 (1983). g , , , pp 247. Thun, M. J., Lakat, M. F, and Altman, R. New Jersy Urea Formaldehyde Foam Insulation Study. New Jersey State Health Dept., 1983. p , 248. Schenker, M. B., Weiss, S. T., and Murawski, B. W Health effects of residents in homes with urea formaldehyde foam insulation: a pilot study Environ. Int. 8: 359-363 (1982). 249 ilb h l p 225. Adams, M. M., Erickson, J. D., Layde, P M., and Oakley, G. P Down's Syndrome: recent trends in the United States. J. Am. Med. Assoc. 246: 758-760 (1981). i 249. Kilburn, K. H., Warshaw, R., Boylen, C. T., Balmer, J., Johnson, S., Seidman, B. S., and DeFloria, G. Toxic effects of formaldehyde and solvents in histology technicians, a prelimi- nary report. J. CONSENSUS WORKSHOP ON FORMALDEHYDE 363 and Toxicology (F A. Patty, Ed.), 2nd Rev. Ed., Vol. II, Interscience, New York, 1963, pp. 1959-1989. Co., Washington, DC, 1983, pp. 26-37. Co., Washington, DC, 1983, pp. 26-37. g pp 228. Golberg, L. Risk assessment with formaldehyde: introductory remarks. In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemi- sphere Publishing Co., New York, 1983, pp. 275-278. 1 , , , pp 207. Dean, J. H., Lauer, L. D., House, R. V, Murray, M. G., Stillman, W S., Trous, R. D. Steinhagen, W H., Phelps, M. C., and Adams, D. 0. Studies of immune function and resistance in B6C3F mice exposed to formaldehyde. Toxicol. Pharmacol., in press. p g pp 229. Neely, W B. The metabolic fate of [14Clformaldehyde intraperi- toneally administered to the rat. Biochem. Pharmacol. 13: 1137-1142 (1964). p 208. Leach, C. L., Sharma, R. R, and Oberg, S. G. Immunotoxic and pathologic effects of various levels of formaldehyde vapour in F344 rats. Toxicologist 3: 73 (1983). ( ) 230. Du Vigneaud, V, Verly, W A., and Wilson, J. Z. Incorporation of the carbon of formaldehyde and formate into the methyl groups of choline. J. Am. Chem. Soc. 72: 2819-2820 (1950). g ( ) 209. Fielder, R. J. Formaldehyde. Her Majesty's Stationery Office, London, 1981. g p 231. Nicholls, P Formate as an inhibitor of cytochrome C oxidase. Biochem. Biophys. Res. Commun. 67: 610-616 (1975). , 210. Koivusalo, M., Koivula, T., and Uotila, L. Oxidation of formalde- hyde by nicotinamide nucleotide dependent dehydrogenases. In: Enzymology of Carbonyl Metabolism: Aldehyde Dehydrogenase and Aldo/Keto Reductase (H. Weiner and B. Wermuth, Eds.), Alan R. Liss, New York, 1982, pp. 155-168. p y 232. Nicholls, P The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain. Biochem. Biophys. Acta 430: 13-29 (1976). p y ( ) 233. Keyhani, J., and Keyhani, E. EPR study of the effect of formate on cytochrome C oxidase. Biochem. Biophys. Res. Commun. 92: 327-333 (1980) , , , pp 211. Ulsamer, A. G., Beall, J. R., Kang, H., and Frazier, J. A. Overview of health effects of formaldehyde. In: Hazard Assess- ment of Chemicals, Current Developments, Vol 3 (J. Saxsena, Ed.), Academic Press, New York, 1984, pp. 337-400. 234. Roe, 0. Species differences in methanol poisoning. Crit. Rev. Toxicol. 10: 275-286 (1982). ( ) 235. Savolainen, H., and Zitting, A. Glial cell effects of subacute formic acid vapour exposure. Acta Pharmacol. CONSENSUS WORKSHOP ON FORMALDEHYDE TR-52, Cincinnati OH, Consumer Protection and Environmental Health Service, 1968, p. 18. alth and Energy Conservation, October 13-16 1981. ll k A D H h L P A d 205. Dally, K. A., Eckamnn, A. D., Hanrahan, L. P, Anderson, H. A., and Kanarek, M. S. A follow-up study of indoor air quality in Wisconsin homes. International Symposium on Indoor Air Pollution, Health and Energy Conservation, October 13-16, 1981. 182. La Belle, C. WV, Long, J. E., and Christofano, E. E. Synergistic effects of aerosols. Arch. Ind. Health 11: 297-304 (1955). i d k i 206. Fassett, D. W Aldehydes and acetals. In: Industrial Hygiene 183. Ionescu, J., Marinescu, D., Tapu, V, and Eskenasy, A. Experi- CONSENSUS WORKSHOP ON FORMALDEHYDE Data Base for Formaldehyde Consensus Workshop The response of guinea pigs to inhalation of formalde- hyde and formic acid alone and with a sodium chloride aerosol. International J. Air Pollution 3: 210-220 (1960). Data Base for Formaldehyde Consensus Workshop 20, 2: 523-560 (1970). ( ) 259. Hoel, D. G. Incorporation of background in dose-response models Fed. Proc. 39: 73-75 (1980). Alarie, Y, Kane, L., and Barrow, C. Sensory irritation: the use of an animal model to establish acceptable exposure to airborne chemical irritants. In: Toxicology: Principles and Practice, Vol. 1 (A. L. Reeves, Ed.), Wiley and Sons, New York, 1982, pp. 48-92. 260. Hoel, D. G. Low-dose and species-to-species extrapolation for chemically induced carcinogenesis. In: Banbury Report I. As- sessing Chemical Mutagens: The Risk to Humans (K. McElheny and S. Abrahamson, Eds.), Cold Spring Harbor, 1979, pp. 135-145. Albert, R. E. Letters: Carcinogen policy at EPA. Science 219: 796-798 (1983). 261. Gaylor, D. W, and Kodell, R. L. Linear interpolation algorithm for low dose risk assessment of toxic substances. J. Environ. Pathol. Toxicol. 4: 305-312 (1980). Albert, R. E., Sellakumar, A. R., Laskin, S., Kuschner, M., Nelson, N., and Snyder, C. A. Gaseous formaldehyde and hydrogen chloride induction of nasal cancer in the rat. J. Natl. Cancer Inst. 68: 597-603 (1982). 262. Hughes, D. H., Bruce, R. D., Hart, R. W, Fishbein, L., Gaylor, D. W, Smith, J. M., and Carlton, W W A report on the workshop on biological and statistical implications of the EDO, study and related data bases. Fundamentals Appl. Toxicol. 3: 129-136 (1983). Alderson, T. Mechanism of formaldehyde induced mutagenesis. The uniqueness of adenylic acid in the mediation of the mutagenic activity of formaldehyde. Nature 187: 485-489 (1960). Alexandersson, R., Kolmodin-Hedman, B., and Hedenstierna, G. Exposure to formaldehyde: effects on pulmonary function. Arch. Environ. Health 37: 279-283 (1982). 263. Krewski, D., Crump, K. S., Farmer, J., Gaylor, D. W, Howe, R., Portier, C., Salsburg, D., Sielken, R. L., and VanRyzin, J. A comparison of statistical methods for low dose extrapolation utilizing time-to-tumor data. Fundamentals Appl. Toxicol. 3: 140-158 (1983). Altshuller, A. P, Cohen, I. R., Meyer, M. E., and Wartburg, A. F, Jr. Analysis of aliphatic aldehydes in source effluents and in the atmosphere. Anal. Chim. Acta 25: 101-117 (1961). 264. Sielken, R. L., and Smith, L. A. Cancer dose-response models. In: Developments in the Science and Practice of Toxicology (A. W Hayes, R. C. Schnell and T. S. Miya, Eds.), Elsevier Science Publ., Amsterdam, 1983, pp. 173-180. Altshuller, A. R, and McPherson, S. P Spectrophotometric analysis of aldehydes in the Los Angeles atmosphere. J. Air Pollution Control Assoc. 13: 109-111 (1963). Amdur, M. 0. CONSENSUS WORKSHOP ON FORMALDEHYDE Histotech. 6: 73-76 (1983). 226. National Toxicology Program (NTP). NTP reproduction and development toxicology program. In: NTP Annual Report, 1983. 227 H k H d'A Chi T Y d S h it M C Di t ib ti f 227. Heck, H. d'A., Chin, T. Y, and Schmitz, M. C. Distribution of [14C]formaldehyde in rats after inhalation exposure. In: Formal- dehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing 250. Bokina, A. I., Eksler, N. D., Semenenko, A. D., and Merkur'ye- va, R. V Investigation of the mechanism of action of atmo- spheric pollutants on the central nervous system and compara- CONSENSUS WORKSHOP ON FORMALDEHYDE 364 tive evaluation of methods of study Environ. Health Perspect. 13: 37-42 (1976). participation in the panel. In some cases, studies in the data base were specifically cited as references in the individual panel reports, and no attempt has been made to remove these previously cited studies from the appendix. Hard copies of all the reports in this computerized data base have been retained by the NCTR Clearing- house on Formaldehyde, as part of the agreement with the EPA. ( ) 251. Merkur'eva, R. V, and Tsapkova, N. N. Hygienic significance of a system of biochemical criteria for the evaluation of hepatotoxic and neurotoxic effects of some chemical factors in the environ- ment. J. Hyg. Epidemiol. Microbiol. Immunol. 26: 336-341 (1982). ( ) 252. Streisinger, G. Extrapolation from species to species and from various cell types in assessing risks from chemical mutagens. Mutat. Res. 114: 93-105 (1983). Data Base for Formaldehyde Consensus Workshop ( ) 253. Lyon, M. F, Adler, I. D., Bridges, B. A., Ehrenberg, L., Golberg, L., Kilian, D. J., Kondo, S., Moustacchi, E., Putra- ment, A., Sankaranarayanan, K., Sobels, F H., Sram, R. J., Streisinger, G., and Sundaram, K. Committee 4 Final Report. Estimation of genetic risks and increased incidence of genetic disease due to environmental mutagens. Mutat. Res. 115: 255-291 (1983). Ablashi, D. V, Levine, P H., Prasad, U., and Pearson, G. R. Meeting Report: Fourth International Symposium on Nasopharyngeal Carci- noma Application of Field and Laboratory Studies to the Control of NPC. Cancer Res. 43: 2375-2378 (1983). Ablashi, D. V, Levine, P H., Prasad, U., and Pearson, G. R. Meeting Report: Fourth International Symposium on Nasopharyngeal Carci- noma Application of Field and Laboratory Studies to the Control of NPC. Cancer Res. 43: 2375-2378 (1983). Acheson, E. D., Winter, P D., Hadfield, D., and Macbeth, R. G. Is nasal adenocarcinoma in the Buckinghamshire furniture industry declining? Nature 299: 263-265 (1982). ( ) 254. Casanova-Schmitz, M., Starr, T. B., and d'A. Heck, H. Differen- tiation between metabolic incorporation and covalent binding in the labeling of macromolecules in the rat nasal mucosa and bone marrow by inhaled ["'C] and [3H] formaldehyde. Toxicol. Appl. Pharmacol., in press. Agatha, G., and Schubert, H. Studies on allergen identification in the allergy to p-tertiary-butylphenol-formaldehyde resin (translation available). Dermatol. Monatsschr. 165: 37-345 (1979). , p 255. Ehrenberg, L., Moustacchi, E., and Osterman-Golkar, S. Dosi- metry of genotoxic agents and dose respose relationships of their effects. Mutat. Res. 123: 121-182 (1983). Agathos M., and Bernecker, H. A. Handdermatitis bei medizinischem Personal (Dermatitis of the hands among medical personnel). Dermatosen 30: 43-47 (1982). ( ) 256. Crump, K. S., Hoel, D. G., Langley, C. H., and Peto, R. Fundamental carcinogenic processes and their implications for low-dose risk assessment. Cancer Res. 36: 2973-2979 (1976). Aggarwal, A. R., and Garg, R. L. Formalin toxicity in hydatid liver disease. Anaesthesia 38: 662-665 (1983). 257. Guess, H. A., Crump, K. S., and Peto, R. Uncertainty estimates for low-dose-rate extrapolations of animal carcinogenicity data. Cancer Res. 37: 3475-3483 (1977). Ahmad, I., and Whitson, T. C. Formaldehyde: how much of a hazard? Ind. Med. Surg. 42: 26-27 (1973). ( ) 258. Peto, R. Carcinogenic effects of chronic exposure to very low levels oftoxic substances. Environ. Health Perspect. 22:155-159 (1978). Akabane, J. Aldehydes and related compounds. International En- cyclopedia of Pharmacology and Therapeutics, Sect. CONSENSUS WORKSHOP ON FORMALDEHYDE Pesticide Toxic Chem. News 10(36): 3 (1982). Anon. Government agencies clash over formaldehyde in mobile homes. Pesticide Tbxic Chem. News 10(45): 17-18 (1982). Anon. Any Questions. Brit. Med. J. 9: 778 (1979). Anon. Key chemicals: formaldehyde. Chem. Eng. News 60(13): 26 (1982). Anon. Formaldehyde study. Pesticide Tbxic Chem. News 10(50): 2 (1982). Anon. EPA foramldehyde position challenged. Chem. Eng. News 60(21): 24 (1982). Archer, M. C., and Labuc, G. E. On the mode of action of N-nitrosomethylbenzylamine, an esophageal carcinogen in the rat. In: Banbury Report 12, Nitrosamines and Human Cancer (P N. Magee, Ed.), Cold Springs Harbor Laboratory, 1982, pp. 87-101. Anon. No cancer link found in formaldehyde study. Chem. Eng. News 60(22): 8 (1982). Anon. Court overturns ban on urea-formaldehyde foam. Chem. Eng. News 61(17): 8 (1983). Ariens, E. J., Hanselaar, A. G. J. M., Henderson, P Th., and Simonis, A. M. Beware of formaldehyde in disguise. Eur. J. Clin. Pharmacol. 23: 373-375 (1982). Anon. Chemical profile: formaldehyde. Chem. Marketing Reptr. January 23, 1978, p. 9. Ashby, J., and Lefevre, P A. Formaldehyde generators: relationship between stability, lipophilicity and carcinogenic potency. Carcinogene- sis 3: 1273-1276 (1982). Anon. Formaldehyde debate moves into center stage in Washington: study said to clear chemical. Chem. Marketing Repr. 221: 7, 56 (1982). Anon. Formaldehyde: Court gives safety agency until May 5 to appeal reversal of foam insulation ban. Chem. Regulation Reptr. 7: 178-179 (1983). Ashford, N. A., Ryan, C. W, and Caldart, C. C. A hard look at federal regulation of formaldehyde; a departure from reasoned decision making. Harvard Environ. Law Rev. 7: 297-370 (1983). Anon. Urea-formaldehyde foam gets the axe for home insulation. Chem. Week 130(9): 12-13 (1982). Auerbach, C. The mutagenic mode of action of formalin. Science 110: 419-420 (1949). Anon. More grief for formaldehyde. Chem. Week 131(18): 13-14 (1982). Auerbach, C., and Moser, H. An analysis of the mutagenic action of formalin. Heredity 4: 272-273 (1950). Anon. Washington Notes: get the urea-formaldehyde out. Consumer Repts. 47: 266 (1982). Auerbach, C., Moutschen-Dahmen, M., and Moutschen, J. Genetic and cytogenetical effects of formaldehyde and related compounds. Mutat. Res. 39: 317-362 (1977). Anon. Re-regulations offormaldehyde in Japan. INDA. Japan Branch, Mitsuwa Building, Osaka, Japan (1975). Ayer, H. E., and Yeager, D. W Irritants in cigarette smoke plumes. Am. J. Publ. Health 72: 1283-1285 (1982). g p Anon. The health hazards of formaldehyde. Lancet i: 926-927 (1981). Bardana, E. J., and Andrach, R. H. CONSENSUS WORKSHOP ON FORMALDEHYDE 365 (ACGIH). Formaldehyde: TLVS-Threshold limit values for chemical substances in work air adopted by ACGIH for 1982, pp. 2-9, 19-19, 40-41. Anon. High threshold for TSCA Section 4(F) actions recommended by OTS Clay. Pesticide Toxic Chem. news 9(44): 18 (1981). Anon. Toxic Substances Office developing study plan on formaldehyde. Pesticide Toxic Chem. News 10(5): 8-9 (1981). American Society for Testing and Materials (ASTM). Standard test method for concentration of formaldehyde solutions. ANSI/ASTM D2194-65 (reapproved 1974), pp. 337-338. Anon. EPA permethrin decision sets policy that positive mouse tests do not matter. Pesticide Toxic Chem. News 10(12): 6-9 (1982). Andersen, I. Formaldehyde in the indoor environment-health implica- tions and the setting of standards. In: Indoor Climate: Effects on Human Comfort, Performance and Health in Residential, Commercial, and Light-Industry Buildings (P. 0. Fanger and 0. Valbjorn, Eds.), Proceedings of the First International Indoor Climate Symposium, Copenhagen, August 30-September 1, 1978, Copenhagen: Danish Building Research Institute, 1979, pp. 65-87. Anon. Formaldehyde should not be a priority TSCA chemical, Todhunter says. Pesticide Toxic Chem. News 10(13): 20-22 (1982). Anon. Objections to formaldehyde risk assessment lead to resignation. Pesticide Toxic Chem. News 10(22): 22 (1982). Anon. Formaldehyde seen as reflecting change in EPA risk assess- ments. Pesticide Toxic Chem. News 10(26): 3-5 (1982). Andersen, I. B., Lundqvist, G. R., and Mohave, L. Indoor air pollution due to chipboard used as a construction material. Atm. Environ. 9: 1121-1127 (1975). Anon. Panel of MDS tells congressional subcommittee of formalde- hyde effects. Pesticide Toxic Chem. News 10(27): 29 (1982). Anon. Formaldehyde hearing sees selective use of peer review by EPA. Pesticide Toxic Chem. News 10(28): 3-9 (1982). Andersen, K. E., Jhorth, N., Bundgaard, H., and Johansen, M. Formaldehyde in a hypoallergenic non-woven textile acrylate tape. Contact Dermatitis 9: 228 (1983). Anon. National Center for Toxicological Research to review formal- dehyde. Pesticide Toxic Chem. News 10(28): 9-10 (1982). Andersen, K. E., Bundgaard, H., and Johansen, M. Allergic contact dermatitis from formaldehyde in Fucidin ointment. Contact Dermati- tis 9: 78-79 (1983). Anon. Regulatory caution before regulating formaldehyde sought. Pesticide Toxic Chem. News 10(28): 25-26 (1982). Anon. Formaldehyde controversy continues to simmer. Pesticide Toxic Chem. News 10 (31): 4-42 (1982) Andersen, S. K., Jensen, 0. M., and Oliva, D. Formaldehydek- sponering og lungecancer blandt danske laeger (Exposure to formalde- hyde and cancer of the lung in Danish doctors). Ugeskr. Laeger 144: 1571-1573 (1982). Anon. Todhunter sees "threshold" debate on carcinogens as irrelevant. Appendix 1: Data Base of the Consensus Workshop on Formaldehyde American Association of Textile Chemists and Colorists (AATCC). Formaldehyde odor in resin treated fabric, determination of: sealed jar method. AATCC Technical Manual, AATCC, Research Triangle Park, NC, 1978, Test Method 112-1978, pp. 89-90. The literature comprising the data base, listed following this introduction, was made available, as reprints, as they were acquired, to the panel chairmen up to six months prior to the workshop. They were also available for all workshop participants at the meeting. The data base was organized by author as well as by a keyword index of important topics, and it was complemented by materials that the different participants brought with them and used as part of their American Conference of Governmental Industrial Hygienists (ACGIH); Formaldehyde. In: Documentation of the Threshold Limit Values, 4th Ed. American Conference of Governmental Industrial Hygienists, Cincinnati, Ohio, 1980, pp. 197-199. American Conference of Governmental and Industrial Hygienists CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE Occupational asthma secondary to low molecular weight agents used in the plastic and resin industries. Eur. J. Resp. Dis. 64: 241-251 (1983). Anon. U.S. formaldehyde policy. Lancet i: 1233 (1983). Anon. Formaldehyde and cancer. Lancet ii: 26 (1983). Anon. Formaldehyde study shows gene effect in anatomy students. New Physicians 6: 17 (1982). Barlow, S. Work and sex. Medical World 119: 16-17 (1981). Barlow, S. M., and Sullivan, F M. Formaldehyde. In: Reproductive Hazards of Industrial Chemicals: An Evaluation of Animal and Human Data (S. M. Barlow and E M. Sullivan, Eds.), Academic Press, New York, 1982, Chapter 24, pp. 334-345. Anon. Pending formaldehyde notice limits EPA investigation. Pesti- cide Tbxic Chem. News 9(30): 20-21 (1981). Anon. Formaldehyde carcinogenicity said to have been confirmed. Pesticide Ibxic Chem. News 9(41): 3-4 (1981). Barnes, E. C., and Speicher, H. W The determination of formalde- hyde in air. J. Ind. Hyg. Tox. 24: 10-17 (1942). Anon. EPA Administrator Gorsuch given 60-days notice on formal- dehyde, DEHP Pesticide Tbxic Chem. News 9(42): 11-12 (1981). Barrow, C. S. Sensory irritation. CIIT Activities, Chemical Industry Institute of Toxicology 1: 3-6 (1981). Anon. OTS recommends that formaldehyde not be given priority attention. Pesticide Tbxic Chem. News 9(44): 9-10 (1981). Barrow, C. S. Sensory irritation of inhaled aldehydes; structure CONSENSUS WORKSHOP ON FORMALDEHYDE 366 activity studies. CIIT Activities, Chemical Industry Institute of Toxicology 2:3-5 (1982). Bourne, H. G., Jr., and Seferian, S. Formaldehyde in wrinkle-proof apparel produces ... tears for milady. Ind. Med. Surg. 28: 232-233 (1959). Bourne, H. G., Jr., and Seferian, S. Formaldehyde in wrinkle-proof apparel produces ... tears for milady. Ind. Med. Surg. 28: 232-233 (1959). activity studies. CIIT Activities, Chemical Industry Institute of Toxicology 2:3-5 (1982). Barrow, C. S., and Steinhagen, W H. Sensory irritation by inhaled formaldehyde in B6C3F1 mice and F-344 rats following single or repeated exposure (abstract). Toxicologist 1: 5-6 (1981). Brabec, M. J. Aldehydes and acetals. In: Patty's Industrial Hygiene and Toxicology, Vol. IIA. John Wiley & Sons, New York, 1981, Chapter 37, pp. 2629-2699. Barrow, C. S., and Steinhagen, W H., and Chand, J. C. F. form aldehyde sensory irritation. In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Corp., Washington, D. C. 1983, Chap- ter 3, pp. 16-25. Breysse, P A. Small plants and their medical problems-the furni- ture industry: the environmental problems of urea-formaldehyde structures-formaldehyde exposure in mobile homes. CONSENSUS WORKSHOP ON FORMALDEHYDE Occupational Safety and Health Symposia, 1979, Contract #210-79-0009, National Institute of Occupational Safety and Health, Divison of Technical Services, Cincinnati, Ohio, 1980, pp. 56-63. Basmadzieva, K., Balabaeva, L., Kolev, K., Agirova, M., Carakciev, D., Krasteva, S., and Ivanova, H. The effect of formaldehyde on animals with experimental emphysema. Khigiena Zdraveopazvane (Hygiene and Sanitation) 24: 361-368 (1981). Breysse, P A. The health cost of tight homes. J. Am. Med. Assoc. 245: 267-268 (1981). Battelle Columbus Laboratories. Final report on a chronic inhalation toxicology study in rats and mice exposed to formaldehyde. Battelle Columbus Laboratories, Columbus, Ohio, 1981. Breysse, P. A. Formaldehyde in mobile and conventional homes. Environmental Health and Safety News, Vol. 25, Dept. of Environ- mental Health, School of Public Health and Community Medicine, University of Washington, Seattle, Washington, 1977, pp. 1-19. Baumann, H. Formaldehyd in UF-Schaum. Plastica 30: 72-75 (1977). Baumann, K., and Angerer, J. Occupational chronic exposure to organic solvents. VI. Formic acid concentration in blood and urine as an indicator of methanol exposure. International Arch. Occup. Environ. Health 42: 241-249 (1979). Breysse, P A. Formaldehyde exposure following urea formaldehyde insulation. Environmental Health and Safety News, Vol. 26, Dept. of Environmental Health, School of Public Health and Community Medicine, University of Washington, Seattle, Washington, 1978, pp. 1-13. Baur, X., and Fruhmann, G. Berufsbedingtes Asthma bronchiale allergischer und irritativer Genese (Bronchial asthma of allergic or irritative origin as an occupational disease). Praxis Klinik Pneumol. 33 (Suppl. 1): 317-322 (1979). Breysse, P A. Formaldehyde exposure in mobile homes and conven- tional homes. Proceedings ofthe 43rd Annual Educational Conference of the National Environmental Health Association, June 23-28, 1979, pp. 1-16. Benbrook, C. M. Letters: Carcinogen policy at EPA. Science 219: 798 (1983). Breysse, P A. Formaldehyde exposure and pericarditis. Environmen- tal Health and Safety News, Vols. 28 and 29, Dept. of Environmental Health, School of Public Health and Community Medicine, University of Washington, Seattle, Washington, 1980-1981, pp. 1-15. Bender, J. R., Mullin, L. S., Graepel, G. J., and Wilson, W E. Eye irritation response of humans to formaldehyde. Am. Ind. Hyg. Assoc. J. 44: 463-465 (1983). Berger, J. M., and Lamm, S. H. Correspondence: Health hazards of formaldehyde. Lancet i: 1264 (1981). Brockman, H. E. Report on formaldehyde using the AD-3 test system (Heterokaryon-12) of Neurospora crassa. NIH Contract No. 273-79-C-0015, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 1980. CONSENSUS WORKSHOP ON FORMALDEHYDE Bergmann, K., and Schneider, W Gas chromatographic method to determine formaldehyde traces in automobile exhaust gases. Chrom- atographia 15: 631-634 (1982). Brockman, H. E., Hung, C. Y, and DeSerres, E J. Potent mutagenic- ity of formaldehyde in a nucleotide excision repair-deficient hetero- karyon of Neurospora crassa (abstract). Environ. Mutagen. 3: 379-380 (1981). Bergreen, P W, Ayala, A. G., and Johnson, D. E. Effect of topical formaldehyde on canine bladder. Urology 7: 279-283 (1976). Beritic, T., Kovac, S., and Dimov, D. Formaldehyde in present day environmental toxicology (translation available). Arhiv Hig Rada Toksikol. (Archives of Industrial Hygiene and Toxicology) 32: 363-394 (1981). Bross, I. D. J., Viadana, E., and Houten, L. Occupational cancer in men exposed to dust and other environmental hazards. Arch. Environ. Health 33: 300-307 (1978). Bernstein, E Kutane Sensibilisierung gegen Formalin als beruffiche Krankheit (Cutaneous sensitivity to formalin-solution of formalde- hyde as an occupational disease). Dermatol. Wochensch. 95: 1683-1686 (1932). Brunn, W, and Klostermeyer, H. Detection and determination of protein-bound formaldehyde. II. Improved recovery of formaldehyde by reduction with sodium cyanoborhydride (NACNBH3) (translation available). Z. Lebensmittel-Untersuchung Forsch. 176:367-370 (1983). Bilimoria, M. H. The detection of mutagenic activity of chemicals and tobacco smoke in a bacterial system (abstract). Mutat. Res. 31: 328 (1975). Brusick, D. J., Myhr, B. C., Stetka, D. G., and Rundell, J. 0. Genetic and transforming activity of formaldehyde. Litton Bionetics' Report, Kensington, Maryland, April, 1980. Blackwell, M., Kang, H., Thomas, A., and Infante, P Formaldehyde: evidence of carcinogenicity. Am. Ind. Hyg. Assoc. J. 42: A34-A46 (1981). Bruynzeel, D. P, Van Ketel, W G., von Blomberg-van der Flier, M., and Scheper, R. J. Angry back or the excited skin syndrome: a prospective study. J. Am. Acad. Dermatol. 8: 392-397 (1983). Blair, A. Formaldehyde. A presentation to the National Cancer Advisory Board Meeting of Oct,ober 5, 1981 (unpublished comments). Bryson, D. D. Correspondence: Health hazards of formaldehyde. Lancet i: 1263-1264 (1981). Buck, H. M. Spectrophysics and photochemistry of the formaldehyde molecule. Part II. Rec. J. Roy. Netherlands Chem. Soc. 101: 225-233 (1982). Boettcher, B., Nanra, R. S., Roberts, T. K., Mallan, M., and Watterson, C. A. Specificity and possible origin of anti-N antibodies developed by patients undergoing chronic haemodialysis. Vox Sang. 31: 408-415 (1976). Budiansky, S. News: formaldehyde cancer risk: court not convinced of dangers. Nature 304: 105 (1983). Boggs, P. B. Letter to the Editor: Occupational asthma and rhinitis caused by urea formaldehyde. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 367 Clark, R. P Formaldehyde in pathology departments. J. Clin. Pathol. 36: 839-846 (1983). Clark, R. P Formaldehyde in pathology departments. J. Clin. Pathol. 36: 839-846 (1983). Cassidy, S. L., Dix, K. M., and Jenkins, T. Evaluation of a testicular sperm head counting technique using rats exposed to dimethoxyethyl phthalate (DMEP), glycerol A-monochlorohydrin (GMCH), epich- lorohydrin (ECH), formaldehyde (FA), or methyl methanesulphonate (MMS). Arch. Toxicol. 53: 71-78 (1983). Clarke, W J. Subchronic study report on formaldehyde. Tracor-Jitco Inhalation Carcinogenesis Bioassay; Battelle Pacific Northwest Laboratories, Richland, Washington, November, 1981, pp. 1-41. Celanese Corporation. Considerations in establishing an appropriate TLV for formaldehyde. Presented to ACGIH Subcommittee on Jan. 20, 1983, Celanese Chemical Co., Inc., New York, 1983. Clay, D. EPA staff memo on applicability of TSCA 4(F) to formal- dehyde. Inside EPA., September 25, 1981, pp. 4-5. Cleveland, W S., Graedel, T. E., and Kleiner, B. Urban formaldehyde: Observed correlation with source emissions and photochemistry. Atmos. Environ. 11: 357-360 (1977). Chaigneau, M., Lemoan, G., Chastagnier, M., and Chuong, P H. On new results dealing with the persistence of residuals in medico- surgical materials sterilized with formaldehyde (translation available). Ann. Pharm. Franc. 40: 431-438 (1982). Cockcroft, D. W, Hoeppner, V H., and Dolovich, J. Occupational asthma caused by cedar urea formaldehyde particle board. Chest 82: 49-53 (1982). Chanet, R., Izard, C., and Moustacchi, E. Genetic effects of formaldehyde in yeast. I. Influence of the growth stages on killing and recombination. Mutat. Res. 33: 179-186 (1975). Coene, R. F Formaldehyde: evidence of carcinogenicity. Vet. Human Toxicol. 23: 282-285 (1981). Chanet, R., Magana-Schwencke, N., and Moustacchi, E. Genetic effects of formaldehyde in yeast: current status and limitations of the radiation equivalence concepts. In: Radiobiological Equivalents of Chemical Pollutants: Proceedings of the Advisory Group Meeting on Radiobiological Equivalents of Chemical Pollutants. International Atomic Energy Agency, Vienna, Austria, 1980, pp. 45-59. Coldiron, V R., Ward, J. B., Jr., Trieff, N. M., Janssen, H. E., Jr., and Smith, J. H. Occupational exposure to formaldehyde in a medical center autopsy service. J. Occup. Med. 25: 544-548 (1983). Collins, C. J., and Guild, W R. Irreversible effects of formaldehyde on DNA. Biochim. Biophys. Acta 157: 107-113 (1968). Chanet, R., and Von Borstel, R. C. Genetic effects of formaldehyde in yeast. III. Nuclear and cytoplasmic mutagenic effects. Mutat. Res. 62: 239-253 (1979). CONSENSUS WORKSHOP ON FORMALDEHYDE Commission for the Study of the Carcinogenic, Mutagenic, and Teratogenic Effects of Chemical Compounds on Health Risks Related to Possible Methods of Formaldehyde Use. Opinion of the commission, meeting of June 1, 1981 (unpublished comments). Chang, J. C. F, and Barrow, C. S. Tolerance and cross-tolerance to the sensory irritants chlorine and formaldehyde in F-344 rats (abstract). Toxicologist 3(1): 73 (1983). Connor, T. H. Detection of chemical mutagens in the urine of humans: studies of exposure to formaldehyde and cigarette smoke (abstract). Dissertation Abstr. Intern. 43: 667 (1982). Chang, J. C. F, Gross, E. A., Swenburg, J. A., and Barrow, C. S. Dose comparison between B6C3F1 mice and F-344 rats following formaldehyde inhalation (abstract). Toxicologist 2(2): 11 (1982). Connor, T. H., Barrie, M. D., Theiss, J. C., Matney, T. S., and Ward, J. B., Jr. Mutagenicity of formalin in the Ames assay. Mutat. Res. 119: 145-149 (1983). Chang, J. C. F, Gross, E. A., Swenberg, J. A., and Barrow, C. S. Nasal cavity deposition, histopathology, and cell proliferation after single or repeated formaldehyde exposures in B6C3F1 mice and F-344 rats. Toxicol. Appl. Pharmacol. 68: 161-176 (1983). Consumer Product Safety Commission (CPSC). Urea-formaldehyde foam insulation; public hearings. Fed. Reg. 44: 69578-69583 (1979). Consumer Product Safety Commission (CPSC). Evaluation of health risks of formaldehyde by government scientists. Fed. Reg. 45: 34031-34033 (1980). Chang, J. C. F, Steinhagen, W H., and Barrow, C. S. Effect of single or repeated formaldehyde exposure on minute volume of B6C3F1 mice and F-344 rats. Toxicol. Appl. Pharmacol. 61: 451-459 (1981). Consumer Product Safety Commission (CPSC). 16 CFR Part 1405, Urea-formaldehyde foam insulation; Proposed notice to purchasers. Fed. Reg. 45: 39434-39444 (1980). Chang, J. C. F, Steinhagen, W H., and Barrow, C. S. Effect of single or repeated formaldehyde exposure on tidal volume in B6C3F1 mice and Fischer-344 rats (abstract). Fed. Proc. 40 (Part 1): 739 (1981). Consumer Product Safety Commission (CPSC). Part IV, Urea- formaldehyde foam insulation; proposed ban; denial of petition. Fed. Reg. 46: 11189-11211 (1981). Chaw, Y F M., Crane, L. E., Lange, P, and Shapiro, R. Isolation and identification of cross-links from formaldehyde-treated nucleic acids. Biochemistry 19: 5525-5531 (1980). Consumer Product Safety Commission (CPSC). Part IV, Ban of urea-formaldehyde foam insulation, withdrawal of proposed informa- tion labeling rule, and denial of petition to issue standard. Fed. Reg. 47: 14366-14419 (1982). Chemical Industry Institute of Toxicology (CIIT). Statement concern- ing research findings, October, 1979. CONSENSUS WORKSHOP ON FORMALDEHYDE Chest 83: 584 (1983). Bundgaard, H. Formaldehyde pro-drugs as potential antitumor agents. Arch. Pharmaci Chemi, Sci. Ed. 9: 133-136 (1981). Boleij, J. S. M., and Brunekreef, B. Indoor air pollution. Publ. Health Rev. 10: 169-198 (1982). Campbell, M. A., and Fantel, A. G. Teratogenicity of acetaldehyde in vitro: Relevance to the fetal alcohol syndrome. Life Sci. 32: 2641-2647 (1983). Bonashevskaya, T. I. Amygdaloid lesions after exposure to formal- dehyde. Arkhiv Anat. Gistol. Embriol. 65: 56-59 (1973). Boreiko, C. J., Ragan, D. L., Abernethy, D. J., and Frazelle, J. H. Initiation of C3H/1OT1/2 cell transformation by N-methyl-N-nitro-N- nitrosoguanidine and Aflatoxin Bi. Carcinogenesis 3: 391-395 (1982). Casanova-Schmitz, M., and Heck, H. DA. Metablism of formaldehyde in the rat nasal mucosa in vivo (abstract). Ibxicologist 3(1): abstract 243 (1983). Cooper, P Genetic effects of formaldehyde. Food Cosmet. Toxicol. 17: 300-301 (1979). CONSENSUS WORKSHOP ON FORMALDEHYDE Effect of different aldehydes on tracheal mucosa. Arch. Otolaryngology 93: 496-500 (1971). Ettinger, I., and Jeremias, M. A study of the health hazards involved in working with flameproofed fabrics. Monthly review, New York State Department of Labor, Division of Industrial Hygiene, July, 1955, pp. 1-2. Decoufle, P Cancer risks associated with employment in the leather and leather products industry. Arch. Environ. Health 34: 33-37 (1979). Della Porta, G., Cabral, J. R., and Parmiani, G. Studio della tossicita transplacentare e di carcerogenesi in ratti trattati con esametilenete- tramina (Transplacental toxicity and carcinogenesis studies in rats treated with hexamethylenetetramine). Tumori 56: 325-334 (1970). Fall, M., and Pettersson, S. Ureteral complications after intravesical formalin installation. J. Urol. 122: 160-162 (1979). Farrelly, J. G., and Steward, M. L. The metabolism of a series of methylalkylnitrosamines. Carcinogenesis 3: 1299-1302 (1982). Della Porta, G., Colhaghi, M. I., and Parmiani, G. Noncarcinogenicity of hexamethylenetetramine in mice and rats. Food Cosmet. Toxicol. 6: 707-715 (1968). Fassbinder, W, and Koch, K. M. A specific innunohaemolytic anemia induced by formaldehyde sterilization of dialysers. In: Biocompat- ability in Hemodialysis (Contributions to Nephrology; Vol. 36). Workshop on Biocompatability in Hemodialysis, Gernreid, March 18-20, 1982 (C. A. Baldamus, K. M. Koch, and W Schoeppe, Ed.), Karger, Basel, Switzerland, 1982, pp. 51-67. Deppe, H. J. Emission of organic substances from wood raw materials. Holz-Zentralbl. 108: 123-126 (1982). Dmitriev, M. T., Zarubin, G. P, and Mishchikhin, V A. Prediction of indoor air pollution levels during the use of synthetic polymer materials. Gig. Sanitar. 12: 55-58 (1982). Fassbinder, W, Pilar, J., Scheuermann, E., and Koch, M. Formalde- hyde and the occurrence of anti-N-like cold agglutinins in RDT patients. In: Proc. Eur. Dialysis Transplant Assoc. 9: 333-338 (1976). Dormans, J. A., and Van Logten, M. J. The effects of ophthalmic preservatives on corneal epithelium of the rabbit: a scanning electron microscopical study Toxicol. Appl. Pharmacol. 62: 251-261 (1982). Fassbinder, W, Seidl, S., and Koch, K. M. The role of formaldehyde in the formation of haemodialysis-associated anti-N-like antibodies. Vox Sang. 35: 41-48 (1978). Dost, F. N. Assessment of potential toxic hazards of formaldehyde. In: 13th Symposium on Particle Board. Washington State University, Pullman, Washington, 1979, pp. 317-327. Fassett, D. W Formaldehyde. In: Industrial Hygiene and Toxicology, Vol. II (F A. Patty, Ed.), Interscience, New York, 1958, pp. 1970-1972. Drill, V A., Friess, S. L., Hays, H. W, Loomis, T. A., and Shaffer, C. B. CONSENSUS WORKSHOP ON FORMALDEHYDE 368 Eckmann, A. D., Daily, K. A., Hanrahan, L. P, and Anderson, H. A. Comparison of the chromotropic acid and modified pararosaniline methods for the determination of formaldehyde in air. Environ. Intern. 8: 159-166 (1982). Couch, D. B., Allen, P F., and Eales, H. C. The mutagenicity of formaldehyde to Salmonella typhimurium (abstract). Environ. Mutagen. 4: 336-337 (1982). Cralley, L. V The effect of irritant gases upon the rate of ciliary activity. J. Ind. Hyg. Toxicol. 24: 193-198 (1942). Eells, J. T., McMartin, K. E., Black, K., Virayotha, V, Tisdell, R. H., and Tephly, T. R. Formaldehyde poisoning: rapid metabolism to formic acid. J. Am. Med. Assoc. 246: 1237-1238 (1981). Csiba, A., Trezl, L., Tyhak, E. Graber, H., Vari, E., Teglas, G., and Rusznak, I. Assumed role of L-argTinine in mobilization ofendogenous formaldehyde. Acta Physiol. Acad. Sci. Hung. 59: 35-43 (1982). Egle, J. L., Jr. Retention of inhaled formaldehyde, propionaldehyde, and acrolein in the dog. Arch. Environ. Health 25: 119-124 (1972). Dagani, D., and Archer, M. C. Colorimetric determination of acetalde- hyde in the presence of formaldehyde. Anal. Biochem. 87: 455-459 (1978). Engle, H. O., and Calnan, C. D. Resin dermatitis in a car factory. Brit. J. Ind. Med. 23: 62-66 (1966). Englesberg, E. The mutagenic action of formaldehyde on bacteria. J. Bacteriol. 63: 1-11 (1952). Dahl, A. R., and Hadley, W M. Formaldehyde production promoted by rat nasal cytochrome P-450-dependent monooxygenases with nasal decongestants, essences, solvents, air pollutants, nicotine and cocaine as substrates. Toxicol. Appl. Pharmacol. 67: 200-205 (1983). Environmental Protection Agency (EPA), Office of Toxic Substances. Priority review level 1: Formaldehyde (draft). EPA, Washington, DC, February 19, 1981. Dahlquist, I., Fregert, S., and Gruvberger, B. Detection of formalde- hyde in corticoid creams. Contact Dermatitis 6: 494 (1980). Environmental Protection Agency (EPA), Office of Toxic Substances. Options paper on formaldehyde (unpublished). EPA, Washington, DC, September 11, 1981. Dalbey, W, and Nettesheim, P Influence of nitrogen dioxide or formaldehyde on incidence of diethylnitroamine-induced tumors in hamster respiratory tract (abstract). Toxicologist 1: 138 (1981). Epstein, E., and Maibach, H. I. Formaldehyde allergy: incidence and patch test problems. Arch. Dermatol. 94: 186-190 (1966). Dalbey, W E. Formaldehyde and tumors in hamster respiratory tract. Toxicology 24: 9-14 (1982). Epstein, S. S., Arnold, E., Andrea, J., Bass, W, and Bishop, Y. Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol. 23: 288-325 (1972). Dalhamn, T., and Rosengren, A. CONSENSUS WORKSHOP ON FORMALDEHYDE CIIT Docket #11109, Chemical Industry Institute of Toxicology, Research Triangle Park, NC, 1979. Consumer Product Safety Commission (CPSC). 16 CFR Part 1306, Ban of urea-formaldehyde foam insulation: confirmation of effective date. Fed. Reg. 47: 57488-57489 (1982). Chemical Industry Institute of Toxicology (CIIT). Progress report on CIIT formaldehyde studies, January 16, 1980. CIIT Docet #32510, Chemical Industry Institute of Toxicology, Research Triangle Park, NC (1980). Consumer Product Safety Commission (CPSC). Questions and an- swers on urea-formaldehyde foam insulation. CPSC, Washington, DC, March, 1982. Chemical Industry Institute of Toxicology (CIIT). Progress report on CIIT formaldehyde studies, December, 1980. CIIT Docket #112DO, Chemical Industry Institute of Toxicology, Research Triangle Park, NC (1980). Consumer Product Safety Commission (CPSC), Environmental Pro- tection Agency (EPA), Dept. of Health Education & Welfare, Dept. of Agriculture. Scientific basis for identification of potential carcinogens and estimation of risks: Request for comments on report. Fed. Reg. 44: 39858-39879 (1979). Chemical Industry Institute of Toxicology (CIIT). Summary of final report on formaldehyde study. CIIT Activities, Chemical Industry Instituted of Toxicology 2(3): 1, 9 (1982). Chen, T.-M., and Chafetz, L. Selective determination of free urinary formaldehyde after oral dosage with methenamine mandelate. Invest. Urol. 10: 212-214 (1972). Cooke, A., Oliver, R. F, and Edward, M. An in vitro cytotoxicity study of aldehyde-treated pig dermal collagen. Brit. J. Exptl. Pathol. 64: 172-176 (1983). Chikamoto,, T., Ezaka, S., Morimoto, K., Oki, H., Tamura, Y, and Sakoda, K. Aldehyde distribution in the atmosphere. Kyoto Pref. Inst. Hyg. Environ. Sci. 23: 96-100 (1978). Coon, R. A., Jones, R. A., Jenkins, L. J., Jr., and Siegel, J. Animal inhalation studies on ammonia, ethylene glycol, formaldehyde, di- methylamine, and ethanol. Toxicol. Appl. Pharmacol. 16: 646-655 (1970). Chin, T. Y and Heck, H. DA. Disposition of 14C formaldehyde in Fischer-344 rats following inhalation exposure (abstract). Toxicologist 1: 78 (1981). Cooper, P Genetic effects of formaldehyde. Food Cosmet. Toxicol. 17: 300-301 (1979). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 369 librari (Methyl bromide, ethylene oxide, formaldehyde: biological, toxicological problems, and problems related to the treatment of library materials) (translation available). Nuovi Ann. Microbiol. 29: 131-167 (1978). Figueroa, W G., Raszkowski, R., and Weiss, W Lung cancer in chloromethyl methyl ether workers. New Eng. J. Med. 288:1096-1097 (1973). Fisher, A. A. Dermatitis due to formaldehyde-releasing agents in cosmetics and medicaments. Current Contact News 655, 658, 664, 708 (1978). Gamble, J. F, McMichael, A. J., Williams, T., and Battigelli, M. Respiratory function and symptoms: an environmental-epidemiological study of rubber workers exposed to a phenol-formaldehyde type resin. Am. Ind. Hyg. Assoc. J. 37: 499-513 (1976). Fisher, A. A., Kanof, N. B., and Biondi, E. M. Free formaldehyde in textiles and paper: clinical significance. Arch. Dermatol. 86: 753-756 (1962). Garfield, E. Risk analysis, Part 2. How we evaluated the health risks of toxic substances in the environment. Cur. Contents 22(35): 5-11 (1982). Fleig, I., Petri, N., Stocker, W G., and Thiess, A. M. Cytogenetic analyses of blood lymphocytes of workers exposed to formaldehyde in formaldehyde manufacturing and processing. J. Occup. Med. 24: 1009-1012 (1982). Garry, V F, Kreiger, R. A., and Wiencke, J. K. The mutagenic and cytotoxic effects of formaldehyde in cultured human lymphocytes (Abstract). Environ. Mutagen. 3: 341 (1981). Fontignie-Houbrechts, N. Genetic effects of formaldehyde in the mouse. Mutat. Res. 88: 109-114 (1981). Garry, V F, Oatman, L., Pleus, R., and Gray, D. Formaldehyde in the home: some environmental disease perspectives. Minnesota Med. 63: 107-111 (1980). Fontignie-Houbrechts, N., Moutschen-Dahmen, J., Moutschen- Dahmen, M., Degraeve, N., and Gloor, H. Genetic effects in the mouse of formaldehyde in combination with adenosine and hydrogen peroxide. Mutat. Res. 104: 371-376 (1982). Gaylor, D. W The use of safety factors for controlling risk. J. Toxicol. Environ. Health 11: 329-336 (1983). Food and Drug Administration (FDA). FDA Talk Paper: Formal- dehyde, T80-27, May 21, 1980. FDA, Rockville, Maryland. Gelfand, H. H. Respiratory allergy due to chemical compounds encountered in the rubber, lacquer, shellac, and beauty culture industries. J. Allergy 34: 374-381 (1963). Food and Drug Administration (FDA). FDA Talk Paper: Formal- dehyde, T82-40, June 17, 1982. FDA, Rockville, Maryland. Georghiou, P E., Harlick, L., Winsor, L., and Snow, D. Temperature dependence of the modified pararosaniline method for the determina- tion of formaldehyde in air. Anal. Chem. 55: 567-570 (1983). Food and Drug Administration. Consensus workshop on formaldehyde; Meeting. Fed. Reg. 48: 55034-55035 (1983). Gibson, J. E. CONSENSUS WORKSHOP ON FORMALDEHYDE Applied research: a case study with formaldehyde (Abstract). The First World Congress on Toxicology and Environmen- tal Health, American College of Toxicology, Washington, DC, May 27-30, 1982. Food and Drug Administration. Consensus workshop on formaldehyde; Meeting. Fed. Reg. 47: 36201-36203 (1982). Fornace, A. J., Jr. Detection of DNA single-strand breaks produced during the repair of damage by DNA-protein cross-linking agents. Cancer Res. 42: 145-149 (1982). Gibson, J. E. Mechanisms of formaldehyde toxicity and carcinogenic- ity in laboratory animals. International Particleboard Series, Sympo- sium No. 16, Washington State University, Pullman, Washington, march 30, 1982. Fornace, A. J., Jr., Lechner, J. F, Grafstron, R. C., and Harris, C. C. DNA repair in human brochial eptihelial cells. Carcinogenesis 3: 1373-1377 (1982). Gilli, G., Corrao, G., and Scursatone, E. Dust as carrier of formaldehyde: low environmental concentrations and allergic phe- nomena. Tinctoria 78: 101-105 (1981). Frankel, L. S., McCallum, K. S., and Collier, L. Formation of bis(chloromethyl) ether from formaldehyde and hydrogen chloride. Environ. Sci. Technol. 8: 356-359 (1974). Glass, W I. An outbreak of formaldehyde dermatitis. New Zealand Med. J. 60: 423-427 (1961). Frazelle, J. H., Abernethy, D. J., and Boreiko, C. J. Weak promotion of C3H/1OT1/2 cell transformation by repeated treatments with formaldehyde. Cancer Res. 43: 3236-3239 (1983). Godish, T. Formaldehyde and building-related illness. J. Environ. Health 44: 116-121 (1981). French, D., and Edsall, J. T. The reactions of formaldehyde with amino acids and proteins. In: Advances in Protein Chemistry, Vol. II (M. L. Anson and J. T. Edsall, Eds.), Academic Press, New York, 1945, pp. 277-335. Godish, T. Residential formaldehyde control revisited. Natural Re- sources Notes, Ball State University, Dept. of Natural Resources, No. 3, Muncie, Indiana, 1982, pp. 1-6. Godish, T. Interpretation of formaldehyde sampling results. Natural Resources Notes, Ball State University, Dept. of Natural Resources, No. 4, Muncie, Indiana, 1982, pp. 1-4. Frenk, E. Pruriginous eruptions of epidemic character in a foundry using synthetic resins. Dermatologica 129: 436-439 (1964). Friedman, G. D., and Ury, H. K. Initial screening for carcinogenicity of commonly used drugs. J. Natl. Cancer Inst. 65: 723-733 (1980). Godish, T. Recognition of building-related illness. Natural Resources Notes, Ball State University, Dept. of Natural Resources, No. 5, Muncie, Indiana, 1982, pp. 1-4. Frigas, E., Filley, W V, and Reed, C. E. Asthma induced by dust from urea-formaldehyde foam insulating material. Chest 79: 706-707 (1981). Godish, T. Are residents of urea-formaldehyde foam insulated houses uniquely victimized? CONSENSUS WORKSHOP ON FORMALDEHYDE Potential health effects from inhalation of low levels of airborne formaldehyde. Drill, Friess, Hays, Loomis & Shaffer, Inc., Arlington, Virginia, March, 1982. Fassett, D. W Aldehydes and acetals. In: Industrial Hygiene and Toxicology II., 2nd Ed. (Rev.) (F A. Patty, Ed.), John Wiley & sons, New York, Chapter 43, 1963, pp. 1959-1989. Du Vigneaud, V, Verly, W G., and Wilson, J. E. Incorporation of the carbon of formaldehyde and formate into the methyl groups of choline. J. Am. Chem. Soc. 72: 2819-2820 (1950). Federal Panel on Formaldehyde. Report of the Federal Panel on Formaldehyde. Environ. Health Perspect. 43: 139-168 (1982). Feigal, R. J. Potential contraindications for the use of formaldehyde in dentistry. Northwest Dentistry 53(5): 13-16 (1982). Dubreuil, A., Bouley, G., Godin, J., and Boudene, C. L. Inhalation, en continu, de faibles doses de formaldehyde: etude experimentale chez le rat. Eur. J. Ibxicol. 9: 245-250 (1976). Feldman, M. Y Reactions of nucleic acids and nucleoproteins with formaldehyde. Prog. Nucleic Acid Res. Mol. Biol. 13: 1-48 (1973). Dumas, T. Determination of formaldehyde in air by gas chroma- tography, J. Chromatog. 247: 289-295 (1982). Feldman, M. Y., Balabanova, H., Bachrach, U., and Pyshnov, M. Effect of hydrolyzed formaldehyde-treated RNA on neoplastic and normal human cells. Cancer Res. 37: 501-506 (1977). Dunn, D. W, Johnson, M. L., Hedley, W H., Pate, J. B., Barrett, G. J., and McKinnery, W N. Reducing plant pollution exposure: control practices at formaldehyde production plants. Chem. Eng. Progr. 79(3): 35-38 (1983). Feldman, Y G. Biological action of certain products from atmospheric photochemical reactions (abstract), Gig. Sanitar.; Chem. Abstr. 76: #122530N (1972). Eberhartinger, C., and Ebner, H. Beitrag zur Kenntnis der Formalin- Kontakt-Allergie (An addition to formalin contact allergy knowledge). Berufsdermatosen 12: 301-306 (1976). Feldman, Y. G., and Bonashevskaya, T. I. On the effects of low concentrations of formaldehyde. Hygiene and Sanitation 36: 174-180 (1971). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 370 Halperin, W E., Goodman, M., Stayner, L., Elliott, L. J., Keenlyside, R. A., and Landrigan, P J. Nasal cancer in a worker exposed to formaldehyde. J. Am. Med. Assoc. 249: 510-512 (1983). Goldman, M., and Stein, A. A. Formaldehyde-health effects, regula- tion and environmental control. J. Histotechnol. 4: 13-16 (1981). Goldman, P, Flach H-D., Hey, W, Hochadel, H. Petri, N. Strass- burger, K. J., and Thiess, A. M. Formaldehyd-morbiditatsstudie (Formaldehyde morbidity study). Zentralbl. Arbeitsmedizin, Arbeits- schutz, Prophyl. Ergon. 32: 250-258 (1982). Hanselaar, A. G. J. M., Ariens, E. J., Henderson, P Th., and Simonis, A. M. Vergorgen Formaldehyde (Hidden formaldehyde). Pharmaceut. Weekblad 117: 505-507 (1982). Hanst, P L., Wong, N. W, and Bragin, J. A long-path infra-red study of Los Angeles Smog. Atmospheric Environment 16: 969-981 (1982). Goldschmidt, B. M., VanDuuren, B. L., and Frenkel, K. The reaction of 14C-labelled bis-(chloromethyl)ether with DNA (abstract). Proc. Am. Assoc. Cancer Res. 16: 66 (1975). Harrington, J. M. Health and safety in medical laboratories. Bull. WHO 60: 9-16 (1982). Goodman, D. G., Ward, J. M., Squire, R. A., Chu, K. C., and Linhart, M. S. Neoplastic and noneoplastic lesions in aging F-344 rats. Toxicol. Appl. Pharmacol. 48: 237-248 (1979). Harrington, J. M., and Oakes, D. Mortality study of British pathologists 1974-80 (submitted for publication). Goodman, J. I., and Tephly, T. R. A comparison of rat and human liver formaldehyde dehydrogenase. Biochim. Biophys. Acta 252: 489-505 (1971). Harrington, J. M., and Shannon, H. S. Mortality study of pathologists and medical laboratory technicians. Brit. Med. J. 4: 329-332 (1975). Harris, C. C., Grafstrom, R. C., Lechner, J. F, and Autrup, H. Metabolism of N-nitrosamines and repair of DNA damage in cultured human tissues and cells. In: Banbury Report 12, Nitrosamines and Human Cancer (P N. Magee, Ed.), Cold Spring Harbor Laboratory, New York, 1982, pp. 121-139. Gori, G. B. The regulation of carcinogenic hazards. Science 208: 256-261 (1980). Gorst, D. W, Riches, R. A., and Renton, P H. Formaldehyde induced anti-N: a possible cause of renal graft failure. J. Clin. Pathol. 30: 956-959 (1977). Harris, J. C., Rumack, B. H., and Aldrich, F D. Toxicology of urea formaldehyde and polyurethane foam insulation. J. Am. Med. Assoc. 245: 243-246 (1981). Grafstrom, R. C., Fornace, A. J., Jr., and Harris, C. C. Effect of formaldehyde on DNA damage and repair in human bronchial epithelial cells and fibroblasts (abstract). Proc. Am. Assoc. CONSENSUS WORKSHOP ON FORMALDEHYDE Cancer Res. 23: 69 (1982). Harrison, P B., Jansson, K., Kronenberg, H., Mahony, J. F, and Tiller, D. Cold agglutinin formation in patients undergoing haemo- dialysis. A possible relationship to dialyser re-use. Austral. New Zealand J. Med. 5: 195-197 (1975). Graftstrom, R. C., Fornace, A. J., Jr., Autrup, H., Lechner, J. F, and Harris, C. C. Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells. Science 220: 216-218 (1983). Hatch, G. C., Conklin, P M., Christensen, C. C., Casto, B. C., and Nesnow, S. Synergism in the transformation of hamster embryo cells treated with formaldehyde and adenovirus. Environ. Mutagen. 5: 49-57 (1983). Gralla, E. J., Heck, H. D'A., Hrubesh, L. W, and Meadows, G. W A report of the review of the formaldehyde exposure made by the CIIT ad hoc analytical chemistry investigative team held at the Battelle Memorial Columbus (Ohio) Laboratory, 505 King Avenue, Columbus, Ohio, on January 11, 1980. CIIT Docket #62620, Chemical Industry Institute of Toxicology, Research Triangle Park, NC, 1980, pp. 1-11. Hatch, T. F, and Gross, P Pulmonary Deposition and Retention of Inhaled Aerosols. Academic Press, New York, 1964, pp. 45-68. Hawthorne, A. R., and Gammage, R. B. Formaldehyde release from simulated wall panels insulated with urea-formaldehyde foam insula- tion. J. Air Pollution Control Assoc. 32: 1126-1131 (1982). Grant, W M. Formaldehyde. In: Toxicology of the Eye, 2nd ed. (W M. Grant, Ed.), Charles C Thomas, Springfield, IL, 1974, pp. 502-507. Green, M. A., and Egle, J. L., Jr. Effects of intravenous acetaldehyde, acrolein, formaldehyde and propionaldehyde on arterial blood pressure following acute guanethidine treatment. Res. Commun. Chem. Pathol. Pharmacol. 40: 337-340 (1983). Hays, S. M. Formaldehyde as a feed additive found to make cattle grow faster. Arkansas Democrat, September 12, 1982, p. 3J. Heck, H. D'A. Biochemical toxicology of inhaled formaldehyde. CIIT Activities, Chemical Industry Institute of Toxicology 2(3): 3-7 (1982). Greenberg, S. R. Formaldehyde: medical ally or adversary. Proc. Inst. Med. Chicago 34: 105-107 (1981). Heck, H. D'A., and Casanova-Schmitz, M. Reactions of formaldehyde in the rat nasal mucosa. In: Formaldehyde-Toxicology-Epidemiology- Mechanisms (J. J. Clark, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker, New York, 1983, pp. 211-223. Greenberg, S. R. The renal response to formalinization. Urol. Intern. 37: 45-48 (1982). Greenberg, S. R. Nucleic acid relationshops in formalin-injured renal tubules. Proc. Inst. Med. Chicago 35: 83-84 (1982). Heck, H. D'A., and Casanova-Schmitz, M. Hagino, N. Ovulation and mating behavior in female rats under various environmental stresses or androgen treatment. Japan. J. Physiol. 18: 350-355 (1968). CONSENSUS WORKSHOP ON FORMALDEHYDE Natural Resources Notes, Ball State University, Dept. of Natural Resources, No. 6, Muncie, Indiana, 1982, pp. 1-2. Frind, H., and Hensel, R. Bestimmung der personlichen Formal- dehydbelastung (Determination of the individual load by formal- dehyde). Fresenius Z. Anal. Chem. 312: 237-240 (1982). Gofmekler, V A. Effect on embryonic development of benzene and formaldehyde in inhalation experiments. Hygiene and Sanitation (USSR) 33: 327-332 (1968). Fujiwara, M., Tomita, S., and Nishimura, T. Micro determination of formaldehyde in vaccines. Japan. J. Bacteriol. 37: 789-791 (1982). Gofmekler, V A., and Bonashevskaya, T. I. Experimental studies of teratogenic propertis of formaldehyde, based on pathological evalua- tions. Hygiene and Sanitation (USSR) 34: 266-268 (1969). Fuks, A. B., Bimstein, E., and Bruchim, A. Radiographic and histologic evaluation of the effect of two concentrations of formocresol on pulpotomized primary and young permanent teeth in monkeys. Pediatric Dent. 5: 9-13 (1983). Goh, K. O., and Cestero, R. V M. Letters to the Editor: Health hazards of formaldehyde. J. Am. Med. Assoc. 247: 2778 (1982). Galli, C. L., Ragusa, C., Resmini, P, and Marinovich, M. Ibxicologi- cal evaluation in rats and mice of the ingestion of a cheese made from milk with added formaldehyde. Food Chem. Ibxicol. 21: 313-317 (1983). Golberg, L. A code of ethics for scientists reporting and reviewing information on chemicals. Fundamentals Appl. Toxicol. 2: 289-292 (1982). Goldmacher, V S., and Thilly, W G. Formaldehyde is mutagenic for cultured human cells. Mutat. Res. 116: 417-422 (1983). Gallo, F P Bromuro di metile ossido di etilene formaldeide: problemi biologici, tossicologici e problemi correlati al trattamento dei materiali CONSENSUS WORKSHOP ON FORMALDEHYDE 371 of 101 agents in a quantitative mammalian cell mutation system, CHO/HGPRT (Abstract). Fed. Proc. 37: 1384 (1978). Helwig, H. Wie ungefahrlich ist Formaldehyd? (How safe is formal- dehyde?) Deut. Med. Wochenschr. 102: 1612- 1613 (1977). Hemminki, K. Reactions of formaldehyde with guanosine. Toxicol. Letters 9: 161-164 (1981). Hurni, H., and Ohder, H. Reproduction study with formaldehyde and hexamethylenetetramine in beagle dogs. Food Cosmet. Toxicol. 11: 459-462 (1973). Hemminki, K. Urinary sulfur containing metabolites after adminis- tration of ethanol, acetaldehyde and formaldehyde to rats. Toxicol. Letters 11: 1-6 (1982). Infante, P F Documentation of excess nasal cancer among workers exposed to formaldehyde. U.S. Department of Labor, Memo, January 1982, to T. G. Auchter, Occupational Safety and Health Administra- tion, Washington, D.C. Hemminki, K. Carcinogen adducts excreted in urine: Experimental studies with formaldehyde and dimethylnitrosamine. In: Prevention of Occupational Cancer. International Symposium, Occupational Safety and Health Series 46, International Labour Office, Geneva, Switzer- land, 1982, pp. 459-465. Infante, P F, Ulsamer, A. G., Groth, D., Chu, K. C., and Ward, J. Correspondence: health hazards of formaldehyde. Lancet ii: 980-981 (1981). Hemminki, K., Mutanen, P., Saloniemi, I., Niemi, M-L., and Vainio, H. Spontaneous abortions in hospital staff engaged in sterilising instruments with chemical agents. Brit. Med. J. 285: 1461-1463 (1982). Interagency Regulatory Liaison Group (IRLG). Meeting Report, IRLF visit to CIIT, January 17-18, 1980, Research Triangle Park, NC. Interagency Regulatory Liaison Group (IRLG). Work group on task assessment scientific bases for identification of potential carcinogens and estimation of risks. J. Natl. Cancer Inst. 63: 241-268 (1979). Hendrick, D. J., and Lane, D. J. Formalin asthma in hospital staff. Brit. Med. J. 1: 607-608 (1975). Hendrick, D. J., and Lane, D. J. Occupational formalin asthma. Brit. J. Ind. Med. 34: 11-18 (1977). International Agency for Research on Cancer. Background and purpose of the IARC programme on the evaluation of the carcinogenic risk of chemicals to man. IARC Monograph: Some Fumigants, the Herbicides 2,4-D and 2,4,5-T, Chlorinated Dibenzodioxins and Miscellaneous Industrial Chemicals, Vol. 15, IARC, Lyon, France, 1977, pp. 11-25. Hendrick, D. J., Rando, R. J., Lane, D. J., and Morris, M. J. Formaldehyde asthma: challenge exposure levels and fate after five years. J. Occup. Med. 24: 893-897 (1982). Henschler, D. Zur Frage eines Karzinogenen Risikos durch Formal- dehyd (The carcinogenic risk of formaldehyde) (abstract). Zentralbl. Arbeitsmed. Arbeitsschutz Prophyl. 30: 53 (1980). International Agency for Research on Cancer. Formaldehyde (gas) (Group 2B). CONSENSUS WORKSHOP ON FORMALDEHYDE In: IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Supplement 4, IARC, Lyon, France, 1982, pp. 131-132. Herskowitz, I. H. Mutation rate in D. melanogaster males treated with formaldehyde and 2,4-dinitrophenol (abstract). Genetics 36: 554-555 (1951). International Agency for Research on Cancer. Formaldehyde. In: IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, Some Industrial Chemicals and Dyestuffs, Vol. 29, IARC, Lyon, France, 1982, pp. 345-389. Herskowitz, I. H. Formaldehyde-induced mutation in mature sper- matozoa and early developmental stages of Drosophila melanogaster (abstract). Genetics 38: 668-669 (1953). Irr, J. D. Mouse lumphoma L5178Y cell TK locus assay for mutagenicity: a study of formaldehyde. Haskell Laboratory Report No. 581-80, E. I. DuPont de Nemours Co., Haskell Laboratory for Toxicology and Industrial Medicine, Elkton Road, Newark, Delaware, 1980. Hileman, B. Formaldehyde: how did EPA develop its formaldehyde policy? Environ. Science Technol. 16: 543A-547A (1982). Hinds, M. D. Product safety agency bans use of formaldehyde foam insualtion. The New York Times, February 23, 1982, p. 15. Ishchenko, V N., and Pushkina, I. K. Evaluation of working conditions in a plant engaged in processing phenol-formaldehyde resins. Gig. Sanitar. 11: 98-100 (1978). Hodgson, A. T., Geisling, K. L., Remijn, B., and Girman, J. R. Validation of a passive sampler for determining formaldehyde in residential indoor air. Lawrence Berkeley Laboratory, University of California/Berkeley, Report No. LBL-14626, EEB-VENT 82-10, Berkeley, California, 1982. Jaeger, R. J., and Gearhart, J. M. Respiratory and metabolic response of rats and mice to formalin vapor. Toxicology 25: 299-309 (1982). Hollowell, C. D., Berk, J. V, Lin, C.-I., and Turiel, I. Indoor air quality in energy-efficient buidlings. Lawrence Berkeley Laboratory, University of California! Berkeley, Report No. LBL-8892, EEB- VENT 79-2; Berkeley, California, 1979. Jensen, D. E., Lotlikar, P D., and Magee, P. N. The in vtiro methylation of DNA by microsomally-activated dimethynitrosamine and its correlation with formaldehyde production. Carcinogenesis 2: 349-354 (1981). Hollowell, C. D., Berk, J. V, and Traynor, G. W Impact of reduced infiltration and ventilation on indoor air quality in residential buildings. ASHRAE Trans. 85(Part 1): 816-827 (1979). Jensen, 0. M. Correspondence: Cancer risk from formaldehyde. Lancet ii: 480-481 (1980). Hooper, K. The hazard evaluation system and information service: a physician's resource in toxicology and occupational medicine. Western J. Med. 137: 560-571 (1982). Jensen, 0. M., and Andersen, S. K. Correspondence: Lung cancer risk from formaldehyde. Lancet i: 913 (1982). CONSENSUS WORKSHOP ON FORMALDEHYDE Covalent binding of formaldehyde with macromolecules in the rat nasal mucosa (abstract). Toxicologist 3(1): 61 (1983). Grossman, L. I. Paresthesia from N2 or N2 substitute. Oral Surg., Oral Med., Oral Pathol. 45: 114-115 (1978). Heck, H. D'A., and Casanova-Schmitz, M. Biochemical toxicology of formaldehyde (in press). Grover, S. Effect of foam insulation ban worries formaldehyde firms. Wall Street Journal, May 21, 1982, p. 33. Heck, H. D'A., Chin, T. Y., and Schmitz, M. C. Distribution of 14C formaldehyde in rats after inhalation exposure. In: Formaldehyde Toxicity, (J. E. Gibson, Ed.), Hemisphere Publishing, Washington, DC, 1983, pp. 26-37. Guess, H., Crump, K., and Peto, R. Uncertainty estimates for low-dose-rate extrapolations of animal carcinogenicity data. Cancer Res. 37: 3475-3483 (1977). Heck, H. D'A., Schmitz, M. C., and White, E. L. Disposition and analysis of formaldehyde in F-344 rats after inhalation exposure (abstract). Toxicologist 2(2): 158 (1982). Gunby, P Fact or fiction about formaldehyde? J. Am. Med. Assoc. 243: 1697-1703 (1980). Gupta, K., and Cohn, M. Health sciences analysis of comments on the proposed ban of UFFI. Memorandum to: Harry Cohen, Program Manager, Consumer Product Safety Commission, Bethesda, Mary- land, February 19, 1982. Heck, H. D'A., White, E. L., and Casanova-Schmitz, M. Determina- tion of formaldehyde in biological tissues by gas chromatography/mass spectrometry. Biomed. Mass Spectrometry 9: 347-353 (1982). Helander, I. Contact urticaria from leather containing formaldehyde. Arch. Dermatol. 113: 1443 (1977). Guseva, V A. Study of the gonadotropic effect in male rats under the action of formaldehyde during its simultaneous administration from air and water (translation available). Gigiena I Sanitariya 10: 102-103 (1972). Heling, B., Ram, Z., and Heling, I. The root treatment of teeth with toxavit. Oral Surg., Oral Med., Oral Pathol. 43: 306-309 (1977). Helmes, C. T., Atkinson, D. L., Jaffer, J., Sigman, C. C., Thompson, K. L., Kelsey, M. I., Kraybill, H. F, and Munn, J. I. Evaluation and classification of the potential cacinogenicity of organic air pollutants. J. Environ. Sci. Health A17: 321-389 (1982). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE Johansen, M., and Bundgaard, H. Kinetics of formaldehyde release from the cosmetic preservative Germall 115. Arch. Pharmici Chemi, Sci. Ed. 9: 117-122 (1981). Horton, A. W., Tye, R., and Stemmer, K. L. Experimental carcinogenesis of the lung. Inhalation of gaseous formaldehyde or an aerosol of coal tar by C3H mice. J. Natl. Cancer Inst. 30: 31-48 (1963). Johansson, E. B., and Tjalve, H. The distribution of 14C dimethyl- nitrosamine in mice. Autoradiographic studies in mice with inhibited and noninhibited dimethylnitrosamine metabolism and a comparison with the distribution of 14C formaldehyde. Toxicol. Appl. Pharmacol. 45: 565-575 (1978). Howell, E. D., and Perkins, H. A. Anti-N-like antibodies in the sera of patients undergoing chronic hemodialysis. Vox Sang. 23: 291-299 (1972). Hsiao, S.-H., and Villaume, J. E. Occupational health and safety and environmental aspects of urea-formaldehyde resins: Final Report- April 1978, Vol. 6, Contract No. DAMD-17-77-C-7020, Science Information Services Department, The Franklin Institute Research Laboratories, Philadelphia, Pennsyvania, 1978. Johnson, E. M. Screening for teratogenic potential: are we asking the proper question? Teratology 21: 259 (1980). Johnson, E. M., and Gabel, B. E. G. Application of the hydra assay for rapid detection of developmental hazards. J. Am. Coll. Toxicol. 1: 57-71 (1982). Hsie, A. W, O'Neill, J. P, San Sebastian, J. R. S., Couch, D. B., Fuscoe, J. C., Sun, W N. C., Brimer, P A., Machanoff, R., Riddle, J. C., Forbes, N. L., and Hsie, M. H. Mutagenicity of carcinogens: study Johnson, E. M., Gorman, R. M., Gabel, B. E. G., and George, M. E. The hydra attenuata system for detection of teratogenic hazards. Teratog. Carcinog. Mutagen. 2: 263-276 (1982). CONSENSUS WORKSHOP ON FORMALDEHYDE 372 Jordan, W F, Sherman, W T., and King, S. E. Threshold responses in formaldehyde-sensitive subjects. J. Am. Acad. Dermatol. 1: 44-48 (1979). Kligerman, A. D., Wilmer, J. L., and Erexson, G. L. Analyses of cytogenetic damage in rat lymphocytes after inhalation of toxicants (abstract). Environ. Mutagen. 5: 400 (1983). Kallos, G. J., and Solomon, R. A. Investigations of the formation of bis-chloromethyl ether in simulated hydrogen chloride-formaldehyde atmospheric environments. Am. Ind. Hyg. Assoc. J. 34: 469-473 (1973). Kline, B. S. Formaldehyd poisoning: with report of a fatal case. Arch. Int. Med. 36: 220-228 (1925). Knecht, U., and Woitowitz, H. J. Felduntersuchungen zur Belastung der Raumluft durch Formaldehyd im Kliniken und Instituten (Field investigations on the burdening of indoor-air by formaldehyde in hospitals and institutes). Offent. CONSENSUS WORKSHOP ON FORMALDEHYDE Effects of formaldehyde on the health of workers in the crease-resistant clothing industry (translation available). Pracovni Lekarstvi 23: 374-375 (1971). Kennedy, E. R., and Hill, R. H., Jr. Determination offormaldehyde in air as an oxazolidine derivative by capillary gas chromatography. Anal. Chem. 54: 1739- 1742 (1982). Kreiger, N. Letters to the Editor: Formaldehyde and nasal cancer mortality. Can. Med. Assoc. J. 128: 248-249 (1983). Kreiger, R. A., and Garry, V F Formaldehyde-induced cytotoxicity and sister chromatid exchanges in human lymphocyte cultures. Mutat. Res. 120: 51-55 (1983). Kensler, C. J., and Battista, S. P Components of cigarette smoke with ciliary-depressant activity: their selective removal by filters containing activated charcoal granules. New Engl. J. Med. 269: 1161-1166 (1963). Kring, E. B., Thornley, G. D., Dessenberger, C., Lautenberger, W J., and Ansul, G. R. A new passive colorimetric air monitoring badge for sampling formaldehyde in air. Am. Ind. Hyg. Assoc. J. 43: 786-795 (1982). Kerfoot, E. J., and Mooney, T. F., Jr. Formaldehyde and parafor- maldehyde study in funeral homes. Am. Ind. Hyg. Assoc. J. 36: 533-537 (1975). Krivanek, N. D., McAlack, J. W, and Chromey, N. C. Mouse skin painting-initiation-promotion study with formaldehyde solutions, preliminary results (abstract). Toxicologist 3(1): abstract 573 (1983). Kerns, W D., Pavkov, K. L., Donofrio, D. J., Gralla, E. J., and Swenberg, J. A. Carcinogenicity of formaldehyde in rats and mice after long term inhalation exposure. Cancer Res. 43: 4382-4392 (1983). Kuhn, M. Belastung der Raumluft durch Formaldehyd (Room air load of formaldehyde). Swiss Chem. 4: 63-66 (1982). Khishin, A. F E. The requirement of adenylic acid for formaldehyde mutagenesis. Mutat. Res. 1: 202-205 (1964). Kuhn, M., and Wanner, H. U. Pollution of indoor air by materials. Sozial. Praventivmed. 27: 260-261 (1982). Kilburn, K. H., Warshaw, R., Boylen, C. T., Balmes, J., Johnson, S., Seidman, B., and Deflorio, G. Ibxic effects of formaldehyde and solvents in histology technicians: a preliminary report. J. Histotechnol. 6: 73-76 (1983). Kulle, T. J., and Cooper, G. P Effects of formaldehyde and ozone on the trigeminal nasal sensory system. Arch. Environ. Health 30: 237-243 (1975). Kimmelman, B. B. Letter to the Editor: potential hazards of formaldehyde. J. Am. Dental Assoc. 104: 288 (1982). Kumar, S. Effect of intravesical formalin on the urothelium. Brit. J. Urology 51: 375-377 (1977). Kimmelman, B. B., and Hillman, E. E. Formaldehyde vapor in the dental environment: absence ofpotential hazard. J. Dentistry Children 50: 55-57 (1983). CONSENSUS WORKSHOP ON FORMALDEHYDE Gesundh.-Wesen 41: 715-723 (1979). Kamchatnov, V P, and Gayazova, S. S. Temperature asymmetry in workers exposed to fornaldehyde vapor. Hygiene and Sanitation (USSR) 36: 286-287 (1971). Knight, B. W Health implications from exposure to urea formaldehyde foam insulation. Communicable Disease Report, Alabama Department of Public Health, Bureau of Epidemiology and Consultation 14: 1-5 (1982). Kamei, H., Yamamoto, T., Yoshida, T., Kuroiwa, Y, Kawai, Y, Kawai, M., Kuroiwa, S., Masahide, K., and Kaneko, Y Toxicological studies on effects of formaldehyde on bronchial tissue. Eisei Kagaku 27: 356-362 (1981). Koivusalo, M., Koivula, T., and Uotila, L. Oxidation of formaldehyde by nicotinamide nucleotide dependent dehydrogenases. In: Enzymol- ogy of Carbonyl Metabolism: Aldehyde Dehydrogenase and Aldo/Keto Reductase (H. Weiner and B. Wermuth, Eds.), Alan R. Liss Inc., New York, 1982, pp. 155-168. Kane, L. E., and Alarie, Y Sensory irritation to formaldehyde and acrolein during single and repeated exposures in mice. Am. Ind. Hyg. Assoc. J. 38: 509-522 (1977). Kaplan, W D. Formaldehyde as a mutagen in Drosophila. Science 108: 43 (1948). Kojima, S., Kaniwa, M.-A., and Nakamura, A. Investigations on the underlying principles of test methods for formaldehyde release from plywoods and furnitures (translation available). Eisei Kagaku 28: 205-218 (1982). Kaplan, W D., and Pelc, S. R. Autoradiographic studies ofDrosophila gonads following the feeding of C'4 labelled formaldehyde (abstract). Genetics 40: 578 (1955). Konopinski, V J. Formaldehyde in office and commercial environ- ments. Am. Ind. Hyg. Assoc. J. 44: 205-208 (1983). Karol, M. H., and Lee, K. Experimental sensitization to formaldehyde (abstract). Toxicologist 3(1): Abstract 339 (1983). Kopylova, L. S., Gleiberman, S. E., Likhtman, T. V, Semenova, T. F., and Alekseeva, M. I. Safety study of a method for disinfecting artificial pulmonary ventilation apparatus with aerosols containing formaldehyde (translation available). Anesteziol. Reanimatol. 2: 33-37 (1980). Kato, N., Miyawaki, N., and Sakazawa, C. Formaldehyde dehydroge- nase from formaldehyde-resistant Debaryomyces vanriji FT-1 and Pseudomonas putida F61. Agric. Biol. Chem. 47: 415-416 (1983). Kayser, R., Sterling, D., and Viviani, D. Intermedia priority pollutant guidance documents. Report of Office of Toxics Integration and Office ofPesticides and lbxic Substances, Environmental Protection Agency, Washington, D.C., July, 1982. Kornbrust, D. J., and Bus, J. S. The role of glutathione and cytochrome P-450 in the metabolism of methyl chloride. Toxicol. Appl. Pharmacol. 67: 246-256 (1983). Kemp, W M. Formaldehyde report-executive summary. Oklahoma State Department of Health, P 0. Box 53551, Oklahoma City, Oklahoma, 1982. Kratochvil, I. CONSENSUS WORKSHOP ON FORMALDEHYDE 373 Loomis, T. A. Formaldehyde toxicity Arch. Pathol. Lab. Med. 103: 321-324 (1979). sodimethylamine (NDMA) in the rat (abstract). Proc. Am. Assoc. Cancer Res. 23: 79 (1982). Loomis, T. A. Formaldehyde toxicity Arch. Pathol. Lab. Med. 103: 321-324 (1979). Lapkina, T. I., Saburova, L. M., Rozvadovskii, V D., and Shcher- bakova, L. N. Endogenous and exogenous formaldehyde in acute ischemia of various organs. Bull. Exper. Biol. Med. 93: 182-184 (1982). Ludwig, H. Acute formaldehyde bronchiolitis in a synthetic resin worker. Samml. Vergiftungsfaellen 6: 277-330 (1935). Lundberg, J. M., and Saria, A. Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature 302: 251-253 (1983). Laskin, S., Kuschner, M., Drew, R. T., Cappiello, V R, and Nelson, N. Tumors of the respiratory tract induced by inhalation of bis(chloromethyl) ether. Arch. Environ. Health 23: 135-136 (1971). Lundqvist, K. Formaldehyd Auspaltas ej Fran Fiberskivor (Formal- dehyde is not emitted from fiberboard). Byggmastaren 9: 18-20 (1979). Lawler, P G. Inhalation of formaldehyde vapour: a potential hazard of a method of sterilisation of bacterial filters. Anaesthesia 37: 1102-1103 (1982). Luo, J. E., Nielsen, G. D., and Alarie, Y Formaldehyde: an exception in the series of saturated aldehydes, aliphatic alcohols and alkyl- benzenes (abstract). Toxicologist 3(1): 74 (1983). Lazar, P Reactions to nail hardeners. Arch. Dermatol. 94: 446-448 (1966). Luster, M. I., and Dean, J. H. Symposium summary: immunological hypersensitivity resulting from environmental or occupational ex- posure to chemicals: a state-of-the-art workshop summary. Funda- mentals Appl. Toxicol. 2: 327-330 (1982). Le Botlan, D. J., Mechin, B. G., and Martin, G. J. Proton and carbon-13 nuclear magnetic resonance spectrometry of formaldehyde in water. Anal. Chem. 55: 587-591 (1983). Leach, C. L., Sharma, R. R, and Oberg, S. G. Immunotoxic and pathologic effects of inhalation of various levels of formaldehyde vapour in F-344 rats (abstract). Toxicologist 3(1): 73 (1983). Lynde, C. W, and Mitchell, J. C. Patch tests results in 66 hairdressers, 1973-1981. Contact Dermatitis 8: 302-307 (1982). Lynen, R., Rothe, M., and Gallasch, E. Characterization of formal- dehyde-related antibodies encountered in hemodialysis patients at different stages of immunization. Vox Sang. 44: 81-89 (1983). Lebowitz, M. D. Health effects of indoor pollutants. Ann. Rev. Publ. Health 4: 203-221 (1983). Lee, C. W, Fung, Y S., and Fung, K. W Determination of formaldehyde vapour in the atmospheres of clinical laboratories using chromotropic acid. Analyst 107: 30-34 (1982). Ma, T. CONSENSUS WORKSHOP ON FORMALDEHYDE Micronuclei induced by x-rays and chemical mutagens in meiotic pollen mother cells of Tradescantia. Mutat. Res. 64: 307-313 (1979). Leong, B. K. J., MacFarland, H. N., and Reese, W H., Jr. Induction of lung adenomas by chronic inhalation of bis(chloromethyl) ether. Arch. Environ. Health 22: 663-666 (1971). Ma, T. Thradescantia micronucleus bioassay and pollen tube chromatid aberration test for in situ monitoring and mutagen screening. Environ. Health Perspect. 37: 85-90 (1981). Leung, K. H., Fischer, D. G., and Koren, H. S. Erythromyeloid tumor cells (K562) induce PgE synthesis in human peripheral blood monocytes. J. Immunol. 131: 445-449 (1983). Ma, T.-H. Project summary: T'radescantia mcn-in-tetrad mutagen test for on-site monitoring and further validation. EPA-600/Sl-81-019, Environmental Protection Agency, Washington, DC, April 1981. Levin, D. E., Hollstein, M., Christman, M. F., Schwiers, E. A., and Ames, B. N. A new Salmonella tester strain (TA102) with A-T base pairs at the site of mutation detects oxidative mutagens. Proc. Natl. Acad. Sci. (U.S.) 79: 7445-7449 (1982). Ma, T. 7'radescantia cytogenetic tests (root-tip mitosis, pollen mitosis, pollen mother-cell meiosis). Mutat. Res. 99: 293-302 (1982). Ma, T. 'radescantia micronuclei (trad-mcn) test for environmental clastogens. In: In Vitro Toxicity Testing of Environmental Agents, Part 1 (A. Kolber, et al., Eds.), Plenum Press, New York, 1983, pp. 191-214. Levine, R. J. Mortality of Ontario undertakers. CIIT Activities, Chemical Industry Institute of Toxicology 2(12): 3-5 (1982). Levine, R. J., Corso, R. D. D., Blunden, P B., and Battigelli, M. C. The effects of occupational exposure on the respiratory health of West Virginia morticians. In: Formaldehyde Toxicity (J. E. Gibson, Ed.), Hemisphere Publishing Corp., Washington, DC, 1983, pp. 212-226. Ma, T., Anderson, V A., and Ahmed, I. Environmental clastogens detected by meiotic pollen mother cells of Tradescantia. In: Genotoxic Effects of Airborne Agents (R. R. Tice, D. L. Costa and K. M. Schaich, Eds.), Plenum Press, New York, 1982, pp. 141-157. Levy, S., Nocentini, S., and Billardon, C. Induction of cytogenetic effects in human fibroblast cultures after exposure to formaldehyde or x-rays. Mutat. Res. 119: 309-317 (1983). Ma, T., Anderson, V A., and Sandhu, S. S. A preliminary study of the clastogenic effects of diesel exhaust fumes using the Tradescantia micronucleus bioassay. In: Short-Term Bioassays in the Analysis of Complex Environmental Mixtures. II (M. Waters, S. Sandhu, J. Huisingh, D. Claxton and S. Nesnow, Eds.), Plenum Press, New York, 1982, pp. 351-358. Lewin, T. Formaldehyde issue stirred by U.S. ban. CONSENSUS WORKSHOP ON FORMALDEHYDE Kuschner, M., Laskin, S., Drew, R. T., Cappiello, V, and Nelson, N. Inhalation carcinogenicity of alpha halo ethers: III. Lifetime and limited period inhalation studies with bis(chloromethyl) ether at 0.1 ppm. Arch. Environ. Health 30: 73-77 (1975). Kitchens, J. F., Casner, R. E., Edwards, G. S., Harward, W E. III, and Macri, B. J. Investigation of selected potential environmental contaminants: Formaldehyde. EPA Draft Report #560/2-76-009, Washington, DC, 1979. Kwong, F, Kraske, G., Nelson, A. M., and Klaustermeyer, W B. Acute symptoms secondary to formaldehyde exposure in a pathology resident. Ann. Allergy 50: 326-328 (1983). Kleeberg, U., and Klinger, W Sensitive formaldehyde determination with Nash's reagent and tryptophan reaction. J. Pharmacol. Methods 8: 19-31 (1982). Labuc, G. E., and Archer, M. C. Esophageal and hepatic microsomal metabolism of N-nitroso-methylbenzylamine (NMBZA) and N-nitro- CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE Tubular necrosis compli- cating intravesical formaldehyde treatment. Intern. Surg. 67: 504-505 (1982). Martin, C. N., McDermid, A. C., and Garner, R. C. Testing of known carcinogens and noncarcinogens for their ability to induce unscheduled DNA synthesis in Hela cells. Cancer Res. 38: 2621-2627 (1978). Martin-Amat, G., McMartin, K. E., Hayreh, S. S., Hayreh, M. S., and Tephly, T. R. Methanol poisoning: ocular toxicity produced by formate. Toxicol. Appl. Pharmacol. 45: 201-208 (1978). Merkur'eva, R. V, and Tsapkova, N. N. Hygienic significance of a system of biochemical criteria for the evaluation of hepatotoxic and neurotoxic effects of some chemical factors in the environment. J. Hyg. Epidem. Microbiol. Immunol. 26: 336-341 (1982). Marutzky, R., Mehlhorn, L., and Menzel, W Reducing the formal- dehyde emission from furniture. Holz als Roh- und Werkstoff 39: 7-10 (1981). Meyer, B., and Carlson, N. L. Formaldehyde emission from particle board post-cured by radio frequency heating. Holzforschung 37: 41-45 (1983). Marzulli, F N., and Maibach, H. I. Contact allery: predictive testing in humans. In: Advances in Modern Toxicology, Vol. 4, Dermato- toxicology and Pharmacology (F N. Marzulli and H. I. Maibach, Eds.), Hemisphere Publishing, Washington, DC, 1977, pp. 353-372. Meyer, B., Koshlp, K., Geisling, K. L., and Micksch, R. R. Comparison of wet and dry desiccator test methods for formaldehyde emission from UF-bonded wood products. Forest Prod. J. 33: 35-37 (1983). Marzulli, F N., and Maibach, H. I. Antimicrobials: experimental contact sensitization in man. J. Soc. Cosmet. Chem. 24: 399-421 (1973). Miksch, R. R., Anthon, D. W, Fanning, L. Z., Hollowell, C. D., Revzan, K., and Glanville, J. Modified pararosaniline method for the determination of formaldehyde in air. Anal. Chem. 53: 2118-2123 (1981). Marzulli, F N., and Maibach, H. I. The use of graded concentrations in studying skin sensitizers: experimental contact sensitization in man. Food Cosmet. Toxicol. 12: 219-227 (1974). Miller, S. Risks: how to get more science in assessments. Environ. Sci. Technol. 17: 199A-200A (1983). Mashford, P M., and Jones, A. R. Formaldehyde metabolism by the rat: a re-appraisal. Xenobiotica. 12: 119-124 (1982). Miller, W H. Dynamical effects of symmetry along a reaction path: mode specificity in the unimolecular dissociation of formaldehyde. J. Am. Chem. Soc. 105: 216-220 (1983). Masilungan, A. O., Gerry, R. T., Kamatari, O., and Bakker, J. CEH Report Abstract Formaldehyde. Chemical Industry Division News- letter, SRI International, Menlo Park, California, January-February, 1983, pp. 6-7. Miretskaya, L. CONSENSUS WORKSHOP ON FORMALDEHYDE The New York Times, May 25, 1982, pp. Dl and D9. Lewis B. B., and Chestner, S. B. Formaldehyde in dentistry: a review of mutagnic and carinogenic potential. J. Am. Dental Assoc. 103: 429-434. Ma, T., Kontos, G. J., Jr., and Anderson, V A. Stage sensitivity and dose response of meiotic chromosomes of pollen mother cells of Tradescantia to x-rays. Environ. Exptl. Bot. 20: 169-174 (1980). Lewis, K. J., Ward, M. K., and Kerr, D. N. S. Residual formaldehyde in dialyzers: quantity, location, and the effect of different methods of rinsing. Artificial Organs 5: 269-277 (1981). Ma, T., Sparrow, A. H., Schairer, L. A., and Nauman, A. E Effect of 1,2-dibromoethane (DBE) on meiotic chromosomes of 7Tradescantia. Mutat. Res. 58: 251-258 (1978). Leysen, J., and Laduron, P Characterization of an enzyme yielding formaldehyde from 5-methyltetrahydrofolic acid. FEBS Letters 47: 299-303 (1974). Magana-Schwencke, N., and Ekert, B. Biochemical analysis of damage induced in yeast by formaldehyde: II. Induction of cross-links between DNA and protein. Mutat. Res. 51: 11-19 (1978). Liebling, T., Roserman, K., Pastides, H., and Lemeshow, S. Cancer mortality at a western Massachusetts chemical plant (abstract). Am. J. Epidemiol. 116: 570 (1982). Magana-Schwencke, N., Ekert, B., and Moustacchi, E. Biochemical analysis of damage induced in yeast by formaldehyde: I. Induction of single-strand breaks in DNA and their repair. Mutat. Res. 50: 181-193 (1978). Linkskov, R. Contact urticaria to formaldehyde. Contact Dermatitis 8: 333-334 (1982). Malorny, G., Rietbrock, N., and Schneider, M. The oxidation of formaldehyde to formic acid in the blood. A contribution on the metabolism of formaldehyde (translation available). Exptl. Pathol. Pharmakol. 250: 419-436 (1963). Lipari, F, and Swarin, S. J. Determination offormaldehyde and other aldehydes in automobile exhaust with an improved 2,4-dinitrophenyl- hydrazine method. J. Chromatog. 247: 298-306 (1982). Logan, W S., and Perry, H. 0. Contact dermatitis to resin-containing casts. Clin. Orthoped. Related Res. 90: 150-152 (1973). Malter, K. E. The man on socks: hypersensitivity for materials in CONSENSUS WORKSHOP ON FORMALDEHYDE 374 shoes and clothing. Nederl. Tijdschr. Geneeskunde 125: 649-654 (1981). Recent Advances in Pollutant Monitoring of Ambient Air and Stationary Sources, Raleigh, NC, May 4-7, 1982 (in press). Mann, G. K., and Parida, B. B. Formalin induced sex chromosome breakage in spermatocyte cells ofthe grasshopper, Tirstria pulvinata. Uvarov (abstract). Indian Sci. Congr. Assoc. Proc. 53: 321 (1966). g Matthews, T. G., and Howell, T. C. Visual colorimetric formaldehyde screening analysis for indoor air. J. CONSENSUS WORKSHOP ON FORMALDEHYDE Air Pollution Control Assoc. 31: 1181-1184 (1981). Matthews, T. G., and Howell, T. C. Solid sorbent for formaldehyde monitoring. Anal. Chem. 54: 1495-1498 (1982). Mantel, N., and Schneiderman, M. A. Estimating safe levels, a hazardous undertaking. Cancer Res. 35: 1379-1386 (1975). Marison, I. W, and Attwood, M. M. A possible alternative mechanism for the oxidation of formaldehyde to formate. J. Gen. Microbiol. 128: 1441-1446 (1982). Mattia, M. A. Hazards in the hospital environment. The sterilants: ethylene oxide and formaldehyde. Am. J. Nurs. 83: 240-243 (1983). McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals. Proc. Natl. Acad. Sci. (U.S.) 72: 5135-5139 (1975). Marks, T. A., Worthy, W C., and Staples, R. E. Influence of formaldehyde and Sonacide (potentiated acid glutaraldehyde) on embryo and fetal development in mice. Teratology 22: 51-58 (1980). McNulty, M. J., Casanova-Schmitz, M., and Heck, H. D'A. Metabo- lism of dimethylamine to formaldehyde in the rat nasal mucosa (abstract). Toxicologist 2(2): abstract 275 (1982). Markuson, K. E., Mancuso, T. F, and Soet, J. S. Dermatitis due to the formaldehye resins: prevention and methods of control. Ind. Med. 12: 383-386 (1943). Meadows, G., and Rusch, G. M. The measuring and monitoring of formaldehyde in inhalation test atmospheres. Am. Ind. Hyg. Assoc. J. 44: 71-77 (1983). Marsh, G. M. Proportional mortality patterns among chemical plant workers exposed to formaldehyde. Brit. J. Ind. Med. 39: 313-322 (1982). Mecke, P, Keller, R., Schumacher, G., and Beckert, J. Zur Bedeutung von Gasruckstanden bei der Formaldehyd-sterilization (Significance of gas traces in formaldehyde sterilization). Forum Staedte-Hygiene 33: 120-123 (1982). Marshall, E. EPAs high-risk carcinogen policy: the government is changing the way it identifies dangerous chemicals, with potentially tragic consequences, critics say. Science 218: 975-978 (1982). Marshall, E. Revisions in cancer policy: Rita Lavelle had something to say about cancer risk assessment, House inquiry learns. Science 220: 36-37 (1983). Medford, R. L. Decision briefing package on urea-formaldehyde foam insulation. Office of Program Management, Consumer Product Safety Commission, Washington, DC, February, 1982. Marshall, T. C., Hahn, F F., Henderson, R. F, Silbaugh, S. A., and Wolff, R. K. Subchronic inhalation exposure of guinea pigs to formaldehyde (CH20) (abstract). Toxicologist 3(1): abstract 244 (1983). Melekos, M., and Lalos, J. Letters to the Editor: Intravesical instillation of formalin and its complications. Urology 21: 331-332 (1983). Merimsky, E., Blum, M., and Aviram, A. CONSENSUS WORKSHOP ON FORMALDEHYDE 375 NIOSH. Health Hazard Evaluation Report, TQ 80-118-928, Dept. of Transportation, Augusta, ME, July 1980. NTIS PB83-102855. Morgan, K. T., Patterson, D. L., and Gross, E. A. Frog palate mucociliary apparatus: Structure, function, and response to formal- dehyde gas. Fundamentals Appl. Toxicol. 4: 58-68 (1984). NIOSH. Report: Formaldehyde: evidence of carcinogenicity. NIOSH Current Intelligence Bulletin 34, Cincinnati, Ohio, April 1981. Morin, N. C., and Kubinski, H. Potential toxicity of materials used for home insulation. Ecotoxicol. Environ. Safety 2: 133-141 (1978). NIOSH. Report: Formaldehyde exposures in dialysis units. Dialysis and Transplantation 12: 43-44 (1983). Morrill, E. E., Jr. Formaldehyde exposure from paper process solved by air sampling and current studies. Air Cond., Heat. Vent. 58: 94-95 (1961). NIOSH Center for Disease Control (CDC). Formaldehyde exposures in a gross anatomy laboratory. Colorado Morbidity and Mortality Weekly Report 31: 698-700 (1983). Moss, E., and Lee, W R. Occurrence of oral and pharyngeal cancers in textile workers. Brit. J. Ind. Med. 31: 224-232 (1974). NIOSH. Formaldehyde: evidence of carcinogenicity. NIOSH/OSHA Current Intelligence Bulletin 34, Cincinnati, Ohio, December, 1980. Mudakavi, J. R., and Ravindram, M. Spectrophotometric determina- tion of traces of formaldehyde with ,3-naphtol. Current Sci. 51: 39-41 (1982). National Research Council, Committee on Tbxicology. Formaldehyde- an assessment of its health effects/prepared for the Consumer Product Safety Commission by the Committee on Toxicology. National Academy of Sciences, Washington, DC, March, 1980. Muller, R, Raabe, G., and Schumann, D. Leukoplakia induced by repeated deposition of formalin in rabbit oral mucosa; long term experiments with a new oral tank. Exptl. Pathol. 16: 36-42 (1978). Natvig, H., Andersen, J., and Rasmussen, E. W A contribution to the toxicological evaluation ofhexamethylenetetramine. Food Cosmet. Toxicol. 9: 491-500 (1971). Muller, R. E., and Schurath, U. Generation of formaldehyde in test atmospheres with low concentrations of hydrogen and carbon monoxide. Anal. Chem. 55: 1440-1442 (1983). Neely, W B. The metabolic fate offormaldehyde-14C intraperitoneally administered to the rat. Biochem. Pharmacol. 13: 1137-1142 (1964). Musher, D. M., and Griffith, D. P Generation of formaldehyde from methenamine: effect of pH and concentration, and antibacterial effect. Antimicro. Agents Chemother. 6: 708-711 (1974). Nestler, F H. M. The formaldehyde problem in wood-based products an annotated bibliography. U.S. Department of Agriculture, Forest Service, USDA Forest Service General Technical Report FPL-8, Forest Products Laboratory, Madison, Wisconsin, 1977. Myers, D. R., Pashley, D. H., Whitford, G. M., and McKinney, R. CONSENSUS WORKSHOP ON FORMALDEHYDE V Tissue changes induced by the absorption of formocresol from pulpotomy sites in dogs. Pediatric Dent. 5: 6-8 (1983). Nethercott, J. R., Albers, J., Guirguis, S., Ching, G., Hofstader, S. and From, L. Erythema multiforme exudativum linked to the manufacture of printed circuit boards. Contact Dermatitis 8: 314-322 (1982). Myers, G. E., and Nagaoka, M. Formaldehyde emission: methods of measurement and effect of several particleboard varibles. Wood Sci. 13: 140-150 (1981). Newhouse, M. T. Letter to the Editor: UFFI dust: nonspecific irritant only? Chest 82: 511 (1982). Nagao, T., Ozawa, A., and Sasaki, S. Maintenance of germ-free environment and its clinical utility. Japan. J. Clin. Oncol. 13(Suppl. 1): 103-110 (1983). Newmann, J. Reuse of dialyzers. NAPHT News, National Assoc. of Patients on Hemodialysis and Transplantation, Inc., 156 William St., New York, New York, May 1982, pp. 1-4. Nagornyi, P Hygenic evaluation of working conditions and health status of workers in the production of phenol-formaldehyde polymers. Gig. Truda, Respubl. Mezhvedom. Sborn. 13: 48-52 (1977). Niemela, R., and Vainio, H. Formaldehyde exposure in work and the general environment. Scand. J. Work, Environ. Health 7: 95-100 (1981). Nagornyi, P A., Sudakova, Zh. A., and Shchablenko, S. M. General toxic and allergic effects of formaldehyde. Gig. Truda Profess. Zabol. 1: 27-30 (1979). Nikiforov, B., Balabaeve, L., and Kazakova, B. Effect offormaldehyde on liver function of albino rats with chronic toxic hepatitis. Gig. Sanitar. 9: 73-74 (1980). Nahata, M. C., Cummins, B. A., McLeod, D. C., and Butler, R. Predictability of methenamine efficacy based on type of urinary pathogen and pH. J. Am. Geriat. Soc. 29: 236-239 (1981). Nioshioka, H. Lethal and mutagenic action of formaldehyde in HCR + and HCR- strains ofEscherichia coli. Mutat. Res. 17: 261-265 (1973). Nantel, A. J., Tolszczuk, M., Weber, J. P, Guillot, J. G., and Landry, B. The health problem related to urea-formaldehyde foam insulation of public schools in the province of Quebec (Abstract). Vet. Human Toxicol. 24: 19 (1982). North American Contact Dermatitis Group. Epidemiology of contact dermatitis in North America: 1972. Archiv. Dermatol. 108: 537-540 (1973). Nantel, A. J., Tolszczuk, M., Weber, J. P Guillot, J. G., and Tat-Ha, C. Health problems related to houses insulated with urea-formal- dehyde foam insulation in the province of Quebec (Abstract). Vet. Human Tbxicol. 24: 19 (1982). North Carolina Dept. of Human Resources, Environmental Health Section. The North Carolina Tbxic Substances Management Guide. Formaldehyde, Environmental Health Section, Raleigh, NC, 1981. CONSENSUS WORKSHOP ON FORMALDEHYDE V, and Shvartsman, P Y Study of chromosomal aberrations in human lymphocytes under the influence of formal- dehyde. I. Formaldehyde treatment of lymphocytes in vitro. Tsitolo- giya 24: 1056-1060 (1982). Mathis, J. F An industrial scientist's perspective on scientific peer review. Fundamentals Appl. Toxicol. 2: 280-282 (1982). Moerman, D. G., and Baillie, D. L. Formaldehyde mutagenesis in the nematode: Caenorhabditis elegans. Mutat. Res. 80: 273-279 (1981). Matthews, T. G. Evaluation of a modified CEA Instruments, Inc. Model 555 analyzer for the monitoring of formaldehyde vapor in domestic environments. Am. Ind. Hyg. Assoc. J. 43: 547-552 (1982). Molhave, L. The relation between the free formaldehyde concentration in stored (urea-formaldehyde) chipboard, and the formaldehyde concentration in the air. Holzforsch. Holzverwert. 29: 73-74 (1977). Matthews, T. G., Hawthorne, A. R., Howell, T. C., Metcalfe, C. E., and Gammage, R. B. Evaluation of selected monitoring methods for formaldehyde in domestic environments. Environ. International (in press). Montgomery, S. Paresthesia following endodontic treatment. J. Endodontics 2: 345-347 (1976). Matthews, T. G., Hawthorne, A. R., Schrimsher, J. M., Corey, M. D., and Daffron, C. R. Formaldehyde surface emission monitor. Pro- ceedings, Environmental Protection Agency National Symposium on Morgan, K. T., Patterson, D. L., and Gross, E. A. Formaldehyde and the nasal mucociliary apparatus. In: Formaldehyde: Toxicology, Epidermiology, Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker Inc., New York, 1983, pp. 193-209. CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE A., Alder, V V, and Poverennyi, A. M. Influence of formaldehyde and products of its interaction with amines on processes of DNA transcription in vitro. Biochemistry (USSR) 47: 1509-1515 (1982). Osterloh, J., Kaysen, G., and Becker, C. E. Handling formalin in dialysis units. Dialysis Transplantation 12: 353-361 (1983). Palese, M., and Tephly, T. R. Metabolism of formate in the rat. J. Toxicol. Environ. Health 1: 13-24 (1975). Pruett, J. J. Scheuenstuhl, H., Michaeli, D., and Nevo, Z. The incorporation and localization of aldehydes (highly reactive cigarette smoke components) into cellular fractions of cultured human lung cells. Arch. Environ. Health 35: 15-20 (1980). Paliard, F., Roche, L., Exbrayat, P, and Sprunck, H. Chronic asthma due to formaldehyde. Bull. Offic. Soc. Travail Lyon 10: 528-530 (1949). Parkinson, D. K. Scientific objectivity: a labor perspective. Food Appl. Toxicol. 2: 278-279 (1982). Pushkina, N., Gofmekler, V A., and Klevtsova, G. N. Changes in content of ascorbic acid and nucleic acids produced by benzene and formaldehyde. Bull. Exptl. Biol. Med. 66: 868-869 (1968). Partanen, T., Kauppinen, T., Nurminen, M., Jickels, J., Hernberg, S., and Thiel, K. Formaldehyde exposure and risk of respiratory cancer: an epidemiologic study plotocol. Institute of Occupational Health, Helsinki, Finland, November 1982. Ragan, D. L., and Boreiko, C. J. Initiation of C3H/1OT1/2 cell transformation by formaldehyde. Cancer Lett. 13: 325-331 (1981). Ranly, D. M., and Fulton, R. An autoradiographic study of the response of rat molar pulp to formocresol using 3H-thymidine. Pediactric Dent. 5: 20-24 (1983). Pattison, E. M. Is there a formaldehyde-disulfiram reaction? J. Studies Alcohol 43: 1257-1259 (1982). Pereira, M. A., Chang, L. WV, McMillan, L., Ward, J. B., and Legator, M. S. Battery of short-term tests in laboratory animals to corroborate the detection of human population exposures to genotoxic chemicals (abstract). Environ. Mutagen. 4: 317 (1982). Rapoport, I. A. Mutations under the influence of unsaturated aldehydes. Doklady Akad. Nauk SSSR 61: 713-715 (1948); Chem. Abstr. 43: 1115-1116 (1949). Re, M. R., and Crovato, E. Esposizione a formaldeide: valore e significato della progettazione statistica I-II guidizio di rispetto dello standard (Exposure to formaldehyde: value and significance of statistical projections. I-The evaluation under consideration of the standard. Ann. Inst. Super. Sanita 17: 511-514 (1981). Perera, F., and Petito, C. Formaldehyde: a question of cancer policy Science 216: 1285-1291 (1982). Pertosovskii, A. L., Charnikova, G. A., Kremko, L. M., and Sidenko, A. T. CONSENSUS WORKSHOP ON FORMALDEHYDE Nova, M. M. H., and Touraine, R. G. Asthma au formol (Asthma from formaldehyde). Soc. Med. Trav. 17: 293-294 (1956). National Cancer Advisory Board. General criteria for assessing the evidence for carcinogenicity of chemical substances: Report of the Subcommittee on Environmental Carcinogenesis. National Cancer Advisory Board. J. Natl. Cancer Inst. 58: 461-465 (1977). Obe, G., and Beek, B. Mutagenic activity of aldehydes. Drug Alcohol Dependence 4: 91-94 (1979). Odom, R. G., and Maibach, H. I. contact urticaria: a different contact dermatitis. In: Advances in Modern Tbxicology, Vol. 4, Dermato- toxicology and Pharmacology (F N. Marzulli and H. I. Maibach, Eds.), Hemisphere Publishing, Washington, DC, 1977, pp. 441-452. NIOSH. Health Hazard Evaluation Report, HHE 80-181-909, Ralston Purina Co., Cincinnati, OH. NTIS PB82-25828 6, 1981, pp. 1-9. NIOSH. Health Hazard Evaluation Report, HETA 81-141-892, McKeesport Hospital, McKeesport, PA, June 1981. NTIS PB82-25845 0, pp. 1-10. Ogden, D. A., Myers, L. E., Eskelson, C. D., and Ziegler, E. J. latrogenic administration of formaldehyde to hemodialysis patients. Trans. Dialysis Transplant Forum, 141-146 (1973). NIOSH. Health Hazard Evaluation Report, HETA 81-084-917, Kutztown State College, Kutztown, PA, July 1981. NTIS PB83-10280 6. Oldham, J. D., Hart, I. C., and Bines, J. A. Formaldehyde-treated proteins for dairy cows-effects on blood hormone concentrations. Brit. J. Nutr. 48: 543-547 (1982). NIOSH. Health Hazard Evaluation Report, HETA 81-002-875, Atlanta Jewish Federation, Atlanta, GA. NTIS PB83-10444, May 1981. Ols0n, J. H., and D0ssing, M. Formaldehyde induced symptoms in day care centers. Am. Ind. Hyg. Assoc. J. 43: 366-370 (1982). NIOSH. Health Hazard Evaluation Report, HETA 81-098-941, Lab-Crest Scientific Glass Co., Subsidiary of Fischer & Porter Co., Warminster, PA, August 1981. NTIS PB83-12645 8, pp. 1-7. O'Quinn, S. E., and Kennedy, C. B. Contact dermatitis due to formaldehyde in clothing textiles. J. Am. Med. Assoc. 194: 123-126 (1965). CONSENSUS WORKSHOP ON FORMALDEHYDE 376 and n-butyraldehyde by ammonical silver nitrate. Current Sci. 52: 749-751 (1982). Orringer, E. P, and Mattern, W D. Formaldehyde-induced hemolysis during chronic hemodialysis. New Engl. J. Med. 294: 1416-1420 (1976). Prokopenko, Yu. I., Il'in, V P, and Zhurkov, V S. Effect of long-wave ultraviolet irradiation on mutagenic and embryotoxic effects of chemical substances in an experiment. Gig. Sanitar. 7: 12-15 (1981). OSHA. General Industry. OSHA Safety and Health Standards (29 CFR Part 1910). Occupational Safety and Health Administration, US Government Printing Office, Revised January 1976, pp. 504-505, 509. Protasova, I. A., Semin, Y. CONSENSUS WORKSHOP ON FORMALDEHYDE Chromatographic determination of dicyandiamide formaldehyde resin aerosol in the air of the workplace on the skin of workers and on work clothing (translation available). Gig. Truda Profess. Zabol. 12: 57-58 (1982). Rea, W J., Peters, D. W, Smiley, R. E., Edgar, R., Greenberg, M., and Fenyves, E., Recurrent environmentally triggered thrombo- phlebitis: a five year follow up. Ann. Allergy 47: 338-344 (1981). Petersen, N. J., Carson, L. A., Doto, I. L., Aguero, S. M., and Favero, M. S. Microbiologic evaluation of a new glutaraldehyde-based disinfectant for hemodialysis systems. Trans. Am. Soc. Artificial Internal Organs 28: 287-290 (1982). Richter, G. Testing formaldehyde and mercury (II) chloride in petrolatum. Dermatol. Monatsschr. 168: 49-50 (1982). Roffael, E., Miertzsch, H., and Menzel, W Nachtragliche Behandlung von Spanplatten zur Verminderung ihers Formaldehydabgabepoten- tials (Supplemental treatment of particleboards for the reduction of their potential for emitting formaldehyde). Adhasion 3: 18-23 (1982). Peto, R. Carcinogenic effects of chronic exposure to very low levels of toxic substances. Environ. Health Perspect. 22: 155-159 (1978). Pirila, V, and Kilpio, 0. On dermatitis caused by formaldehyde and its compounds. Ann. Med. Intern. Fenn. 38: 38-51 (1949). Rooke, J. A., Brookes, I. M., and Armstrong, D. G. The digestion of untreated and formaldehyde-treated soya-bean and rapeseed meals by cattle fed a basal silage diet. J. Agr. Sci. (Cambridge) 100:329-342 (1983). Pisati, G., Brini, D., and Cirla, A. H. Alergia alla formaleide in una fabbrica di resine sinthetiche (Formaldehyde allergy in a synthetic resin factory). Med. Lavoro 1: 88-91 (1980). Rosenkranz, H. S. Formaldehyde as a possible carcinogen. Bull. Environ. Contam. Toxicol. 8: 242-244 (1972). Place, A. R., Benyajati, C., and Sofer, W The molecular consequences of formaldehyde and ethyl methanesulfonate mutagenesis in Droso- phila: analysis of mutants in the alcohol dehydrogenase gene. In: Methods in Protein Sequence Analysis (M. Elzinga, Ed.), Humana Press, Clifton, NJ, 1982, pp. 373-379. Ross, W E., and Shipley, N. Relationship between DNA damage and survival in formaldehyde-treated mouse cells. Mutat. Res. 79: 277-283 (1980). Ross, W E., McMillan, D. R., and Ross, C. F. Comparison of DNA damage by methylmelamines and formaldehyde. J. Natl. Cancer Inst. 67: 217-221 (1981). Plesner, B. H., and Hansen, K. Formaldehyde and hexamethylene- tetramine in Styles' cell transformation assay. Carcinogenesis 4: 457-459 (1983). Rostenberg, A., Bairstow, B., and Luther, T. W A study ofeczematous sensitivity to formaldehyde. J. Invest. Dermatol. 19: 459-462 (1952). Plunkett, E. R., and Barbela, T. Summary Report... CONSENSUS WORKSHOP ON FORMALDEHYDE A., and Honchar, P Health effects associated with urea-formaldehyde foam insulation in Connec- ticut. J. Environ. Health 41: 270-272 (1979). Shiba, M., Marchok, A. C., and Klein-Szanto, A. J. P An open-ended rat tracheal implant model: toxic effects of formaldehyde on the respiratory epithelium. Toxicol. Letters 16: 241-248 (1983). Sargenti, A. Letter to the Editor: Potential hazards of formaldehyde. J. Am. Dent. Assoc. 104: 288 (1982). Shumilina, A. V Menstrual and reproductive functions in workers with occupational exposure to formaldehyde. Gig. Truda Profess. Zabol. 12: 18-21 (1975). Savolainen, H. Neurotoxicity ofindustrial chemicals and contaminants: aspects of biochemical mechanisms and effects. New toxicology for old. Arch. Tbxicol. 5: 71-83 (1982). Siegel, D. M., Frankos, V H., Schneiderman, M. A. Formaldehyde risk assessment for occupationally exposed workers. 3rd Annual Meeting of the American College of Toxicology, December 10, 1982, abstract #117, Washington, D. C. Savolainen, H. Dose-dependent effects of peroral dimethylformamide administration on rat brain. Acta Neuropathol. 53: 249-252 (1981). Savolainen, H., and Zitting, A. Glial cell effects of subacute formic acid vapour exposure. Acta Pharmacol. Toxicol. 47: 239-240 (1980). Sim, V M., and Pattle, R. E. Effect of possible smog irritants on human subjects. J. Am. Med. Assoc. 165: 1908-1913 (1957). Schoenberg, J. B., and Mitchell, C. A. airway disease caused by phenolic (phenol-formaldehyde) resin exposure. Arch. Environ. Health 30: 574-577 (1975). Singh, H. B., Salas, L. J., and Stiles, R. E. Distribution of selected gaseous organic mutagens and suspect carcinogens in ambient air. Environ. Sci. Technol. 16: 872-880 (1982). Schorr, W F Allergic skin reactions from cosmetic preservatives. American Perfumer Cosmetics 85: 39-47 (1970). Skiest, E. N. Technical efforts of the formaldehyde institute-an overview-14th Symposium on Particleboard, Washington State University, Pullman, Washington, 1980, pp. 137-144. Schreiber, H., Bibbo, M., Weid, G. L., Saccomanno, G., and Nettesheim, P Bronchial metaplasia as a benign or premalignant lesion. Acta Cytol. 23: 496-503 (1979). Skog, E. A toxicological investigation of lower aliphatic aldehydes. I. Toxicity of formaldehyde, acetaldehyde, propionaldehyde and butyral- dehyde; as well as of acrolein and crotonaldehyde. Acta Pharmacol. 6: 299-318 (1950). Schubert, H., and Agatha, G. Zur Allergennatur der para-tert. Butyl-phenolformaldehydharze (The allergenic nature of para-tert- butylphenolformaldehyde resins). Dermatosen 27: 49-52 (1979). Skvortsova, R. I., Poznyakoevskii, V M., and Rudakov, S. A. State of some metabolic functions in workers manufacturing phenol-formal- dehyde resins. Gig. Sanitar. 8: 69-71 (1980). Schuck, E. A., Stephens, E. R., and Middleton, J. T. CONSENSUS WORKSHOP ON FORMALDEHYDE 377 Sentein, P Action of glutaraldehyde and formaldehyde on segmen- tation mitoses. Exptl. Cell Res. 95: 233-246 (1975). 26-week inhalation toxicity study with formaldehyde in the monkey, rat and hamster (Abstract). The Toxicologist 3(1), abstract #291 (1983). Septon, J. C., and Ku, J. C. Workplace air sampling and polarographic determination of formaldehyde. Am. Ind. Hyg. Assoc. J. 43: 845-852 (1982). Rycroft, R. J. G. Contact sensitization to 2-monomethylolyphenol in phenol formaldehyde resin as an example of the recognition and prevention of industrial dermatoses. Clin. Exper. Dermatol. 7: 285-290 (1982). Shafaiziev, U. Yu., and Shipovskikh, G. P Working conditions of health of workers employed in processing of plastic resins in Uzbekistan (abstract). Referat. Zhur. Khim. Abstract 181420 (1972). Sakula, A. Correspondence: formalin asthma in hospital laboratory staff. Lancet ii: 816 (1975). Shaldon, S., Chevallet, M., Maraoui, M., and Mion, C. Dialysis associated auto-antibodies. Proc. Dialysis Transplant Assoc. 13: 339-346 (1976). Saladino, A. J., Willey, J. C., Lechner, J. F, and Harris, C. C. Altered human respiratory epithelial cell growth patterns induced by aldehydes and peroxides in vitro (abstract). Fed. Proc. 42: 513 (1983). Shapiro, J. L. Letters: Carcinogen policy at EPA. Science 219: 798 (1983). Salem, H., and Cullumbine, H. Inhalation toxicities of some aldehydes. Tbxicol. Appl. Pharmacol. 2: 183-187 (1960). Sheldrick, J. E., and Steadman, T. R. Final report on product/industry profile and related analysis for formaldehyde and formaldehyde- containing consumer products: Parts I, II, III. Consumer Product Safety Commission, CPSC-C-78-0091 Task 9, Subtask 9.01, revised February 5, 1979, Washington, D. C. Sandler, S. G., Sharon, R., Bush, M., Stroup, M., and Sabo, B. Formaldehyde-related antibodies in hemodialysis patients. Iransfusion 19: 682-687 (1979). Sankaranarayanan, K. Determination and evaluation of genetic risks to humans from exposure to chemicals. Progr. Mutat. Res. 3: 289-321 (1982). Shelley, W B. Immediate sunburn-like reaction in a patient with formaldehyde photosensitivity. Arch. Dermatol. 118: 117-118 (1982). Shellow, H., and Altman, A. T. Dermatitis from formaldehyde resin textiles. Arch. Dermatol. 94: 799-801 (1966). Sanotskii, I. V, Fomenko, V N., Sheveleva, G. A., Sal'nikova, L. S., Nakoryakova, M. V, and Pavlova, T. Y Study of the effect of pregnancy on the sensitivity of animals to chemical agents. Gig. TIruda Profess. Zabol. 1: 25-28 (1976). Sheveleva, G. A. Investigation of the specific effect of formaldehyde on the embryogenesis and progeny of white rats. Tbksikol. Novykh Prom. Kim. Veshch. 12: 78-86 (1971). Sardinas, A. V, Most, R. S., Guilietti, M. CONSENSUS WORKSHOP ON FORMALDEHYDE Are embalmer's at risk. Am. Ind. hyg. Assoc. J. 38: 61-62 (1977). Rudzki, E., and Schubert, H. Contact eczema incidence in the German Democratic Republic and the People's Republic of Poland-a comparison. II. Skin diseases from rubber and other occupational contactants. Dermatol. Monatsschr. 167: 665-671 (1981). Polakoff, P L. Odor pollution: don't deny what the nose knows (odors in the workplace can irritate, envigorate, and even warn us of impending danger). Occupational Health Safety 52: 26-28 (1983). Popa, V, Teculescu, D., Stanescu, D., and Gavrilescu, N. Bronchial asthma and asthmatic bronchitis determined by simple chemicals. Dis. Chest 56: 395-404 (1969). Ruempling, D. R., Morton, T. H., Jr., and Anderson, M. W Electrosurgical pulpotomy in primates-a comparison with formo- cresol pulpotomy. Pediatric Dent. 5: 14-18 (1983). Porter, J. A. H. Correspondence: acute respiratory distress following formalin inhalation. Lancet i: 603-604 (1975). Rumack, B. H. Position paper: urea-formaldehyde foam. Rocky Mountain Poison Center, Denver, Colorado, November, 1978. Poverenny, A. M., Siomin, Y A., Saenko, A. S., and Sinzinis, B. I. Possible mechanisms of lethal and mutagenic action of formaldehyde. Mutat. Res. 27: 123-126 (1975). Rusch, G. M., Clary, J. J., Rinehart, W E., and Bolte, H. F. a 26-week inhalation toxicity study with formaldehyde in the monkey, rat, and hamster. Toxicol. Appl. Pharmacol. 68: 329-343 (1983). Prasad, T. R., Ram, B. S., and Rao, T. N. Potentiometric study of the kinetics of oxidation of formaldehyde, acetaldehyde propionaldehyde Rusch, G. M., Clary, J. J., Rinehart, W E., and Bolte, H. F. A CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 378 Temeharoen, P, and Thilly, W G. Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium. Mutat. Res. 119: 89-93 (1983). Temeharoen, P, and Thilly, W G. Toxic and mutagenic effects of formaldehyde in Salmonella typhimurium. Mutat. Res. 119: 89-93 (1983). Snyder, R. D., and Smith, P D. Mutagen sensitivity of Drosophila melanogaster V Identification of second chromosomal mutagen sensitive strains. Molecular Gen. Genet. 188: 249-255 (1982). Sobels, F. H. Organic peroxides and mutagenic effects in Drosophila. Nature 177: 979-980 (1956). Tephly, T. R., Watkins, W D., and Goodman, J. I. The biochemical toxicology of methanol. In: Essays in Toxicology, Vol. 5. (W J. Hayes, Jr., Ed.), Academic Press, New York, 1974, pp. 149-177. Spassowski, M. Estimation of formaldehyde in the urine of workers as an indication of the hygiene of their working conditions (abstract). Bull. Hyg. (London) 40: 807-808 (1965). Thun, M. J., Lakat, M. F., and Altman, R. Symptom survey of residents of homes insulated with urea-formaldehyde foam. Environ. Res. 29: 320-334 (1982). Spellman, G. G. Formaldehyde poisoning successfully treated with hemodialysis. J. Iowa Med. Soc. 73: 175-176 (1983). Todhunter, J. A. Letters to the Editor: Carcinogen policy at EPA. Science 219: 794 (1983). Spengler, J. D., and Sexton, K. Indoor air pollution: a public health perspective. Science 221: 9-17 (1983). Todhunter, J. A. Memorandum: review of data available to the administrator concerning formaldehyde and di( 2-ethylhexyl) phthalate (DEHP). Memorandum to Anne M. Gorsuch, February, 1982, Office of Pesticides and Toxic Substances, Environmental Protection Agency, Washington, DC. Starr, T. B. Mechanisms of formaldehyde toxicity and risk evaluation. In: Formaldehyde: Ibxicology, Epidemiology, Mechanisms (J. J. Clary, J. E. Gibson and R. S. Waritz, Eds.), Marcel Dekker Inc., New York, 1983, pp. 237-258. Tou, J. C., and Kallos, G. J. Study of aqueous HCl and formaldehyde mixtures for formation of bis(chloromethyl) ether. Am. Ind. Hyg. Assoc. J. 35: 419-422 (1974). Storrs, E J., and Bell, D. E. allergic contact dermatitis to 2-bromo- 2-nitropropane-1,3-diol in a hydrophilic ointment. J. Am. Acad. Dermatol. 8: 157-170 (1983). Thain, R. (EPA). Health risk and Economic impact assessments of suspected carcinogens: interim procedures and guidelines FRL 546-2. Fed. Reg. 41: 21402-21406 (1976). Strittmatter, R, and Ball, E. G. Formaldehyde dehydrogenase, a glutathione-dependent enzyme system. J. Biol. Chem. 213: 455-461 (1955). Traynor, G. W, Allen, J. R., Apte, M. G., Dillworth, J. E, Girman, J. R., Hollowell, C. D., and Koonce, J. CONSENSUS WORKSHOP ON FORMALDEHYDE R., Jr. Indoor air pollution from portable kerosene-fired space heaters, wood-burning stoves, and wood-burning furnaces, LBL-14027, Lawrence Livermore Laboratory, Berkeley, California, NTIS DE82 013014, March, 1982. Stupfel, M. Recent advances in investigation of toxicity of automotive exhaust. Environ. Health Perspect. 17: 253-285 (1976). Sulaiman, S. T., and Amin, D. Indirect amplification method for determining formaldehyde and chloralhydrate by differential pulse polarography. Microchem. J. 28: 168-173 (1983). Irezl, L., Rusznak, I., Tihak, E., Szarvas, T., and Szende, B. Spontaneous N(E)-methylation and N(E) -formylation reactions between L-lysine and formaldehyde inhibited by L-ascorbic acid. Biochem. J. 214: 289-292 (1983). Sun, M. Study shows formaldehyde is carcinogenic. Science 213: 1232 (1981). Sun, M. OSHAs new thoughts on cancer policy. Science 217: 35 (1982). Sun, M. A firing over formaldehyde. Science 213: 630-631 (1981). Triebig, V G., Trautner, P, and Lutjen-Drecoll, E. Untersuchung zur Abschatzung einer Formaldehydeinwirkung im anatomischen Pra- pariersaal. Arbeitsmed. Sozialmed. Praventivmed. 15:264-266 (1980). Sun, M. Formaldehyde ban is overturned. Science 220: 699 (1983). Sundin, B. Formaldehyde emission from particleboard and other building materials: a study from the Scandinavian countries. Washing- ton State University, 12th Symposium on Particleboard, 12: 251-273 (1978). Tsuchiya, K., Hayashi, Y, Onodera, M., and Hasegawa, T. Toxicity of formadehyde in experimental animals - concentrations of the chemical in the elution from dishes of formadehyde resin in some vegetables. Keio Journal Med. 24: 19-37 (1975). Suskov, I. I., and Sazonova, L. A. Mutagenic effects of synthetic resins in man (abstract). Mutat. Res. 97: 224 (1982). Tuazon, E. C., Winer, A. M., Graham, R. A., and Pitts, J. N., Jr. Atmospheric measurements of trace pollutants by kilometer-path- length FT-IR spectroscopy. In: Advances in Envrionmental Science and Toxicology, Vol. 10 (J. N. Pitts, Jr., R. L. Metcalf, and D. Grosjean, Eds.), John Wiley & Sons, New York, 1980, pp. 259-300. Swarin, S. J., and Lipari, F Determination of formaldehyde and other aldehydes by high performance liquid chromatography with fluores- cence detection. J. Liquid Chromat. 6: 425-444 (1983). Swenberg, J. A. Formaldehyde induced carcinogenesis of the nasal cavity. Thirty-second Annual Meeting, American College ofVeterinary Pathologists, Pulmonary Pathology, November 10-13, 1981, Monterey, California. Turiel, I., Hollowell, C. D., Miksch, R. R., Rudy, J. V, and Young, R. A. The effects of reduced ventilation on indoor air quality in an office building. Atmos. Environ. 17: 51-64 (1983). Tuss, H., Neitzert, V, Seiler, W, and Neeb, R. CONSENSUS WORKSHOP ON FORMALDEHYDE Eye irritation response at low concentrations of irritants. Arch. Environ. Health 13: 570-575 (1966). Slizynska, H. Cytological analysis of formaldehyde induced chromo- somal changes in Drosophila melanogaster. Proc. Royal Soc. Edinburgh B66: 288-304 (1957). Schutte, W C., Cole, R. S., Long, K. R., and Frank, C. W Formaldehyde levels in homes insulated with urea-formaldehyde foam. Environ. Monitoring Assessment 1: 257-261 (1982). Smith, D. L., Bolyard, M., and Kennedy, E. R. Instability of formaldehyde air samples collected on a solid sorbent. Am. Ind. Hyg. Assoc. J. 44: 97-99 (1983). Sejersted, 0. M., Jacobsen, D., Ovrebo, S., and Jansen, H. Formate concentrations in plasma from patients poisoned with methanol. Acta Med. Scand. 213: 105-110 (1983). Smith, R. G., Bryan, R. J., Feldstein, M., Levadie, B., Miller, F A., Stephens, E. R., and White, N. G. Tentative method of analysis for formaldehyde content of the atmosphere (colorimetric method). Health Lab. Sci. 7 (Suppl.): 87-91 (1970). Sellakumar, A. R., Albert, R. E., Rusch, G. M., Katz, G. V, Nelson, N. and Kuschner, M. Inhalation carcinogenicity of formaldehyde and hydrogen chloride in rats (abstract). Proc. Am. Assoc. Cancer Res. 21: 106 (1980). Smyth, H. F., Seaton, J., and Fischer, L. The single dose toxicity of some glycols and derivatives. J. Ind. Hyg. Toxicol. 23: 259-268 (1941). Sellakumar, A. R., Laskin, S., Kuschner, M., Rusch, G., Katz, G. V, Snyder, C. A., and Albert, R. E. Inhalation carcinogenesis by dimethylcarbamoyl chloride in Syrian golden hamsters. J. Environ. Pathol. Toxicol. 4: 107-115 (1980). Sneddon, I. B. Letters to the Editor: dermatitis in an intermittent haemodialysis unit. Brit. Med. J. 1: 183-184 (1968). CONSENSUS WORKSHOP ON FORMALDEHYDE 379 Whittinghill, M., and Lewis, B. M. Clustered crossovers from male Drosophila raised on formaldehyde media. Genetics 46: 459-462 (1961). Usdin, V R., and Arnold, G. B. Final Report: Transfer of formal- dehyde to guinea pig skin. Gillette Research Institute (Contract No. 78-391), Rockville, Maryland (1979). Vainio, H. Inhalation anesthetics, anticancer drugs and sterilants as chemical hazards in hospitals. Scand. Work, Environ. Health 8: 94-107 (1982). Wiberg, G. S., and Baranowski, E. Health implications of urea- formaldehyde foam insulation. Can. J. Publ. Health 72: 335-338 (1981). Van Der Wal, J. F Formaldehyde measurements in Dutch houses, schools and offices in the years 1977-1980. Atmos. Environ. 16: 2471-2478 (1982). Willdns, R. J., and MacLeod, H. D. Formaldehyde induced DNA- protein crosslinks in Escherichia coli. Mutat. Res. 36: 11-16 (1976). Williams, R. T. The metabolism of some aliphatic aldehydes, ketones and acids: Aliphatic aldehydes. In: Detoxification Mechanisms: The Metabolism and Detoxication of Drugs, Toxic Substances and Other Organic Compounds, 2nd ed. (R. T. Williams, Ed.), John Wiley & Sons, New York, 1959, pp. 88-90. Van Duuren, B. L., Sivak, A., Goldschmidt, B. M., Katz, C., and Melchionne, S. Carcinogenicity of halo-ethers. J. Natl. Cancer Inst. 43: 481-486 (1969). Van Netten, C. Analysis of sources contributing to elevated formal- dehyde concentrations in the air in a new elementary school. Can. J. Publ. Health 74: 55-59 (1983). Williford, B. Letters to the Editor: Potential hazards offormaldehyde. J. Am. Dental Assoc. 104-288 (1982). Vicini, J. L., Clark, J. H., and Crooker, B. A. Effectiveness of acetic acid and formaldehyde for preventing protein degradation in the rumen. J. Dairy Sci. 66: 350-354 (1983). Willingmyre, G. T. Reuse of single-use hemodialyzers: technical, legal, medical and economic implications. Health Industry Manu- facturers Association Report, Washington, D. C., March 1979. Volkova, Z. A., and Sidorova, E. A. Formaldehyde content in the blood ofworkers in contact with urea-formaldehyde resins (translation available). Gig. Truda Profess. Zabol. 15: 44-46 (1971). Wills, J. H. Nasal carcinoma in woodworkers: a review. J. Occup. Med. 24: 526-530 (1982). Wong, 0. Epidemiologic studies of occupational cancers: approaches and data sources (abstract). Abstracts of Papers of the American Chemical Society, 182: abstract 24 (1981). Von Einbrodt, H. J., Prajsnar, D., and Erpenbeck, J. Der Formal- dehyd- und Ameisensaurespiegel im Blut und Urin beim Menschen nach Formaldehyde Exposition (The formaldehyde and formic acid levels in the blood and urine of man upon exposure to formaldehyde). Zentralbl. Arbeitsmed. CONSENSUS WORKSHOP ON FORMALDEHYDE Arbeitsschutz und Prophyl. 26: 154-158 (1976). Wood, R. W Determinants of irritant termination behavior. Toxicol. Appl. Pharmacol. 61: 260-268 (1981). Yager, J. W, Cohn, K., Spear, R., Fisher, J. M., and Morse, L. Exposure to formaldehyde in a gross anatomy laboratory. Internal Project Report, Northern California Occupational Health Center, School of Public Health, University of California, Berkeley, California, December, 1982. Walker, J. F. Formaldehyde. Kirk-Othmer Encyclopedia of Chemical Technology, 2nd ed., Vol. 10, John Wiley & Sons, New York, 1966, pp. 77-99. Walrath, J., and Fraumeni, J. F, Jr. Mortality patterns among embalmers. Intern. J. Cancer 31: 407-411 (1983). Yefremov, G. G. State of the upper respiratory tract in formaldehyde production workers (by the data of the a special investigation). Zhur. Ushnykh. Nos. Forl. Bolez. 30: 11-15 (1970). Watanabe, F., and Sugimoto, S. Studies on the carcinogenicity of aldehydes (Part II). Seven cases of transplantable sarcomas of rats developed in the area of repeated subcutaneous injections of urotropin (Hexamethylene tetramine). Gann 46: 365-367 (1955). Yodaiken, R. E. The uncertain consequences of formaldehyde toxicity. J. Am. Med. Assoc. 246: 1677-1678 (1981). York, M., Lawrence, R. S., and Gibson, G. B. An in vitro test for the assessment of eye irritancy in consumer products-preliminary findings. Intern. J. Cosmet. Sci. 4: 223-234 (1982). Watanabe, F, and Sugimoto, S. Study on the carcinogenicity of aldehyde (Report 3). Four cases of sarcomas in rats appearing in areas of repeated subcutaneous injection of acetaldehyde. Gann 47: 599-601 (1956). Young, E. Overgevoeligheid voor formaldehyde (Hypersensitivity to formaldehyde). Nederlands Tijdschr. Geneeskunde 126: 985-989 (1982). Watanabe, F, Matsunaga, T., Soejima, T., and Iwata, Y Study on aldehyde carcinogenicity. Communication I. Experimentally induced rat sarcomas by repeated injection of formalin. Gann 45: 451-452 (1954). Zapp, J. A., Jr. HMPA: a possible carcinogen. Science 190: 422 (1975). Zarubin, G. R, Dmitriev, M. T., and Mishchikhin, V A. Hygienic prediction of indoor air pollution by harmful substances released from polymeric materials. Gig. Sanitar. 4: 51-54 (1981). Waydhas, C., Weigl, K., and Sies, M. The disposition of formaldehyde and formate arising from drug N-demethylations dependent on cytochrome P-450 in hepatocytes and in perfused rat liver. Eur. J. Biochem. 89: 143-150 (1978). Zitting, A., and Savolainen, H. Biochemical effects of subacute formic acid vapor exposure. Res. Commun. Chem. Pathol. Pharmacol. 27: 157-162 (1980). Weber-Tschopp, A., Fischer, T., and Grandjean, E. Irritating effects of formaldehyde on men (Summary only translated). Intern. Arch. Occup. Environ. CONSENSUS WORKSHOP ON FORMALDEHYDE Health 39: 207-218 (1977). Zitting, A., Savolainen, H., and Nickels, J. Biochemical and toxicological effects of single and repeated exposures to polyacetal thermodegradation products. Environ. Res. 29: 287-296 (1982). Weber-Tschopp, A., Jermini, C., and Grandjean, E. Luftverunreini- gung und Belastigung durch Zigaretten Rauch (Air pollution and irritation due to cigarette smoke) (abstract). Sozial- Praeventivmed. 21: 101-106 (1976). CONSENSUS WORKSHOP ON FORMALDEHYDE Method for determination of formaldehyde in air in the PPTV-range by HPLC after extraction as 2,4-dinitrophenyl-hydrazone. Fresenius Z. Anal. Chem. 312: 613-617 (1982). Swenberg, J. A., Barrow, C. S., Boreiko, C. J., Heck, H. D'A., Levine, R. J., Morgan, K. T., and Starr, T. B. Nonlinear biological responses to formaldehyde and their implications for carcinogenic risk assessment. Carcinogenesis 4: 945-952 (1983). Uehara, M. Follicular contact dermatitis due to formaldehyde. Dermatologica 156: 48-54 (1978). Swenberg, J. A., Gross, E. A., Morgan, K. T., Chang, J. C. F., and Barrow, C. S. Effects of formaldehyde concentrations on deposition and cell proliferation (abstract). Proc. Am. Assoc. Cancer Res. 23: 55 (1982). Uehleke, H. Wo liegen Sicherheit und Gefahren von Formaldehyd? Umweltmedizin 3: 48-51 (1981). United States of America Standards Institute. USA standard acceptable concentrations of formaldehyde. United States of America Standards Institute, New York, New York, USAS Z37.16 (1967). Swenberg, J. A., Kerns, W D., Mitchell, R. I., Gralla, E. J., and Pavkov, K. L. Induction of squamous cell carcinomas of the rat nasal cavity by inhalation exposure to formaldehyde vapor. Cancer Res. 40: 3398-3402 (1980). United States Court of Appeals. Gulf South Insulation v. United States Consumer Product Safety Commission, C. P Chemical Company, Inc. v. Consumer Product Safety Commission, Public Citizen v. Consumer Product Safety Commission, The Formaldehyde Institute, Inc. v. United States Consumer Product Safety Commis- sion. Nos. 82-4218, 82-4136, 82-4311, 82-4135. United States Court of Appeals, Fifth Circuit, Apr: 3632-3645 (1983). Swenberg, J. A., Kerns, W, Pavkov, K., Mitchell, R., and Gralla, E. J. Carcinogenicity of formaldehyde vapor: interim findings in a long-term bioassay of rats and mice. In: Mechanisms of Toxicity and Hazard Evaluation (B. Holmstedt, R. Lauwerys, M. Mercier and M. Roberfroid, Eds.) Elsevier/North Holland, 1980, pp. 283-286. Szabad, J., Soos, I., Polgar, G., and Hejja, G. Testing the mutagenicity of malondialydehyde and formaldehyde by the Drosophila mosaic and the sex-linked recessive lethal tests. Mutat. Res. 113: 117-133 (1983). Uotila, L., and Koivusalo, M. Formaldehyde dehydrogenase from human liver. J. Biol. Chem. 249: 7653-7663 (1974). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 380 Jack H. Dean Department of Cell Biology Chemical Industry Institute of Toxicology P 0. Box 12137 Research Triangle Park, NC 27709 Frederick J. de Serres National Institute of Environmental Health Sciences A2-02, P 0. Box 12233 Research Triangle Park, NC 27709 Frederick J. de Serres National Institute of Environmental Health Sciences A2-02, P 0. Box 12233 Research Triangle Park, NC 27709 Joseph Arcos Existing Chemical Assessment Division Office of Ibxic Substances United State Environmental Protection Agency 401 M St., S. W Washington, DC 20460 John F. Gamble NIOSH/DRDS Epidemiology Investigation Branch 944 Chestnut Ridge Road Morgantown, WV 26505 Ruth E. Billings University of Texas Health Science Center Department of Pharmacology P 0. Box 20708 Houston, TX 77225 Richard Gammage Rm. S-256, Bldg. 45005 Oak Ridge National Laboratory P 0. Box X Oak Ridge, TN 37830 David Gaylor Division of Biometry National Center for Toxicological Research Jefferson, AR 72079 Craig Boreiko Chemical Industry Institute of Toxicology P 0. Box 12137 Research Triangle Park, NC 27709 James E. Gibson Chemical Industry Institute of Tbxicology P 0. Box 12137 Research Triangle Park, NC 27709 Sarah H. Broman Mental Retardation and Learning Disorders Section Developmental Neurology Branch NINCDS, CDNCP, NIH 7550 Wisconsin Ave., Rm. 8C-06 Bethesda, MD 20205 Leon Golberg Division of Occupational Medicine Box 2914 Duke University Medical Center Durham, NC 27710 Charles C. Brown National Cancer Institute Landow Building, Rm. 5C03 Bethesda, MD 20205 Richard Griesemer Biology Division P 0. Box 4, Bldg. 9207 Oak Ridge National Laboratory Oak Ridge, TN 37830 Frank W Carlborg 400 South Ninth Street Saint Charles, IL 60174 K. C. Gupta Consumer Products Safety Commission 5401 Westbard Avenue Bethesda, MD 20207 Jean Chen Shih Institute for Tbxicology University of Southern California John Stauffer Pharmaceutical Sciences Center 1985 Zonal Ave. Los Angeles, CA 90033 Henry d'A. Heck Chemical Industry Institute of Tbxicology P 0. Box 12137 Research Tiangle Park, NC 27709 g Murray S. Cohn Consumer Product Safety Commission 5401 Westbard Avenue Bethesda, MD 20207 Michael J. Colligan National Institute for Occupational Safety and Health 4676 Columbia Parkway Cincinnati, OH 45226 Thomas F. Coflins Bureau of Foods Food and Drug Administration FB8, 200 C St., S. W Washington, DC 20204 Peter F. Infante United States Department of Labor/OSHA Health Standards Program-Rm. N3718 200 Constitution Avenue, N. W Washington, DC 20210 Eugene R. Appendix II. List of Panelists Weinstein, I. B. Letters: Carcinogen policy at EPA. Science 219: 794-795 (1983). Weiss, W, Moser, R. L., and Auerbach, C. Lung cancer in chloromethyl ether workers. Am. Rev. Respir. Dis. 120: 1031-1037 (1979). E. A. Acheson MRC Environmental Health Unit Southhampton General Hospital Southhampton, S09 4XT, England Werner, J. Experiences in formaldehyde reduction from health aspects. Inst. Vatten-och Luftvardsforskning, Stockholm, Sweden, 1979. Yves Alaire Graduate School of Public Health University of Pittsburgh Pittsburgh, PA 15261 Yves Alaire Graduate School of Public Health University of Pittsburgh Pittsburgh, PA 15261 Wessel, M. R. The state of the science conference: a new approach to scientific decision making. Fundamentals Appl. Toxicol. 2: 283-288 (1982). CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE 381 Michael D. Lebowitz Department of Internal Medicine University of Arizona Health Sciences Center Division of Respiratory Sciences, Rm. 2332 Tucson, AZ 85724 Keith R. Long Institute of Agricultural Medicine and Environmental Healt University of Iowa Oakdale, IA 52319 Mary F Lyon MRC Radiobiology Unit Harwell Didcot Oxon OR1l ORD, England Howard Maibach University of California Medical School San Francisco, CA 94143 Thomas A. Marks Teratology and Reproduction Upjohn Co. 301 Henrietta St. Kalamazoo, MI 49001 J. Justin McCormick Michigan State University Carcinogenesis Lab, Fee Hall East Lansing, MI 48824 Edward A. Mortimer, Jr. Dept. of Epidemiology and Community Health Case Western Reserve University School of Medicine Cleveland, OH 44106 Paul Nettesheim Laboratory of Pulmonary Function and Toxicology National Institute of Environmental Health Sciences P 0. Box 12233 Research Triangle Park, NC 27709 Ian Nisbet Clement Associates, Inc. 1515 Wilson Blvd., Suite 700 Arlington, VA 22209 Godfrey P Oakley, Jr. Birth Defects Branch Chronic Disease Division Centers for Disease Control 1600 Clifton Rd., N. E. Atlanta, GA 30333 Roy Patterson Department of Medicine Northwestern University Medical School 303 E. Chicago Ave. Chicago, IL 60611 Jack Pepys 34 Ferncroft Ave. Hampstead London NW3 7PE, England Kenneth J. Rothman Epidemiology Department Harvard School of Public Health 677 Huntington Avenue Boston, MA 02115 Marc Schenker Occupational and Environmental Health Unit Department of Medicine, TB 136 University of California Davis Davis, CA 95616 Marvin A. Schneiderman 6503 Halbert Road Bethesda MD 20817 James A. Swenberg Chemical Industry Institute of Toxicology P 0. Box 12137 Research Tiangle Park, NC 27709 J. Justin McCormick Michigan State University Carcinogenesis Lab, Fee Hall East Lansing, MI 48824 Thomas R. Tephly The Toxicology Center Department of Pharmacology University of Iowa Iowa City, IA 52242 Edward A. Mortimer, Jr. Dept. of Epidemiology and Community Health Case Western Reserve University School of Medicine Cleveland, OH 44106 Edward A. Mortimer, Jr. Dept. of Epidemiology and Community Health Case Western Reserve University School of Medicine Cleveland, OH 44106 Benjamin F Trump Department of Pathology University of Maryland School of Medicine Baltimore, MD 21201 Paul Nettesheim Laboratory of Pulmonary Function and Toxicology National Institute of Environmental Health Sciences P 0. Box 12233 Research Triangle Park, NC 27709 Andrew G. Ulsamer Consumer Product Safety Commission 5401 Westbard Ave. Bethesda, MD 20207 Ian Nisbet Clement Associates, Inc. 1515 Wilson Blvd., Suite 700 Arlington, VA 22209 Arthur C. CONSENSUS WORKSHOP ON FORMALDEHYDE Kennedy National Institute for Occupational Safety and Health 4676 Columbia Parkway Cincinnati, OH 45226 Carole A. Kimmel Perinatal and Postnatal Evaluation Branch Division of Teratogenesis Research National Center for Tbxicological Research Jefferson, AR 72079 CONSENSUS WORKSHOP ON FORMALDEHYDE CONSENSUS WORKSHOP ON FORMALDEHYDE Upton Department of Environmental Medicine New York University Medical Center 550 First Ave. New York, NY 10016 Godfrey P Oakley, Jr. Birth Defects Branch Chronic Disease Division Centers for Disease Control 1600 Clifton Rd., N. E. Atlanta, GA 30333 Jerrold M. Ward National Cancer Institute Frederick Cancer Research Facility Building 538 Frederick, MD 21701 Roy Patterson Department of Medicine Northwestern University Medical School 303 E. Chicago Ave. Chicago, IL 60611 Jack Pepys 34 Ferncroft Ave. Hampstead London NW3 7PE, England Kenneth J. 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Export, PA 15632 Marc Schenker Occupational and Environmental Health Unit Department of Medicine, TB 136 University of California Davis Davis, CA 95616 Mike Wright United Steelworkers of America Safety and Health Department, Rm. 901 Five Gateway Center Pittsburgh, PA 15222 Mike Wright United Steelworkers of America Safety and Health Department, Rm. 901 Five Gateway Center Pittsburgh, PA 15222 Marvin A. Schneiderman 6503 Halbert Road Bethesda, MD 20817 Marvin A. Schneiderman 6503 Halbert Road Bethesda, MD 20817
https://openalex.org/W3132744465	https://www.nature.com/articles/s41598-021-83465-w.pdf	English	null	Insecticide resistance and underlying targets-site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan	Scientific reports	2,021	cc-by	13,937	Insecticide resistance and underlying targets‑site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan Rafi Ur Rahman1, Barbara Souza1, Iftikhar Uddin2, Luana Carrara1, Luiz Paulo Brito1, Monique Melo Costa1, Muhammad Asif Mahmood3, Sozaina Khan4, Jose Bento Pereira Lima1 & Ademir Jesus Martins1,5* Insecticide resistant Aedes populations have recently been reported in Pakistan, imposing a threat to their control. We aimed to evaluate the susceptibility of Aedes aegypti and Aedes albopictus populations from Lahore to WHO-recommended insecticides and to investigate metabolic and target-site resistance mechanisms. For this purpose, we first carried out bioassays with the larvicides temephos and pyriproxyfen, and the adulticides malathion, permethrin, deltamethrin, alpha- cypermethrin, and etofenprox. We looked for Knockdown resistance mutations (kdr) by qPCR, High-Resolution Melt (HRM), and sequencing. In order to explore the role of detoxifying enzymes in resistance, we carried out synergist bioassay with both species and then checked the expression of CYP9M6, CYP9J10, CYP9J28, CYP6BB2, CCAe3a, and SAP2 genes in Ae. aegypti. Both species were susceptible to organophosphates and the insect growth regulator, however resistant to all pyrethroids. We are reporting the kdr haplotypes 1520Ile + 1534Cys and T1520 + 1534Cys in high frequencies in Ae. aegypti while Ae. albopictus only exhibited the alteration L882M. PBO increased the sensitivity to permethrin in Ae. aegypti, suggesting the participation of P450 genes in conferring resistance, and indeed, CYP928 was highly expressed. We presume that dengue vectors in Lahore city are resistant to pyrethroids, probably due to multiple mechanisms, such as kdr mutations and P450 overexpression. Lahore, the second-largest Pakistani city, also known as Pakistan’s cultural capital, has faced several outbreaks of dengue in the last two decades. The biggest of which was in 2011 when more than 20,000 people were hos- pitalized and 350 died1. Dengue virus has four serotypes, and all four have been reported in Lahore2. However, serotype 2 (genotype IV) was more prevalent and was responsible for high mortality and morbidity in Lahore3. The most recent outbreaks in the country took place in 2019 when more than 47,000 patients were admitted to hospitals (up to November) due to mild dengue fever, severe hemorrhagic fever, or shock. Lahore was once again the hotspot of the infection4. g p Dengue virus is transmitted to humans by the bite of a female infected Aedes aegypti Linnaeus, 1762 and Aedes albopictus Skuse, 1895 (Diptera: Culicidae). The recent spread of the vector population and global envi- ronmental conditions, such as the average temperature increase, has promoted the disease’s transmission beyond the tropics5,6. In the absence of a preventive vaccine and potent treatment, effective vector management remains the best option to keep dengue under control7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ The first four segments make the voltage-sensing domain (S1–S4), while the last two segments (S5 and S6) along with their respective P-loops assemble in such a way to form the pore, which is responsible for the action potential of ions, particularly Na+. Upon binding, PY and OC keep this pore or channel open for more extended periods resulting in fast contractions, paralysis, and eventual death, known as the knockdown effect17–19. Pyrethroids are either type I or type II based on their chemical structure. Type I (for example, permethrin) lack an alpha-cyano group, while type II (for example, deltamethrin and cypermethrin) have an alpha-cyano group close to phenyl benzyl alcohol moiety. On the other hand, organophosphates (OPs) and carbamates (CAs) target the acetylcholinesterase (AChE, EC 3.1.1.7) coded by the ace-1 gene18. Insect Growth Regulators (IGRs) mimic the growth hormones, not directly killing the insect but affecting their growth, development, physiology, and behavior, ultimately provoking the insect death20. f g g y gy y g Continuous application of insecticides results in the selection of resistant insects. Resistance mechanisms in arthropod vectors are provoked by behavioral or physiological changes. Physiological responses in the form of target-site modifications alone can be sufficient to confer pyrethroids resistance in Aedes. One or more point mutations can achieve resistance to pyrethroids’ knockdown effect (kdr) in the voltage-gated sodium channel gene (Nav). About 11 kdr mutations alone or in combinations are classically associated with PY and DDT resist- ance in Aedes. Of them, Val410Leu (IS6 segment), Ser989Pro (IIS6 segment), Val1016Ile/Gly (IIS6 segment), and Phe1534Cys (IIIS6 segment) are well studied21. A more recently described mutation, Thr1520Ile (IIIS6 segment), is also associated with PY resistance in Ae. aegypti populations from India22 and Thailand23. The 1534Cys is the only kdr mutation widespread on a global scale. On the other hand, mutation at the 1016 site varies geographically, as Val1016Gly is predominant in populations from Asia while Val1016Ile occurs in Latin American23. Surprisingly, Val1016Ile has also been detected in Vietnam as well24, and Val1016Gly in Panama25. Of other notable mutations, Val410Ile is restricted to Latin America and Ser989Pro to Asia, but they alone do not cause resistance21. The 1534C alone can confer resistance to PYs, and this resistance can reach higher levels when combined with other mutations like 989P, 1016Ile/Gly, or 1520I26. www.nature.com/scientificreports/ was detected in patients from Karachi and Lahore9,10. So far, there is no evidence of Zika and yellow fever trans- mission in Pakistan. Since the beginning of the dengue outbreaks in Lahore, neurotoxic insecticides like pyrethroids (PY), mainly permethrin and deltamethrin, have been used as adulticides, while the organophosphate (OP) temephos as a larvicide. When a case of dengue is reported, comprehensive vector surveillance and health education activities are conducted in all houses at a radius of approximately 200 m from the index house. Potential breeding sites are searched and mechanically eliminated, temephos is applied in containers that cannot be managed physically. Alpha-cypermethrin (10% SC) is applied as a residual spray on the expected vector’s resting sites. All houses or industrial areas present in a 50 m radius are also sprayed with this pyrethroid. Deltamethrin was used for vector control before the introduction of alpha-cypermethrin. In addition to case response, larvicide temephos is used during routine vector surveillance in addition to the mechanical elimination of potential vector breeding sites in the Punjab province. The option of space spray is used during outbreaks only under the advice of a technical committee. All vector control practices, including space spraying and larvicides applications, are applied by the public sector. Community is well educated to eliminate possible breeding places from their houses and workplaces mechanically. Legal action is taken against the occupants of the premises in non-compliance with the instruc- tions regarding eliminating mosquito breeding sites in their premises11. Resistance against pyrethroids in Aedes has been reported from various parts of Punjab12 and Khyber Pakhtunkhwa provinces13. Similarly, in the Indian capital city of New Delhi, which is only 425 km from Lahore, pyrethroid-resistant Ae. aegypti are prevalent14. Resistance to temephos in Aedes has also been shown in several districts of Punjab province, including Lahore and its neighbor districts15. On the other side, Insect Growth Regulators (IGRs) were effective against Aedes in the area, at least until 200816. Neurotoxic insecticides interfere with the central nervous system of insects. Pyrethroids (PYs) and the organochlorines (OCs) target the voltage-sensitive sodium channel (NaV, also commonly referred to as Vssc or Vgsc). This protein comprises four homologous domains (I-IV), each containing six transmembrane subunits of α-helix (S1–S6) and a P-loop connecting S5 and S6 segments. www.nature.com/scientificreports/ Contrary to pyrethroids, target site modification in OP resistance, Gly119Ser (G119S) in the ace-1 gene of other mosquito species is not likely to occur in the genus Aedes due to codon constraints7,27. g Metabolic resistance mechanisms are also very well investigated in Ae. aegypti, aiding in xenobiotic detoxi- fication through enhanced expression or gene alterations28. Enzymes that detoxify neurotoxins are mainly from the cytochrome P450 dependent multi-function oxidases (CYP450 MFOs), esterases (ESTs), or glutathione-S- transferases (GSTs) families28. Around 160 MFOs29 have been reported to have altered expression in the resistant Aedes population, of which CYP9J22, CYP9J26, CYP9J28, CYP9M6, CYP9M9, CYP6BB2, and CYP9J10 have been strongly associated with PY resistance. The esterase gene CCEAe3a is generally overexpressed in OP resist- ant populations7,30. A recent observation was the overexpression of a sensory appendage protein (SAP2) in PY resistant Anopheles gambiae population from Burkina Faso31.i p g p p In this study, we have evaluated the susceptibility/resistance profile of Ae. aegypti and Ae. albopictus popula- tions from Lahore to the insecticides in practice and the alternative compounds IGR pyriproxyfen. We inves- tigated the main molecular mechanisms involved in insecticide resistance in Aedes populations from Lahore for the first time. These results will enrich the knowledge about insecticide resistance in Pakistani Aedes species and contribute to vector control monitoring actions that would ultimately reduce Pakistan’s arboviruses burden. Insecticide resistance and underlying targets‑site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan Rafi Ur Rahman1, Barbara Souza1, Iftikhar Uddin2, Luana Carrara1, Luiz Paulo Brito1, Monique Melo Costa1, Muhammad Asif Mahmood3, Sozaina Khan4, Jose Bento Pereira Lima1 & Ademir Jesus Martins1,5* Both Aedes species are abundant in Pakistan, with Ae. albopictus found at high altitude and in peri-urban areas and Ae. aegypti most abundant in urban settlements. In Lahore, both mosquito species are found, but Ae. aegypti is relatively more abundant8. Apart from dengue, chikungunya 1Laboratório de Fisiologia E Controle de Artrópodes Vetores, Instituto Oswaldo Cruz/FIOCRUZ, Av. Brasil 4365, Rio de Janeiro, RJ 21040‑360, Brazil. 2Community Medicine Department, Bacha Khan Medical College Mardan, Mardan, Pakistan. 3Institute of Public Health, Lahore, Pakistan. 4Zoology Department, Government College University, Lahore, Pakistan. 5Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, INCT-EM, UFRJ, Rio de Janeiro, RJ 21941‑902, Brazil. *email: ademirjr@ioc.fiocruz.br \| https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 www.nature.com/scientificreports/ Results Bi Susceptibility profile to the larvicide organophosphate temephos in Aedes aegypti and Aedes albopictus from Lahore, Pakistan. a Resistance ratios (RR) were calculated based on Ae. aegypti Rockefeller. (PAb) populations were 0.006 mg/L and 0.008 mg/L, with respective RR50 as 1.50 and 2.04, employing Ae. aegypti Rock as a baseline reference. Accordingly, LC90 were 0.011 and 0.014, with RR90 of 2.0 and 2.3 for PAg and PAb, respectively (Table 1). (PAb) populations were 0.006 mg/L and 0.008 mg/L, with respective RR50 as 1.50 and 2.04, employing Ae. aegypti Rock as a baseline reference. Accordingly, LC90 were 0.011 and 0.014, with RR90 of 2.0 and 2.3 for PAg and PAb, respectively (Table 1). Pyriproxyfen. We performed a qualitative bioassay (diagnostic dose assay) with the insect growth regulator (IGR) pyriproxyfen using a diagnostic dose of 0.3 µg/L. There was no mortality in the control condition, which generally lasted for 7 to 10 days until all pupae emerged into adults, indicating completion of tests. At this point, mortality ranged from 99.4% to 100% in the experimental condition, and the higher proportions of mortality occurred in the pupal stage. Rock exhibited the highest proportion of mortality in the larval stage (32.2%), com- pared to PAg (9.03%) and PAb (24.8%). Pyriproxyfen induced 100% of Adult Emergence Inhibition (AEI) in the reference lineage and in both PAg and PAb populations (Fig. 1b). Pyrethroids (rapid knockdown assay). For Rock, PAg and PAb, there was 100% knockdown in 0.1 ppm and 0.4 ppm for permethrin and deltamethrin. In total, we exposed 60 larvae of each strain to both pyrethroids. The susceptibility index (SI) average for deltamethrin was 2.7 in Rock, 3.0 in PAb and 3.7 in PAg, and for perme- thrin, it was 4.0 in Rock, 3.7 in PAb, and 5.3 in PAg (Fig. 1c). Although this data roughly suggested a higher SI to PAg for both pyrethroids, the difference among the strains was not significant, according to the ANOVA test: deltamethrin (P = 0.4640) and permethrin (P = 0.6892). Bioassays with adulticides. Malathion. Both PAg and PAb exhibited 100% mortality showing suscepti- bility to the organophosphate malathion (20 μg/mL), similar to Rock. Pyrethroids. Both species tested were resistant to the WHO diagnostic doses to the four pyrethroids, with the magnitude of resistance varying between species for different insecticides. As a whole, mortality rates were lower for permethrin and etofenprox compared to deltamethrin and alpha-cypermethrin (Fig. 2). Results Bi Bioassays with larvicides. Temephos. Dose–response assays determined the lethal concentrations (LCs) and resistant ratios (RRs) in Aedes aegypti (PAg) and Aedes albopictus (PAb) populations from Lahore, Pakistan, with serial dilutions from 0.001 to 0.018 mg/L (Fig. 1a). The LC50 for Aedes aegypti (PAg) and Aedes albopictus https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Larvicide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. (a) Dose– response bioassay to the organophosphate temephos, with mortality evaluated after 24 h of exposure. (b) Proportional mortality of each life stage to the diagnostic dose of the IGR pyriproxyfen. The adult emergence inhibition (AEI) was 100% in all experimental cases. (c) Susceptibility index (SI) average (with SEM error bars) for the pyrethroids permethrin and deltamethrin. The higher the SI, the less susceptible the population. Ae. aegypti Rockefeller strain (Rock) was used as a reference in all these larvicide bioassays. Figure 1. Larvicide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. (a) Dose–t Figure 1. Larvicide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. (a) Dose– response bioassay to the organophosphate temephos, with mortality evaluated after 24 h of exposure. (b) Proportional mortality of each life stage to the diagnostic dose of the IGR pyriproxyfen. The adult emergence inhibition (AEI) was 100% in all experimental cases. (c) Susceptibility index (SI) average (with SEM error bars) for the pyrethroids permethrin and deltamethrin. The higher the SI, the less susceptible the population. Ae. aegypti Rockefeller strain (Rock) was used as a reference in all these larvicide bioassays. Population LC50 (CI95%) LC90 (CI95%) RR50 a RR90 a Slope Ae. aegypti (PAg) 0.006 (0.004–0.009) 0.011 (0.007–0.016) 1.5 1.8 5.1 Ae. Albopictus (PAb) 0.008 (0.003–0.018) 0.014 (0.005–0.039) 2.0 2.3 4.9 Ae. aegypti (Rock) 0.004 (0.003–0.006) 0.006 (0.005–0.008) – – Table 1. Susceptibility profile to the larvicide organophosphate temephos in Aedes aegypti and Aedes albopictus from Lahore, Pakistan. a Resistance ratios (RR) were calculated based on Ae. aegypti Rockefeller. Population LC50 (CI95%) LC90 (CI95%) RR50 a RR90 a Slope Ae. aegypti (PAg) 0.006 (0.004–0.009) 0.011 (0.007–0.016) 1.5 1.8 5.1 Ae. Albopictus (PAb) 0.008 (0.003–0.018) 0.014 (0.005–0.039) 2.0 2.3 4.9 Ae. aegypti (Rock) 0.004 (0.003–0.006) 0.006 (0.005–0.008) – – Table 1. Susceptibility profile to the larvicide organophosphate temephos in Aedes aegypti and Aedes albopictus from Lahore, Pakistan. a Resistance ratios (RR) were calculated based on Ae. aegypti Rockefeller. Table 1. Results Bi Among pyrethroids, deltamethrin 0.03% caused the maximum mortality rates in both PAg (85.04%) and PAb (78.85%), while we observed minimum mortality rates with permethrin 0.25% for PAg (8.04%) and PAb (4.52%). In all experiments, control tubes showed no mortality, while Rock was 100% susceptible to all insecticides.h y p The pyrethroid with the longest estimated time for 50% of the exposed mosquitoes (KdT50) to be knocked down was permethrin in PAg (145 min) and PAb (289 min), and the shortest KdT50 was induced by deltamethrin in PAg (40.4 min) and PAb (46.8 min). Resistance ratios based on knockdown timings (KdRR50) for the pyre- throids were higher for permethrin in PAb (8.4) and lower for deltamethrin in PAg (1.8) (Table 2). Mechanisms of resistance. Synergistic assay. We tested if the synergist PBO would increase mortality to the pyrethroid permethrin to evaluate the possibility of metabolic resistance mechanisms involvement in permethrin resistance. Permethrin (0.25%) caused 22.3% mortality in PAg and 19.8% in PAb, in this assay, while https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ tificreports/ Figure 2. Adulticide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. PAg and PAb designate Ae. aegypti and Ae. albopictus populations, respectively. Dose-diagnostic tests with the pyrethroids permethrin (0.25%), etofenprox (0.5%), alpha-cypermethrin (0.03%) and deltamethrin (0.03%) in WHO test tubes, and the organophosphate malathion (20 µg) in bottle assays. Bars and errors indicate the mean mortality ± SEM. Populations below 90% of mortality (indicated by the red dotted line) are resistant. Aedes aegypti Rockefeller reached 100% mortality in all assays run in parallel. Figure 2. Adulticide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. PAg and PAb designate Ae. aegypti and Ae. albopictus populations, respectively. Dose-diagnostic tests with the pyrethroids permethrin (0.25%), etofenprox (0.5%), alpha-cypermethrin (0.03%) and deltamethrin (0.03%) in WHO test tubes, and the organophosphate malathion (20 µg) in bottle assays. Bars and errors indicate the mean mortality ± SEM. Populations below 90% of mortality (indicated by the red dotted line) are resistant. Aedes aegypti Rockefeller reached 100% mortality in all assays run in parallel. Figure 2. Adulticide bioassays with Aedes aegypti and Aedes albopictus from Lahore, Pakistan. PAg and PAb designate Ae. aegypti and Ae. albopictus populations, respectively. Dose-diagnostic tests with the pyrethroids permethrin (0.25%), etofenprox (0.5%), alpha-cypermethrin (0.03%) and deltamethrin (0.03%) in WHO test tubes, and the organophosphate malathion (20 µg) in bottle assays. Results Bi Bars and errors indicate the mean mortality ± SEM. Populations below 90% of mortality (indicated by the red dotted line) are resistant. Aedes aegypti Rockefeller reached 100% mortality in all assays run in parallel. Table 2. Susceptibility profile to pyrethroid adulticides in Aedes aegypti and Aedes albopictus from Lahore, Pakistan. a Resistance ratios (RR) were calculated based on Ae. aegypti Rockefeller strain (Rock). Pyrethroids Knockdown time PAg PAb Rock Permethrin KdT50 (CI95%) 145.0 (119.0–175.4) 289.9 (189.7–443.2) 34.4 (28.5–41.4) KdTRR50 a 4.2 8.4 – KdT90 (CI95%) 336.9 (235.0–483.0) 1075.7 (506.2–2283.3) 45.2 (37.5–54.6) KdTRR90 a 7.5 23.8 – Etofenprox KdT50 (CI95%) 120 (93.1–155.4) 246.6 (150.2–405.0) 36.8 (31.9–42.4) KdTRR50 a 3.3 6.7 – KdT90 (CI95%) 264.5 (160.8–434.9) 607.4 (262.8–1403.9) 48.7 (40.7–58.3) KdTRR90 a 5.4 2.3 – Alfa-cypermethrin KdT50 (CI95%) 56.1 (46.3–67.8) 61.0 (49.6–74.8) 21 (15.7–28.5) KdTRR50 a 2.7 2.9 – KdT90 (CI95%) 101.6 (79.7–129.6) 107.5 (83.0–139.3) 30 (23.5–38.4) KdTRR90 a 3.4 3.6 – Deltamethrin KdT50 (CI95%) 40.4 (36.6–45.6) 46.8 (38.6–56.7) 22.3 (17.8–27.9) KdTRR50 a 1.8 2.1 – KdT90 (CI95%) 63.9 (56.6–72.2) 71.2 (59.0–86.1) 29.1 (22.9–37.0) KdTRR90 a 2.2 2.4 – Table 2. Susceptibility profile to pyrethroid adulticides in Aedes aegypti and Aedes albopictus from Lahore, Pakistan. a Resistance ratios (RR) were calculated based on Ae. aegypti Rockefeller strain (Rock). when pre-exposed to 4% PBO, the mortality increased to 100% and 50% in PAg and PAb, respectively. There was no mortality in the control and PBO-only conditions. when pre-exposed to 4% PBO, the mortality increased to 100% and 50% in PAg and PAb, respectively. There was no mortality in the control and PBO-only conditions. Expression analyses. We assessed the levels of expression of five genes related to the metabolism of neurotoxic insecticides (CYP9M6, CYP9J10, CYP9J28, CYP6BB2, and CCEAe3a) and a gene of sensory appendage protein (SAP2) in the whole-body of PAg adult females, in comparison to the housekeeping gene Rps14. Comparing with the profile exhibited by Rock, the only gene with a significant relative fold change expression was the MFO P450 gene CYP9J28, 12.7 × more expressed in PAg (Supplementary Table S1). The SAP2 was nearly four- fold underexpressed in PAg than Rock. The fold change expression of these genes is found in Fig. 3, and more detailed data in Supplementary Table S1. Kdr genotyping and sequencing. Aedes aegypti. We genotyped 45 PAg individuals for four kdr mutations: V410L (IS6 segment), S989P and V1016G (IIS6 segment), and F1534C (IIIS6 segment) with TaqMan SNP assay. Results Bi https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ tificreports/ Figure 3. Fold-change expression of genes related to metabolic resistance of insecticides in Aedes aegypti from Lahore. The gene Rps14 was used as a normalizing reference gene, and fold-change was relative to the Rockefeller strain. Means with standard deviations are represented. Figure 3. Fold-change expression of genes related to metabolic resistance of insecticides in Aedes aegypti from Lahore. The gene Rps14 was used as a normalizing reference gene, and fold-change was relative to the Rockefeller strain. Means with standard deviations are represented. All samples were similar to a wild type for the three SNPs in the IS6 and IIS6 NaV segments (V410, S989 and V1016). HRM analyses for the IIS6 segments were run for the exons 20 and 21 in independent reactions and indicated monomorphic sequences in exon 20 (N = 40 samples) and two variants in the exon 21 (N = 44 samples), though the distinct variant contained only one sample (#PAg_22). Sequencing of the exons 20 to 21 with the intron in between revealed two haplotypes: PAg-2s6_1 and PAg-2s6_2 (GenBank accession numbers MT707209 and MT707210, respectively). The polymorphisms were found only in the intron and justified the distinct vari- ant observed in the HRM analysis for the #Pag_22 sample (Supplementary Figure S2). Sequences spanning exons 20–21 of the NaV gene in Ae. aegypti are phylogenetically divided into two groups, clades A or B23. In this context, both PAg haplotypes belonged to the clade B, as revealed when aligned with homologous sequences available on the GenBank (NCBI). PAg-2s6_1 was similar to a haplotype observed in Ae. aegypti populations from the Americas, Africa, Asia, and Australia (MN602762). PAg-2s6_2 was similar to haplotypes obtained in samples from Brazil, Australia and Kenya (MN602775) and Vietnam (MG257775, LC036556). This haplotype is also similar to the sequence of the reference lab strain LVP (MK977832) originally collected in Sierra Leone in the 1930s (Supplementary Figure S2). pp y g For the IIIS6 segment, however, 44 samples were kdr homozygous (1534 C/C), and one sample (that same #PAg_22) was heterozygote (1534 F/C), as obtained by TaqMan SNP assay for the F1534C SNP. In addition to this TaqMan assay, an HRM analysis in part of the exon 31 for this segment determined three variants (Fig. 4a), which when sequenced (see below), indicated variation in the 1520 (T1520I) in addition to the 1534 (F1534C) site. Discussion Th f There are few insecticide resistance studies in Pakistani Aedes spp12,13,15,16,33–40, in a scenario where vector control on its own has been one of the neglected aspects of arthropod-borne infections. There were interesting projects about IR status and mechanisms selected in Anopheles mosquitoes during the late 1970s and early 1980s39,41–43. Nevertheless, the studies on this particular area were discontinued due to its geopolitical situation and other factors. Lahore, the second-most populous city in Pakistan, has faced various dengue outbreaks in the last dec- ade. Before dengue vector control, several chemicals from pyrethroid, organophosphates, and organochlorine classes were employed in malaria vector control programs. They included deltamethrin, permethrin, malathion, DDT, among others. Here we evaluated the insecticide resistance status of Ae. aegypti and Ae. albopictus from Lahore to the larvicides temephos (organophosphate) and pyriproxyfen (IGR), as well to the adulticides per- methrin, etofenprox, alpha-cypermethrin and deltamethrin (pyrethroids), and malathion (organophosphate). We evidenced that these species were still susceptible to both organophosphates and that the IGR is a suitable additional compound to be used in the region. On the other hand, Ae. aegypti and Ae. albopictus were resistant to all types of pyrethroids here evaluated. In addition to kdr mutations, an overexpressed P450 cyp gene might be playing a role in resistance to pyrethroids. p y g py Temephos has been employed in Punjab, Pakistan, against Aedes spp. since 2011–2012, and resistance to this larvicide was detected in Ae. aegypti from several cities of that district, including Lahore, collected in 201615. Surprisingly, the LC50 of temephos in Ae. aegypti from this same city was around 13X lower in our study than that samples collected 2 years earlier (0.0815 against 0.006 µg/mL). A decrease in temephos resistance was indeed observed in laboratory lines maintained in the absence of selection pressure as well as in natural populations, some years later without the employment of temephos44 (Rahman et al. 45), likely due to a substantial fitness cost associated with physiological changes selected for resistance46. However, this decrease can be relatively slight and requires several generations or years in the field without selection pressure, and to our knowledge, temephos is still applied in Lahore. Further studies with new collections are necessary in order to understand this phenom- enon better. In the case of Ae. albopictus from Lahore, there were records of resistance to the organophosphates larvicides chlorpyrifos, profenofos, and triazophos47, while in our results Ae. Results Bi The genotypes were then determined as 1520 T/T + 1534 C/C (kdr homozygous at 1534) in 18.2% samples, 1520 I/I + 1534 C/C (kdr homozygous in both sites) in 27.3% samples, and 1520 T/I + 1534 C/C or 1520 T/T + 1534 F/C (heterozygous at 1520 or 1534 sites) in 54.5% samples (Fig. 4b). The exon 31 sequencing of the aforementioned #PAg_22 indicated its genotype as 1520 T/T + 1534 F/C. Summing up all of this infor- mation, the allelic frequencies in PAg were: 53.4% (IC), 45.5% (TC) and 1.1% (TF) (Fig. 4c). Three haplotypes were identified in the IIIS6 segment out of 13 sequenced samples (Supplementary Figure S2): the wild-type (PAg_3s6-1) without any nonsynonymous substitution, and two kdr haplotypes: one with the 1534C (PAg_3s6-2) and the other with both 1520I and 1534C kdr mutations (PAg_3s6-3). The PAg_3s6-1 haplotype was observed only under heterozygosis (overlapped peaks in the electropherogram) with PAg_3s6-2, confirming the #PAg_22 genotype (1520 T/T + 1534 F/C). We aligned these haplotypes with 69 other homologous sequences available on the NCBI GenBank and found that PAg_3s6-1 was similar to the wild-type haplotype described in Ae. aegypti populations from all continents. Likewise, PAg_3s6-2 was similar to the 1534C kdr sequences observed worldwide. PAg_3s6-3, with the double mutation 1520I + 1534C, was similar to sequences from Thai and Indian populations (Supplementary Figure S2). Aedes albopictus. HRM analyses for each of the exons 20 and 21 showed two variants in PAb (N = 45 samples) in both reactions. When sequenced, four SNPs were found in the exon 20, indicating five haplotypes (Supple- mentary Figure S2). These observed SNPs were three nonsynonymous substitutions and the mutation L882M. A Blast search with the exon 20 of these five haplotypes matched with identical sequences of Ae. albopictus popula- tions from Brazil and China (Supplementary Figure S2). The L882M substitution was also present in a sample from India (MF776970)32, although in a haplotype distinct to the Pakistani herein observed. g y Concerning the exon 31, we obtained a 257 bp fragment from 13 samples, in which a total of six SNPs were identified, all synonymous substitutions. The sequences of eight haplotypes are available in (Supplementary Figure S2) (GenBank accession numbers: MT740758-MT740765). The haplotypes Pab_3s6-1 and Pab_3s6-2 were identical to sequences from Ae. albopictus from China and Malaysia, available on NCBI GenBank (Sup- plementary Figure S2). https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ Figure 4. Results Bi Kdr genotyping in the NaV IIIS6 segment in Aedes aegypti from Lahore, Pakistan. (a) The high- resolution melting analyses (HRM) difference plot, where each line represents a sample, is grouped into three variants. Sequencing of some samples of each variant, summed with TaqMan genotyping of the F1534C SNP indicated their genotypes, which then rendered the genotypic (b) and allelic frequencies (c), considering the 1520 and 1534 sites. Figure 4. Kdr genotyping in the NaV IIIS6 segment in Aedes aegypti from Lahore, Pakistan. (a) The high- resolution melting analyses (HRM) difference plot, where each line represents a sample, is grouped into three variants. Sequencing of some samples of each variant, summed with TaqMan genotyping of the F1534C SNP indicated their genotypes, which then rendered the genotypic (b) and allelic frequencies (c), considering the 1520 and 1534 sites. https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| https://doi.org/10.1038/s41598-021-83465-w www.nature.com/scientificreports/ we did not evidence resistance to pyrethroid in Pakistani Ae. aegypti and Ae. albopictus larvae. However, both species were resistant to all tested pyrethroid adulticides. Levels of mortality were under 20% in adult bioassays with the pyrethroid type I permethrin, though around 80% to the type II pyrethroid deltamethrin. Resistance to permethrin and deltamethrin was previously recorded in Ae. aegypti from Lahore, collected in 201035. In 2015, both Ae. aegypti and Ae. albopictus from four towns in the Lahore district were found to be resistant to permethrin, lambda-cyhalothrin, and deltamethrin, in addition to DDT36.i Deltamethrin and permethrin resistant Ae. aegypti populations from Lahore showed a significant increase in mortality after being treated with synergist PBO15. Our study corroborated those findings by observing that a pre-exposure to 4% PBO increased mortality to permethrin (from 22 to 100% in Ae. aegypti and from 19.8% to 50% in Ae. albopictus), suggesting an influence of P450 detoxifying enzymes. Indicative of metabolic resistance in PY resistant Ae. aegypti from Lahore was previously shown by biochemical analysis, which indicated higher quantities of esterases, MFOs, GSTs and AchE33. Here we evaluated the expression of genes previously related to metabolic resistance, such as the P450 MFOs (CYP9J28, CYP9M6 and CYP6BB2) and the carboxylesterase CCE- Ae3a52–54. This trend has been identified in resistant Aedes population from Southeast Asia29,54–56, the Caribbean57, Central58, and South America59. Of them, we found CYP9J28 12-fold overexpressed in Ae. aegypti from Lahore. Its role has already been shown in vivo, when the transgenic expression of AaegCYP9J28 increased pyrethroid- resistance in D. melanogaster60. As far as the overexpression of carboxylesterase genes in Ae. aegypti is concerned, they are more associated with OP resistance61. Indeed, as we did not observe resistance to the OPs temephos and malathion, it makes sense that the CCEAe3a gene was not overexpressed in our samples from Lahore. Knockdown resistance mutations in voltage-gated sodium channel (NaV) alone or in combinations have been reported from several Ae. aegypti populations from Southeast and South Asian countries including Malaysia62, Indonesia63, Thailand64, Singapore65, Myanmar66, India67, Sri Lanka68, Laos30, China69,70 and Saudi Arabia71. Here the Ae. aegypti Pakistani populations were genotyped to several kdr SNPs: V410L, described in populations from Latin America72, P989S and V1016G from Asia54,66,73 and F1534C, present in all continents23,74,75. www.nature.com/scientificreports/ Mutations on th it l i bi ti t d i l ti i t t t th id d DDT tl Knockdown resistance mutations in voltage-gated sodium channel (NaV) alone or in combinations have been reported from several Ae. aegypti populations from Southeast and South Asian countries including Malaysia62, Indonesia63, Thailand64, Singapore65, Myanmar66, India67, Sri Lanka68, Laos30, China69,70 and Saudi Arabia71. Here the Ae. aegypti Pakistani populations were genotyped to several kdr SNPs: V410L, described in populations from Latin America72, P989S and V1016G from Asia54,66,73 and F1534C, present in all continents23,74,75. Mutations on these sites alone or in combination are expected in populations resistant to pyrethroids and DDT, as recently reviewed21. We did not detect these classical SNPs at IS6 (V1014L) and IIS6 (S989P and V1016G) NaV segments, nor any additional nonsynonymous substitutions in the IIS6 segment. Interestingly, the sequences of this segment in worldwide Ae. aegypti populations are divided into two clades, A and B, distinguished mostly by indels in the intron between exons 20 and 21. Besides, all IIS6 kdr mutations described so far are in haplotypes from clade A23. Here all IIS6 sequences of Ae. aegypti belonged to clade B. On the other hand, the 1534C kdr mutation in the IIIS6 segment was present in all Ae. aegypti samples from Lahore here evaluated, as indicated by TaqMan genotyping assay specific for the F1534C SNP and confirmed by sequencing. Moreover, HRM analyses displayed a distinct profile that verified the presence of the SNP T1520I. Summing up TaqMan, HRM, and sequencing analyses, we evidenced two kdr haplotypes: T1520 + 1534C (45.5%) and the double kdr 1520I + 1534C (53.4%). The haplotype without kdr T1520 + F1534 (1.1%) was present in only one sample. This partially explains the resistance observed in the Pakistani Ae. aegypti population to the pyrethroids. The F1534C kdr has been described in Ae. aegypti populations from several Asian countries76. In PY-resistant Indian Ae. aegypti populations, a PCR–RFLP analysis followed by sequencing showed the presence of three haplotypes [T1520 + F1534 (21%), T1520 + 1534C (66%), and 1520I + 1534C (13%)] as we have reported in our findings. It was also observed that the F1534C mutation always occurred independently while T1520I was always found in association with F1534C14,23.i In the case of Ae. albopictus, HRM analyses identified distinct variants in the analyses of both IIS6 and IIIS6 NaV segments. www.nature.com/scientificreports/ However, they all accounted for synonymous substitutions, as revealed by sequencing, except for one nonsynonymous in the IIS6 segment: L882M. Likewise, several mutations in PY-resistant Ae. albopictus populations from India were also reported32. Chinese Ae. albopictus presented a different mutation (F1534S) in pyrethroids resistant populations70. It is noteworthy that the diversity of haplotypes in Ae. albopictus was higher than Ae. aegypti, as expected since it is native to Asia while Ae. aegypti is an invasive species77. Consequently, lower diversity is expected in this species, compared to the autochthonous Asian tiger mosquito. Conclusion Ae. aegypti and Ae. albopictus from Lahore, Pakistan, were resistant to pyrethroids while susceptible to organo- phosphates and the IGR. Among compounds we tested herein, both larvicides temephos and pyriproxyfen, as well as the adulticide malathion, should be the most effective against both species. Synergist PBO increased mortality against permethrin, indicating the participation of metabolic resistance mechanisms. In fact, the P450 gene CYP9J28 was overexpressed in Ae. aegypti. Also, the kdr mutations T1520I and F1534C were present under high frequencies. Therefore, resistance to pyrethroids in Ae. aegypti from Lahore is likely related to multiple physiological mechanisms. These results’ implications may be discussed with authorities responsible for Lahore’s vector control actions, aiming to improve strategies against Aedes in Pakistan. Discussion Th f albopictus presented a temephos LC50 value similar to that of Ae. aegypti. Concerning the adulticide malathion, both Ae. aegypti and Ae. albopictus populations from Lahore were susceptible in collections performed in 201536, and maintained this status as we observed here.hh The IGRs emerged as a prominent alternative to neurotoxic larvicides48. The chitin synthesis inhibitor com- pounds like diflubenzuron and buprofezin have been tested in Ae. aegypti from Lahore, with diflubenzuron causing higher mortality at the pupal stage while buprofezin resulting in more larval deaths16. Both Ae. aegypti and Ae. albopictus we evaluated here showed 100% of adult emergence inhibition against the analog of juvenile hormone IGR, pyriproxyfen, with maximum mortality at the pupal stage, as previously observed in 201540. Also, as an alternative to chemicals, the biolarvicide Bti caused 100% mortality in the Aedes field populations from the same city16,34. Although resistance to pyriproxyfen and other IGRs is not common, there are reports of Brazilian and Malaysian populations resistant to pyriproxyfen and methoprene, respectively49,50. Therefore, although IGRs have emerged as a promising alternative to neurotoxicants, their effectiveness must be continuously monitored. Although pyrethroids are not used as larvicides we performed a rapid test with permethrin and deltamethrin f Although pyrethroids are not used as larvicides, we performed a rapid test with permethrin and deltameth adopted as a preliminary method to indicate pyrethroid resistance in mosquito larvae51. Based on a similar a Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ Methods The rabbit blood utilized to feed the mosquitoes was kindly provided by the Institute of Science and Technology in Biological models (ICTB), Fiocruz, under the license CEUA L-004/2018, approved by the Ethics Committee for the Use of Animals in Research of Oswaldo Cruz Institute (CEUA-IOC). y With the purpose of maximizing growth synchronization, we put the Aedes eggs to hatch in 1 L dechlorinated water, and after 3–4 h, 500 larvae were transferred to separate trays and grown to L3/L4 stage under standard lab conditions of temperature 26 °C ± 1 °C, light: dark 12 h: 12 h period and 70 ± 5% relative humidity46. The Ae. aegypti Rockefeller strain, the international reference for susceptibility to insecticides and vigor under laboratory conditions79, was reared in parallel and tested in all experiments as an internal control. We have referred to Ae. aegypti Rockefeller as Rock, and Ae. aegypti and Ae. albopictus Lahore populations as PAg and PAb, respectively. Bioassays with larvae. Temephos. We followed the dose–response WHO protocol to calculate the re- sistance ratios (RR) to temephos in the evaluated populations80. Temephos technical grade (Pestanal Sigma- Aldrich) was dissolved in ethanol to give a series of concentrations ranging from 0.002 to 0.018 mg/L in order to obtain mortality from 5 to 99%, as recommended for probit analysis. Each concentration and a negative con- trol condition (containing 300 µL of ethanol only) was replicated four times, each containing 100 mL solution with ~ 20 late L3 or early L4 larvae, in a disposable plastic cup of 150 mL capacity. Mortality was calculated 24 h after initial exposure. The tests were performed three times with both PAg and PAb populations simultaneously, always in parallel to Rock as an internal control. Pyriproxyfen. For the IGR pyriproxyfen, we performed dose-diagnostic assays. Each assay contained eight treatment replicas with pyriproxyfen (Sigma-Aldrich) at a diagnostic concentration of 0.3 μg/L. This dosage was previously obtained, as twice the CL99 for Rock49. Besides, we included six control replicas (with 1 mL of the solvent instead of the IGR solution) in a 250 mL solution. A 300 mL disposable, transparent plastic cup was used for each replica, into which we added 10 L4 larvae. To nourish the larvae, we introduced 15 mg of fish food (Tetramarine Granules, Tetra) at the start of the assay and 10 mg on the fourth day of the experiment. Methods Sample collection and laboratory rearing. We collected larvae of Aedes aegypti and Aedes albopictus from different Union Councils (UCs)/neighborhoods of Mughulpura and airport areas of Lahore district with the help of sanitary agents and health workers recruited by the local health department for this purpose (Fig. 5). We visited different domestic and industrial areas. Larvae mainly infested uncovered or partially covered water storage tanks, buckets, and small pots made from various materials, including plastic, steel, copper, and cement, which contained water and were located in shaded places. Larvae were brought to the insectary of the Department of Zoology, GC University Lahore, identified into species level with the help of morphological characters78, and provided with fish food (Tetra- Marine Granules, Tetra). Emerged pupae were shifted to clean and disinfected cages, and adults were provided with a 10% sugar solution. Adults were starved overnight but Sample collection and laboratory rearing. We collected larvae of Aedes aegypti and Aedes albopictus from different Union Councils (UCs)/neighborhoods of Mughulpura and airport areas of Lahore district with the help of sanitary agents and health workers recruited by the local health department for this purpose (Fig. 5). We visited different domestic and industrial areas Larvae mainly infested uncovered or partially covered https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ Figure 5. Political map of Pakistan, divided into four provinces, with Lahore magnified in red. The map was generated with a free and open- source software QGIS version 2.18.24 (GNU General Public License), developed by the Open Source Geospatial Foundation Project (http://qgis.org). Figure 5. Political map of Pakistan, divided into four provinces, with Lahore magnified in red. The map was generated with a free and open- source software QGIS version 2.18.24 (GNU General Public License), developed by the Open Source Geospatial Foundation Project (http://qgis.org). allowed to feed on blood using an artificial feeder, Hemotek (PS-6 System, Discovery Workshops, Accrington, UK). Eggs of the F1 generation were shipped to Laficave, IOC/FIOCRUZ, Brazil, following the formalities of the Brazilian Ministry of Agriculture and the relevant authorities in Pakistan. Colonies were raised to F2 and F3 generations in our insectary at Fiocruz in the same fashion as mentioned before, while F1 males were preserved at − 20 °C for genotyping. www.nature.com/scientificreports/ Papers (Whatman grade 1) were impregnated with diagnostic concentrations of four pyrethroids following WHO standard protocol and dosages: deltame- thrin 0.03%, permethrin 0.25%, etofenprox 0.5% and alpha-cypermethrin 0.03%81. Solutions were prepared from technical grade insecticides (Pestanal Sigma-Aldrich) in acetone and silicone oil (used as a carrier) and evenly applied to a filter paper (Whatman grade 1). Papers were allowed to air dry for 72 h before use. Papers impregnated with just solvent were used as the control. In each assay, we put 15–20 female mosquitoes, 3–5 days old, non-blood-fed, in a resting tube (tube without insecticide) to acclimatize for 30 min. After that, they were gently blown into tubes having insecticides-impregnated papers, and knockdown was checked every 5 min for 2 h. This differed from the WHO protocol81, which recommends exposure time of 60 min. In the case of Rock, mortality was checked every two minutes. After they were exposed to insecticides, mosquitoes were shifted back to resting tubes, provided 10% sugar water, and mortality evaluated after 24 h. Organophosphate. For the OP malathion bioassays, we used CDC bottle tests83, impregnating 250 mL glass bot- tles (Wheaton) with 20 µg/mL malathion (Cheminova Brasil Ltda, São Paulo) dissolved in acetone. For impreg- nating the bottles, 1 mL of the insecticide solution was distributed evenly to all parts of the bottle, including its cap, and left to air dry for at least 24 h before the test. In each bottle, 20–25 female mosquitoes, three to five days old, non-blood fed, were left for one hour, and mortality was recorded. Bottles impregnated with 1 mL acetone were used as control. This experiment was done in four replicates, three separate times. Standard conditions of temperature, humidity, and light–dark periods were maintained throughout the assays, as described earlier. Synergist assay. In order to evaluate the occurrence of metabolic resistance mechanisms to the pyrethroid type I permethrin, we carried out WHO bioassays with the synergist PBO (Piperonyl Butoxide)84. This substance inhibits the action of Multi-Function Oxidases (MFOs) and can revert resistance85. For this purpose, papers were impregnated with technical grade PBO (Endura) and permethrin (Pestanal, Sigma-Aldrich), as described earlier. The test was composed of four conditions: (i) exposure to 4% PBO, (ii) a pre-exposure to 4% PBO and then to 0.25% permethrin, (iii) exposure to 0.25% permethrin only, and (iv) exposure only to solvent control. The susceptible strain, Rock, was carried out parallelly. www.nature.com/scientificreports/ environmental conditions of temperature (26 °C ± 1 °C), light: dark regiments (12 h: 12 h), and relative humidity (75 ± 5%). Unlike neurotoxic insecticides, for IGRs, mortality itself is not the critical parameter to evaluate, but the index of Adult Emergence Inhibition (AEI). Populations are considered to be resistant when AEI is lower than 90%. In addition to AEI, we recorded the mortality observed at each developmental stage: larvae, pupae, and adult (adults that remained attached to the exuviae and died). environmental conditions of temperature (26 °C ± 1 °C), light: dark regiments (12 h: 12 h), and relative humidity (75 ± 5%). Unlike neurotoxic insecticides, for IGRs, mortality itself is not the critical parameter to evaluate, but the index of Adult Emergence Inhibition (AEI). Populations are considered to be resistant when AEI is lower than 90%. In addition to AEI, we recorded the mortality observed at each developmental stage: larvae, pupae, and adult (adults that remained attached to the exuviae and died). Susceptibility Index of Aedes larvae against pyrethroids. We adopted a simplified knockdown assay51 to evaluate susceptibility to the knockdown effect to type I (permethrin) and type II (deltamethrin) pyrethroids in larvae. We prepared solutions with technical grades permethrin (Pestanal, Sigma-Aldrich) and deltamethrin (Pestanal, Sigma-Aldrich) by dissolving each in 1 mL acetone and then ethanol to make 250 ppm stock solutions. From this, we prepared a 20 mL solution for each pyrethroid, with 10 replicas for two concentrations (0.1 ppm and 0.4 ppm) in H2O. Two negative control cups with ethanol (32 µL) in 20 mL of H2O were run in parallel. We used one L4 larva per replica and registered the knockdown every 5 min, for half an hour. The KdT50, i.e., the time when 50% of the larvae were knocked down, was scored according to these categories: 1 (0–5 min), 2 (6–10 min), 3 (11–15 min), 4 (16–20 min), 5 (21–30 min) or 6 (> 30 min). The susceptibility index (SI) for each species against deltamethrin and permethrin was obtained by multiplicating the KdT50 categories of both con- centrations (0.1 and 0.4 ppm). Population with the lower SI is considered more sensitive. The obtained SI values were the mean of three independent assays, with Rock in parallel as an internal control in all of them. Adulticides. Pyrethroids. We followed the WHO-like tube tests procedure, with modifications81,82 for the tarsal contact tests with insecticides-impregnated filter papers. Methods All the cups were covered with gauze to avoid eventual adult escaping. Mortality and life stage transformation were cumulatively recorded bi-weekly. Parallel assays were done with Rock. Data was recorded until all individuals in the control condition emerged into adults. Assays were performed three independent times, under standard https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 3. Primers and probes for TaqMan SNP genotyping qPCR assays for kdr mutations in Aedes aegypti. a Identification of the customized TaqMan SNP Genotyping Assay (Thermo Fischer). NaV Site, segment Assay IDa Variation Primers Probes 410, IS6 AN2XA9W GTA/TTA (Val/Leu) for: GTGGCACATGCTCTTCTTCATT rev: GGCGACAATGGCCAAGATC Val: VIC-TCGTTCTACCTTGTAAAT T-NFQ Leu: FAM-TTCGTTCTACCTTTTAAA TT-NFQ 989, IIS6 AH21C3C TCT/CCT (Ser/Pro) for: TGATCGTGTTCCGGGTATTATGC rev: CCATCACTACGGTGGCCAAAA Ser: VIC-CCCACATGGATTCGAT-NFQ Pro: FAM- CCACATGGGTTCGAT-NFQ 1016, IIS6 AHX1KC9 GTT/ GGT (Val/Gly) for: CGTGCTAACCGACAAATTGTT TCC rev: TTGGACAAAAGCAAGGCT Val: VIC-AGAAAAGGTTAAGTACCT GTGCG-NFQ Gly: FAM- AGAAAAGGTTAAGTACCT GTGCG-NFQ 1534, IIIS6 AHWSL61 TTC/TGC (Phe/Cys) for: TCGCGAGACCAACATCTACATG rev: GATGATGACACCGATGAACAG ATTC Phe: VIC-AACGACCCGAAGATGA- NFQ Cys: FAM-ACGACCCGACGATGA-NFQ Table 3. Primers and probes for TaqMan SNP genotyping qPCR assays for kdr mutations in Aedes aegypti. a dentification of the customized TaqMan SNP Genotyping Assay (Thermo Fischer) Table 3. Primers and probes for TaqMan SNP genotyping qPCR assays for kdr mutations in Aedes aegypti. a Identification of the customized TaqMan SNP Genotyping Assay (Thermo Fischer). Figure 6. Representation of the IIS6 (a) and IIIS6 (b) NaV segments with their respective amino acid (above) and nucleotide (bellow) sequences. The SNPs at 989, 1016, 1520, and 1534 sites are indicated inside brackets with the mutant alternative in red. The shaded colors indicate the amplified sequences in the HRM reactions, with their respective primer sequences underlined and orientation following the arrows. spermathecae, which would mislead interpretations about individual genotypes. Purified DNA was quantified with NanoDrop One (ThermoFisher Scientific) and diluted to 20 ng/µL in ultra-pure water. SNPs genotyping. A TaqMan SNP genotyping qPCR approach was employed for kdr genotyping, essentially as described previously88, for the variations Val410Leu, Ser989Pro, Val1016Gly, and Phe1534Cys in PAg. We performed reactions independently for each SNP, consisting of 1X TaqMan Genotyping Master Mix (Ther- moFisher), 1X of the respective Custom TaqMan SNP Genotyping Assay (Table 3), 20 ng of DNA and ultra-pure water q.s. 10 µL, run in a QuantStudio 6 Flex (Applied Biosystems), under standard conditions. The genotype callings were obtained by the online software Genotype Analysis Module V3.9 (Applied Biosystems, Thermo Fischer cloud platform). As a positive control, we used the DNA of Rockefeller strain (wild-type homozygote genotypes) in all SNP reactions, and the Rock-kdr strains89, which has the kdr homozygote genotypes: 410 Leu/ Leu, 1016 Ile/Ile and 1534 Cys/Cys. www.nature.com/scientificreports/ Each assay consisted of four tubes, and each tube con- tained 20–25 female mosquitoes, 4–6 days old, non-blood-fed. All exposures lasted 1 h, after which mortality was checked in each assay. Mosquitoes were then transferred to resting tubes and provided 10% sugar solution. The number of dead mosquitoes was counted after 24 h, according to the standard criteria of WHO81. Calculations. In the case of both pyrethroids and organophosphates bioassays, we expected 100% mortality of the reference strain and no (zero) mortality in the control. If mortality in the control exceeded 20%, the test should be repeated. If less, Abbott’s correction formula would be applied86. Temephos Lethal Concentrations (LC) were obtained by log x probit transformations followed by linear regression analyses87 with the sum of the values from the three assays. We calculated the resistance ratios (RR) by dividing the LC of both PAg and PAb by the LC of Rock, in the absence of a reference lab strain for Ae. albopictus at that time. p To estimate knockdown time to pyrethroids (KdT50), readings were likewise submitted to probit analysis. Accordingly, knockdown-time Resistant Ratios (KdT-RR50) were calculated by dividing KdT50 of PAg and PAb by the KdT50 of Rock. Exploration of knockdown resistance (kdr) mutations. We evaluated the nucleotide diversity in the genomic region corresponding to IS6, IIS6, and IIIS6 NaV segments to investigate kdr mutations classically found in Ae. aegypti pyrethroid-resistant populations. Genomic DNA was isolated from male mosquitoes (n = 45) of both PAg and PAb with a column-based DNA extraction kit (NuleoSpin, Macherey–Nagel Laboratories), accord- ing to manufacturer’s instructions. We did not use females to avoid eventual amplification of DNA inside their https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ Also, we employed a synthesized DNA fragment (gBlock, IDT) with the sequence of an Asian kdr haplotype (GenBank accession number MN602755)23 as the kdr homozygote geno- types in 989 Pro/Pro and 1016 Gly/Gly SNP reactions. An equimolecular quantity of Rock DNA was mixed with each kdr positive controls to obtain the respective heterozygote controls. HRM reactions. To search for possible SNPs beyond the classical mutations tested with TaqMan qPCR, we developed a high-resolution melting analysis (HRM) for the genomic regions in the IIS6 and IIIS6 NaV segments of PAg and PAb. We performed three HRM reactions, each for part of the NaV gene exons 20, 21 (IIS6), and 31 (IIIS6) (Fig. 6). We focused on these exons because the known kdr sites 989, 1016, and 1534 are respectively placed in the exons 20, 21, and 31 of the Ae. aegypti NaV gene. Reactions were performed with 1X MeltDoctor https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ Table 4. Primers for HRM analyses in Aedes aegypti and Aedes albopictus. All primer sequences are in the 5′-3′orientation. NaV segment Targeted sites Primer sequences Species IIS6 Exon 20 for: TGATCGTGTTCCGGGTATTATGC Both rev: CCATCACTACGGTGGCCAAAA Both Exon 21 for: GCTAACCGACAAATTGTTTCC Ae. aegypti for: GAATGCTTTCTCCCCCAAAC Ae. albopictus rev: TGGACAAAAGCAAGGCTAAG Both IIIS6 Exon 31 for: TGGGAAAGCAGCCGATTCG Both rev: GAACAGATTCAGCGTGAAGAACG Both NaV segment Targeted sites Primer sequences Species IIS6 Exon 20 for: TGATCGTGTTCCGGGTATTATGC Both rev: CCATCACTACGGTGGCCAAAA Both Exon 21 for: GCTAACCGACAAATTGTTTCC Ae. aegypti for: GAATGCTTTCTCCCCCAAAC Ae. albopictus rev: TGGACAAAAGCAAGGCTAAG Both IIIS6 Exon 31 for: TGGGAAAGCAGCCGATTCG Both rev: GAACAGATTCAGCGTGAAGAACG Both Table 4. Primers for HRM analyses in Aedes aegypti and Aedes albopictus. All primer sequences are in the 5′-3′orientation. HRM Master Mix kit (Thermo Fischer), 0.3 µM of each primer (Table 4), 20 ng DNA, and ultra-pure water q.s. 10 µL. The thermocycling program followed the standard qPCR conditions with an additional HRM step in a QuantStudio 6 (Applied Biosystems). The HRM curve analyses were performed with the QuantStudio Real- Time PCR Software v1.3 (ThermoFisher), which grouped the samples into distinct variants. To determine the variant genotypes, we sorted at least three samples of each variant to be sequenced. DNA sequencing. www.nature.com/scientificreports/ The IIS6 and IIIS6 corresponding genomic regions were amplified with Phusion High-Fidel- ity DNA Polymerase PCR kit (New England BioLabs) in a 25 µL reaction, containing 1X Phusion HF buffer, 200 µM dNTP, 3% DMSO, 0.4 U Polymerase, 0.5 µM of each primer, 40 ng DNA and H2O q.s. 20 µL. The prim- ers employed were 5para3: ACAATGTGGATCGCTTCCC x NaV_E21R: GCAATCTGGCTTGTTAACTTG for the IIS6 segment in both species and 31P: TCGCGGGAGGTAAGTTATTG x 31Q: GTTGATGTGCGATGG AAATG for the IIIS6 segment in Ae. aegypti, and 31F: GATCGCGGGAGGTAAGTT X 31R: CCGTCTGCT TGTAGTGATCG in Ae. albopictus (all primers described in 5′ to 3′orientation). Thermocycling conditions were 98 °C/30" for initial polymerase activation, followed by 35 cycles of 98 °C/30" for double-strand denaturation, 60 °C (in IIS6 reactions) or 61 °C (in IIIS6 reactions)/15" for primer annealing, and 72 °C/30" for enzyme exten- sion. The PCR products were purified with magnetic beads Agencourt AMPure XP (Beckman Coulter), follow- ing manufacturer instructions. Purified amplicons (1 µL) were submitted to a Sanger sequencing reaction with the kit BigDye Terminator v3.1 (ThermoFisher), with 1 µM of one of the primers, according to manufacturer’s protocol, and followed to the FIOCRUZ platform of DNA sequencing. Each sample was sequenced in both strands. Sequences were analyzed with Geneious v. Prime90 and submitted to GenBank (accession numbers: MT740753-MT740765 and MT707209-MT707210). We compared our sequences with those available in the GenBank (NCBI Blast) to check their similarity with sequences from worldwide populations. Expression analysis of genes related to detoxification of insecticides. We evaluated the expres- sion profiles of genes previously associated with resistance in Aedes populations from South-east Asia in PAg populations: three were from the CYP9 family (CYP9M6, CYP9J10, and CYP9J28) and one from CYP6 (CYP6BB2). The expression profile of one carboxylesterase (CCEAe3a) and one sensory appendage protein (SAP2) was also investigated31,91,92. The expression levels were relative to the housekeeping gene of the ribosomal protein S14 (RpS14)93. p ( p ) Five-day-old female mosquitos were pooled in a tube. For each population, we used four pools as biological replicates per population. In order to isolate RNA, these mosquitoes were macerated in 300 µL TRIzol (Invit- rogen, CA, USA) and then homogenized with glass beads in TissueLyser II (Qiagen, Venlo, Netherlands). RNA was precipitated with TRIzol (Invitrogen, CA, USA) and chloroform and then washed with ethanol to remove any debris of DNA and protein, according to the manufacturer’s protocol. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 5. Primers employed in gene expression analyses. a All sequences are in 5′-3′ sense. For and rev indicate forward and reverse, respectively. Gene Family Sequences a Reference RpS14 Ribosomal protein, housekeeping gene for: AGGAACTAGCAGAATGGCTCCC rev: ACAGATCCGTGACATGGACGAAG 94 CYP6BB2 Cytochrome P450 for: AGTTCAAGGGCCGAGGATTG rev: CGGATCCACGAAAATTCCGC 58,95,96 CYP9J10 Cytochrome P450 for: AATACGTACGAGGGATCCAAGA rev: CTATCTCCTCCGACCTCGTCCTC 29,95 CYP9J28 Cytochrome P450 for: CTATTTCGGAGTCCTAGTGGCC rev: CTTTGACTCCTCGGTACTTGTCG 29,58,94–97 CYP9M6 Cytochrome P450 for: AGCTTGGCAATGACATCATCAC rev: TAAGTCCCTGAAATCCACCAGTG 29,96 CCEae3a Esterase for: TCTAAGAAACCCGAATATGACG rev: TTGAGGAGGCACGAACAG 57 SAP2 Sensory appendage protein for: TGGAGCCATCAAAGTCATCAA rev: GCGCGATATTGCTCCAGATA This study Table 5. Primers employed in gene expression analyses. a All sequences are in 5′-3′ sense. For and rev indicate forward and reverse, respectively. expression of each gene in PAg compared to Rock. The ΔCt values of each gene were compared between Rock and PAg by unpaired t-test with Welch’s correction, performed with GraphPad Prism 8 v 8.4.2 for Mac (Graph- Pad Software, San Diego, California USA). Received: 20 September 2020; Accepted: 18 January 2021 References Pyrethroid-resistance and presence of two knockdown resist- ance (kdr) mutations, F1534C and a novel mutation T1520I, in Indian Aedes aegypti. PLoS Negl. Trop. Dis. 9, 2 (2015). y p g y p ance (kdr) mutations, F1534C and a novel mutation T1520I, in Indian Aedes aegypti. PLoS Negl. Trop. Dis. 9, 2 (2015). 15. Khan, H. A. A. & Akram, W. Resistance status to deltamethrin, permethrin, and temephos along with preliminary resi mechanism in aedes aegypti (Diptera: Culicidae) from Punjab, Pakistan. J. Med. Entomol. 56, 1304–1311 (2019).l gyp ( p ) j ( ) 16. Jahan, N., Razaq, J. & Jan, A. Laboratory evaluation of chitin synthesis inhibitors (Diflubenzuron and Buprofezin) against Aedes aegypti larvae from Lahore, Pakistan. Pak. J. Zool. 43, 2 (2011). 17 Sil K S t l Ad i I t Ph i l 389 433 (El i A t d 2014) gyp 17. Silver, K. S. et al. Advances in Insect Physiology 389–433 (Elsevier, Amsterdam, 2014). y gy 18. Dong, K. et al. Molecular biology of insect sodium channels and pyrethroid resistance. Insect Biochem. Mol. Biol. 50, 1–17 (2014). 19. Narahashi, T., Ginsburg, K. S., Nagata, K., Song, J.-H. & Tatebayashi, H. Ion channels as targets for insecticides. Neurotoxicology 18. Dong, K. et al. Molecular biology of insect sodium channels and pyrethroid resistance. Insect Biochem. Mol. Biol. 50, 1–17 (2 h h b b h h l f d 18. Dong, K. et al. Molecular biology of insect sodium channels and pyrethroid resistance. In 9. Narahashi, T., Ginsburg, K. S., Nagata, K., Song, J.-H. & Tatebayashi, H. Ion channels as targets for insecticides. Neurotoxicology 19, 581–590 (1998). 20. Mian, L. S. & Mulla, M. S. Residue Reviews 27–112 (Springer, Berlin, 1982). 20. Mian, L. S. & Mulla, M. S. Residue Reviews 27–112 (Springe 1. Scott, J. G. Life and death at the voltage-sensitive sodium channel: Evolution in response to insecticide use. Annu. Rev. Entomol 64, 243–257 (2019). 22. Kushwah, R. B. S., Dykes, C. L., Kapoor, N., Adak, T. & Singh, O. P. Pyrethroid-resistance and presence of two knockdown resist- (kd ) t ti F1534C d l t ti T1520I i I di A d ti PL S N l T p Di 9 3332 (2015) 22. Kushwah, R. B. S., Dykes, C. L., Kapoor, N., Adak, T. & Singh, O. P. References 1. Khanani, M. R., Arif, A. & Shaikh, R. Dengue in Pakistan: Journey from a disease free to a hyper endemic nation. Editorial Board 5, 81 (2011).i 2. Javed, F. T. et al. Prevalence of all four dengue virus serotypes confirmed by using real time RT-PCR among population of Lahore Pakistan. Int. J. Agric. Vet. med. Sci 3, 1–3 (2009).h g 3. Idrees, M. et al. Dengue virus serotype 2 (DEN-2): The causative agent of 2011-dengue epidemic in Pakistan. Am. J. Biomed. Sci 4, 307–315 (2012). 4. OCHA, U. Outbreak update – Dengue in Pakistan, 19 November 2019, <https://reliefweb.int/report/pakistan/outbreak-update-dengu e-pakistan-19-november-2019> (2019). p 5. Powell, J. R., Gloria-Soria, A. & Kotsakiozi, P. Recent history of Aedes aegypti: Vector genomics and epidemiology records. Biosci ence 68, 854–860 (2018). 6. Powell, J. R. Mosquito-borne human viral diseases: Why Aedes aegypti?. Am. J. Trop. Med. Hyg. 98, 1563–1565 (2018). 6. Powell, J. R. Mosquito-borne human viral diseases: Why Aede 7. Moyes, C. L. et al. Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting huma Negl. Trop. Dis. 11, e0005625. https://doi.org/10.1371/journal.pntd.0005625 (2017). g p g j 8. Akhtar, M. S., Aihetasham, A., Saeedb, M. & Abbassa, G. Aedes survey following a dengue outbreak in Lahore, Pakistan, 2011 Dengue 36, 87 (2012). g 9. Aamir, U. B., Badar, N., Salman, M., Ahmed, M. & Alam, M. M. Outbreaks of chikungunya in Pakistan. Lancet. Infect. Dis 17, 483 (2017). 0. Mallhi, T., Khan, Y., Khan, A., Tanveer, N. & Qadir, M. First chikungunya outbreak in Pakistan: A trail of viral attacks. New Microb New Infect. 19, 13–14 (2017). 11. Governament of Punjab, P. Standard operation protocol for dengue control and prevention, <https://phc.org.pk/downloads/GoPb%20 SOPs%20for%20Dengue%20Control%202014.pdf> (2014).i g p 2. Khan, H. A. A., Akram, W., Shehzad, K. & Shaalan, E. A. First report of field evolved resistance to agrochemicals in dengue mos- quito, Aedes albopictus (Diptera: Culicidae), from Pakistan. Parasit. Vect. 4, 146 (2011). 3. Khan, G. Z. et al. Monitoring of resistance status in dengue vector Aedes albopictus (Skuse) (Culicidae: Diptera) to currently used public health insecticides in selected districts of Khyber Pakhtunkhwa-Pakistan. Int. J. Mosquito Res. 4, 123–127 (2017). y 4. Kushwah, R. B. S., Dykes, C. L., Kapoor, N., Adak, T. & Singh, O. P. Pyrethroid-resistance and presence of two knockdown resist 14. Kushwah, R. B. S., Dykes, C. L., Kapoor, N., Adak, T. & Singh, O. P. gyp g p 3. Cosme, L. V., Gloria-Soria, A., Caccone, A., Powell, J. R. & Martins, A. J. Evolution of kdr haplotypes in worldwide populations o Aedes aegypti: Independent origins of the F1534C kdr mutation. PLoS Negl. Trop. Dis. 14, e0008219 (2020). www.nature.com/scientificreports/ The pellet obtained was air-dried and eluted with RNase-free water. The isolated RNA was quantified with Qubit RNA HS Assay kit (Invitrogen), and cDNA was then synthesized in an RT-PCR reaction with SuperScript Vilo MasterMix (Invitrogen), using 5 µL RNA and molecular grade water qi 20 µL. The reagents were incubated at room temperature for 10 min, 42 °C for 1 h, and 85 °C for 10 min. We used the Qubit dsDNA HS Assay kit (Invitrogen) to quantify this cDNA and diluted it to 4 ng/µL for qPCR use.h The qPCR reactions were performed in a 96-Well MicroAmp reaction (Invitrogen) plate, with the kit KAPA SYBR FAST qPCR Master Mix (1x) and ROX LOW (KAPA Biosystems) kit, 0.2 µM of each forward and reversed primers (Table 5), 4 ng/µL cDNA and molecular grade water q.s. 10 µL. Each pool (biological replicate) was divided into four technical replicates. Thermocycling conditions in a QuantStudio 6 Flex Real-Time PCR system (ThermoFisher Scientific) consisted of enzyme activation and initial denaturation at 50 °C and 95 °C for 2 and 3 min, respectively, followed by 40 cycles of denaturation (95 °C for 3 s) and annealing/extension of primers (60 °C for 1 min), with an additional standard melting curve analysis step. Analysis of gene expression. We obtained the Ct values of four replicates for each gene and calculated their means. Replicates presenting different Ct from Grubbs values (outliers)98 or with an unexpected peak in the melting analysis were excluded. Relative quantification analyses were performed using the ΔΔCt method99, where the RpS14 was taken as the reference gene and Rock, reared alongside test population, as the reference strain. For all genes, the Ct threshold was set at 0.2. The 2(−ΔΔCt) equation was applied to define the relative fold-change https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| 2. Kushwah, R. B. S., Dykes, C. L., Kapoor, N., Adak, T. & Singh, O. P. Pyrethroid-resistance and presence of two knockdown resist- ance (kdr) mutations, F1534C and a novel mutation T1520I, in Indian Aedes aegypti. PLoS Negl. Trop. Dis. 9, e3332 (2015). www.nature.com/scientificreports/ & Reisen, W. Status of insecticide resistance in anopheline mosquitoes of Punjab Province, Pakistan. Southeast Asian J. Trop. Med. Public Health 11, 332–340 (1980).lf 38. Rathor, H. R., Nadeem, G. & Khan, I. A. Pesticide susceptibility status of Anopheles mosquitoes in four flood-affected districts of South Punjab Pakistan. Vector Borne Zoon. Dis. 13, 60–66. https://doi.org/10.1089/vbz.2012.1055 (2013). 9. Rowland, M. Location of the gene for malathion resistance in Anopheles stephensi (Diptera: Culicidae) from Pakistan. J. Med Entomol. 22, 373–380 (1985).fi 40. Mahmood, M. A. Bio-efficacy and persistancy of Pyriproxyfen against Aedes aegypti: A control trial at district Lahore, Punjab, Pakistan.h 41. Hemingway, J. The genetics of malathion resistance in Anopheles stephensi from Pakistan. Trans. R. Soc. Trop. Med. Hyg. 77, 106–108 (1983). 2. Reisen, W. K., Mahmood, F. & Parveen, T. Anopheles culicifacies Giles: A release-recapture experiment with cohorts of known age with implications for malaria epidemiology and genetical control in Pakistan. Trans. R. Soc. Trop. Med. Hyg. 74, 307–317 (1980) h l h h l h l h k ( ) with implications for malaria epidemiology and genetical control in Pakistan. Trans. R. Soc. Trop. Med. Hyg. 74, 307–317 (1980). 43. Rathor, H. R. & Toqir, G. Malathion resistance in Anopheles stephensi liston in Lahore Pakistan. Mosq. News 40, 526–531 (1980). 44. Valle, D., Bellinato, D. F., Viana-Medeiros, P. F., Lima, J. B. P. & Martins Junior, A. J. Resistance to temephos and deltamethrin in with implications for malaria epidemiology and genetical control in Pakistan. Trans. R. Soc. Trop. Med. Hyg. 74, 307–317 (1980). 43. Rathor, H. R. & Toqir, G. Malathion resistance in Anopheles stephensi liston in Lahore Pakistan. Mosq. News 40, 526–531 (1980). 44 Valle D Bellinato D F Viana Medeiros P F Lima J B P & Martins Junior A J Resistance to temephos and deltamethrin in 43. Rathor, H. R. & Toqir, G. Malathion resistance in Anopheles stephensi liston in Lahore Pakistan. Mosq. News 40, 526–531 (19 & Toqir, G. Malathion resistance in Anopheles stephensi liston in q p p q ( 44. Valle, D., Bellinato, D. F., Viana-Medeiros, P. F., Lima, J. B. P. & Martins Junior, A. J. Resistance to temephos and deltameth Aedes aegypti from Brazil between 1985 and 2017. Memórias do Inst. Oswaldo Cruz 114, 2 (2019). gyp 45. Rahman, R. U., Cosme, L. V., Costa, M. M., Carrara, L., Lima. J. B. P. & Martins, A. J. www.nature.com/scientificreports/ B., Tyagi, R., Kasai, S. & Scott, J. G. CYP-m regulation. PLoS Negl. Trop. Dis. 12, e0006933 (2018). 54. Kasai, S. et al. Mechanisms of pyrethroid resistance in the dengue mosquito vector, Aedes aegypti: Target site insensitivity, penetra- tion, and metabolism. PLoS Negl. Trop. Dis. 8, 2 (2014).i tion, and metabolism. PLoS Negl. Trop. Dis. 8, 2 (2014). g p 55. Chan, H. H. & Zairi, J. Permethrin resistance in Aedes albopictus (Diptera: Culicidae) and associated fitness costs. J. Med. Entomol. 50, 362–370 (2013). 6. Ishak, I. H. et al. Pyrethroid resistance in Malaysian populations of dengue vector Aedes aegypti is mediated by CYP9 family o cytochrome P450 genes. PLoS Negl. Trop. Dis. 11, e0005302 (2017). 57. Marcombe, S. et al. Exploring the molecular basis of insecticide resistance in the Martinique Island (French West Indies). BMC Genom. 10, 494 (2009). 57. Marcombe, S. et al. Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: A case study in Martinique Island (French West Indies). BMC Genom. 10, 494 (2009). 58. Reid, W. R. et al. Transcriptional analysis of four family 4 P450s in a Puerto Rico strain of Aedes aegypti (Diptera: Culicidae) compared with an Orlando strain and their possible functional roles in permethrin resistance. J. Med. Entomol. 51, 605–615 (2014). 59 C t M d M A li ã d i tê i i ti id i l i d p p l õ d A d pti Li 1762 58. Reid, W. R. et al. Transcriptional analysis of four family 4 P450s in a Puerto Rico strain of Aedes aegypti (Diptera: Culicidae) compared with an Orlando strain and their possible functional roles in permethrin resistance. J. Med. Entomol. 51, 605–615 (2014). 59. Costa, M. d. M. Avaliação da resistência a inseticidas e mecanismos selecionados em populações de Aedes aegypti Linnaeus 1762 (Diptera, Culicidae) da fronteira entre Brasil e Guiana Francesa, (2017). 59. Costa, M. d. M. Avaliação da resistência a inseticidas e mecanismos selecionado (Diptera, Culicidae) da fronteira entre Brasil e Guiana Francesa, (2017). 9. Costa, M. d. M. Avaliação da resistência a inseticidas e mecanismos selecionados em populações de Aedes aegypti Linnaeus 1762 (Diptera, Culicidae) da fronteira entre Brasil e Guiana Francesa, (2017). Diptera, Culicidae) da fronteira entre Brasil e Guiana Francesa, (201 p f 60. Pavlidi, N. et al. Transgenic expression of the Aedes aegypti CYP9J28 confers pyrethroid resistance in Drosophila melanogaster. Pestic. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 24. Bingham, G., Strode, C., Tran, L., Khoa, P. T. & Jamet, H. P. Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid-resistant Aedes aegypti?. Trop. Med. Int. Health 16, 492–500 (2011). py gyp p 5. Murcia, O. et al. Presence of the point mutations Val1016Gly in the voltage-gated sodium channel detected in a single mosquito from Panama. Parasit. Vect. 12, 62 (2019).h ( ) 26. Fan, Y. & Scott, J. G. The F1534C voltage-sensitive sodium channel mutation confers 7-to 16-fold resistance to pyrethroid insec- ticides in Aedes aegypti. Pest Manag. Sci. 76, 2251–2259 (2020). 27. Essandoh, J., Yawson, A. E. & Weetman, D. Acetylcholinesterase (Ace-1) target site mutation 119S is strongly diagnostic of carba- mate and organophosphate resistance in Anopheles gambiae ss and Anopheles coluzzii across southern Ghana. Malar. J. 12, 404 (2013).h ( ) 8. Hemingway, J., Hawkes, N. J., McCarroll, L. & Ranson, H. The molecular basis of insecticide resistance in mosquitoes. Insec Biochem. Mol. Biol. 34, 653–665 (2004).i 29. Strode, C. et al. Genomic analysis of detoxification genes in the mosquito Aedes aegypti. Insect Biochem. Mol. Biol. 38, 113 (2008). 30. Marcombe, S. et al. Distribution of insecticide resistance and mechanisms involved in the arbovirus vector Aedes aegypti in and implication for vector control. PLoS Negl. Trop. Dis. 13, e0007852 (2019). 31. Ingham, V. A. et al. A sensory appendage protein protects ma V. A. et al. A sensory appendage protein protects malaria vectors f g y pp g p p py ( ) 32. Chatterjee, M. et al. Polymorphisms in voltage-gated sodium channel gene and susceptibility of Aedes albopictus to insecti in three districts of northern West Bengal India. PLoS Negl. Trop. Dis. 12, e0006192 (2018).f 3. Abbas, S., Nasir, S., Fakhar-e-Alam, M. & Saadullah, M. Toxicity of different groups of insecticides and determination of resistance in Aedes aegypti from different habitats. Pak. J. Agric. Sci. 56, 3 (2019). gyf g 34. Jahan, N. & Shahid, A. Evaluation of resistance against Bacillus thuringiensis israelensis WDG in dengue vector from Lahore, Pakistan. Pak. J. Zool. 44, 2 (2012). 35. Jahan, N. & Shahid, A. Evaluation of resistance against deltamethrin and cypermethrin in dengue vector from Lahore, Pakistan. J. Anim. Plant Sci. 23, 1321–1326 (2013). 6. Mohsin, M. et al. Susceptibility status of Aedes aegypti and Aedes albopictus against insecticides at eastern Punjab, Pakistan. MMJ 3, 41–46 (2016). 37. Rathor, H., Toqir, G. References Pyrethroid-resistance and presence of two knockdown resist- ance (kdr) mutations, F1534C and a novel mutation T1520I, in Indian Aedes aegypti. PLoS Negl. Trop. Dis. 9, e3332 (2015). ( ) gyp g p ( ) 23. Cosme, L. V., Gloria-Soria, A., Caccone, A., Powell, J. R. & Martins, A. J. Evolution of kdr haplotypes in worldwide populations of Aedes aegypti: Independent origins of the F1534C kdr mutation. PLoS Negl. Trop. Dis. 14, e0008219 (2020). https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ Insecticide resistance and genetic structure of Aedes aegypti populations from Rio de Janeiro State, Brazil. PLoS Negl Trop Dis 15(2), e0008492 https://doi.org/10.1371/journ al.pntd.0008492 (2021).l p 46. Bellinato, D. F. et al. Resistance status to the insecticides temephos, deltamethrin, and diflubenzuron in Brazilian Aedes aegypti populations. BioMed Res. Int. 20, 16 (2016). p p 7. Bisset, J., Rodriguez, M., Soca, A., Pasteur, N. & Raymond, M. Cross-resistance to pyrethroid and organophosphorus insecticides in the southern house mosquito (Diptera: Culicidae) from Cuba. J. Med. Entomol. 34, 244–246 (1997).f 8. Andrighetti, M. T. M., Cerone, F., Rigueti, M., Galvani, K. C. & Macoris, M. d. L. d. G. Effect of pyriproxyfen in Aedes aegypt populations with different levels of susceptibility to the organophosphate temephos. (2008). l f h b l f d ( l d ) l f d p pf p y g p p p 49. Campos, K. B. et al. Assessment of the susceptibility status of Aedes aegypti (Diptera: Culicidae) populations to pyriproxyfen and malathion in a national wide monitoring of insecticide resistance in Brazil, 2017–2018. (2020).ii 50. Lau, K. W., Chen, C. D., Lee, H. L., Norma-Rashid, Y. & Sofian-Azirun, M. Evaluation of insect growth regulators against f collected Aedes aegypti and Aedes albopictus (Diptera: Culicidae) from Malaysia. J. Med. Entomol. 52, 199–206 (2015). 50. Lau, K. W., Chen, C. D., Lee, H. L., Norma-Rashid, Y. & Sofian-Azirun, M. Evaluation of insect growth regulators against field- collected Aedes aegypti and Aedes albopictus (Diptera: Culicidae) from Malaysia. J. Med. Entomol. 52, 199–206 (2015). 51. Kawada, H. et al. Nationwide investigation of the pyrethroid susceptibility of mosquito larvae collected from used tires in Vietnam. PLoS Negl. Trop. Dis. 3, e391 (2009). 51. Kawada, H. et al. Nationwide investigation of the pyrethroid susceptibility of mosquito larvae collected from used tires in Viet PLoS Negl. Trop. Dis. 3, e391 (2009). g p 52. Smith, L. B., Kasai, S. & Scott, J. G. Pyrethroid resistance in Aedes aegypti and Aedes albopictus: Important mosquito vectors of human diseases. Pestic. Biochem. Physiol. 133, 1–12 (2016). y 3. Smith, L. B., Tyagi, R., Kasai, S. & Scott, J. G. CYP-mediated permethrin resistance in Aedes aegypti and evidence for trans- regulation. PLoS Negl. Trop. Dis. 12, e0006933 (2018). y 53. Smith, L. B., Tyagi, R., Kasai, S. & Scott, J. G. CYP-mediated permethrin resistance in Aede regulation. PLoS Negl. Trop. Dis. 12, e0006933 (2018). 53. Smith, L. www.nature.com/scientificreports/ Transcription of detoxification genes after permethrin selection in the mosquito Aedes aegypti. Insect Mol. Biol. 21, 61–77 (2012). 98 Rohlf F J & Sokal R R Statistical Tables (Macmillan New York 1995) 98. Rohlf, F. J. & Sokal, R. R. Statistical Tables (Macmillan, New York, 1995).fli 99. Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 29, e45–e45 (2001). www.nature.com/scientificreports/ Biochem. Physiol. 104, 132–135 (2012).i 61. Poupardin, R., Srisukontarat, W., Yunta, C. & Ranson, H. Identification of carboxylesterase genes implicated in temephos resistance in the dengue vector Aedes aegypti. PLoS Negl. Trop. Dis. 8, 2 (2014). g gyp g p 2. Ishak, I. H., Jaal, Z., Ranson, H. & Wondji, C. S. Contrasting patterns of insecticide resistance and knockdown resistance (kdr) in the dengue vectors Aedes aegypti and Aedes albopictus from Malaysia. Parasit. Vect. 8, 181 (2015). 63. Wuliandari, J. R. et al. Association between three mutations, F1565C, V1023G and S996P, in the voltage-sensitive sodium channel gene and knockdown resistance in Aedes aegypti from Yogyakarta Indonesia. Insects 6, 658–685 (2015). https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid insecticides in Aedes albopictus populations from Haikou City, Hainan Island China. Infect. Dis. Poverty 5, 31 (2016). i g py in Aedes albopictus populations from Haikou City, Hainan Island China. Infect. Dis. Poverty 5, 31 (2016). p p p y f y 71. Al Nazawi, A. M., Aqili, J., Alzahrani, M., McCall, P. J. & Weetman, D. Combined target site (kdr) mutations play a p in highly pyrethroid resistant phenotypes of Aedes aegypti from Saudi Arabia. Parasit. Vect. 10, 161 (2017). 2 H ddi K l D i f h id i i (V410L) i h di h l f A d i in highly pyrethroid resistant phenotypes of Aedes aegypti from Saudi Arabia. Parasit. Vect. 10, 161 (2017). 72. Haddi, K. et al. Detection of a new pyrethroid resistance mutation (V410L) in the sodium channel of Aedes aegypti: A potential challenge for mosquito control. Sci. Rep. 7, 46549 (2017). 72. Haddi, K. et al. Detection of a new pyrethroid resistance challenge for mosquito control. Sci. Rep. 7, 46549 (2017) 72. Haddi, K. et al. Detection of a new pyrethroid resistance mutation (V410L) in the sodium channel of Aedes aegypti: A pot challenge for mosquito control. Sci. Rep. 7, 46549 (2017). g q p ( ) 73. Stenhouse, S. A. et al. Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand. Parasit. Vect. 6, 253 (2013). 4. Fan, Y. et al. Evidence for both sequential mutations and recombination in the evolution of kdr alleles in Aedes aegypti. PLoS Negl Trop. Dis. 14, e0008154 (2020). p 5. Moyes, C. L. et al. Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans. PLoS Negl. Trop. Dis. 11, 2 (2017). g p ( ) 76. Amelia-Yap, Z. H., Chen, C. D., Sofian-Azirun, M. & Low, V. L. Pyrethroid resistance in the dengue vector Aedes aegypti in Southeast Asia: Present situation and prospects for management. Parasit. Vect. 11, 332 (2018). 77. Powell, J. R. & Tabachnick, W. J. History of domestication and spread of Aedes aegypti—a review. Mem. Inst. Oswaldo Cruz 108, 11–17 (2013).i 78. Rueda, L. M. www.nature.com/scientificreports/ Pictorial keys for the identification of mosquitoes (Diptera: Culicidae) associated with dengue virus transmis (Walter Reed Army Inst Of Research Washington Dc Department Of Entomology, 2004). 79. Kuno, G. Early history of laboratory breeding of Aedes aegypti (Diptera: Culicidae) focusing on the origins and use of selected strains. J. Med. Entomol. 47, 957–971 (2014). 79. Kuno, G. Early history of laboratory breeding of Aedes aegypti (Diptera: Culici strains. J. Med. Entomol. 47, 957–971 (2014). 80. WHO. Vol. WHO/VBC/81.805 4 (World Health Organization, Geneva, 1981). 81. Organization, W. H. Monitoring and managing insecticide resistance in Aedes mosquito populations: interim guidance for e mologists. (2016). g 82. Brito, L. P., Carrara, L., Freitas, R. M., Lima, J. B. P. & Martins, A. J. Levels of resistance to pyrethroid among distinct kdr alleles in Aedes aegypti laboratory lines and frequency of kdr Alleles in 27 natural populations from Rio de Janeiro Brazil. BioMed Res. Int. 20, 18 (2018). 83. Brogdon, W. & Chan, T. Guideline for evaluating insecticide resistance in vectors using the CDC bottle bioassay. (2011). O T d f d l ( ) 84. Organization, W. H. Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. (2016). g p g q ( ) 85. Casida, J. E. Mixed-function oxidase involvement in the biochemistry of insecticide synergists. J. Agric. Food Chem. 18, 753 (1970).f ( ) 86. Abbott, W. S. A method of computing the effectiveness of an insecticide. J. Econ. Entomol 18, 265–267 (1925). f 87. Finney, D. Probit Analysis (Cambridge University Press, Cambridge, 1971). f 7. Finney, D. Probit Analysis (Cambridge University Press, Cambridge, 1971). 88. Linss, J. G. B. et al. Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations. Parasit. Vect. 7, 25 (2014).fi 9. Brito, L. P. et al. Assessing the effects of Aedes aegypti kdr mutations on pyrethroid resistance and its fitness cost. PLoS ONE 8, 2 (2013).t 90. Kearse, M. et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649. https://doi.org/10.1093/bioinformatics/bts199 (2012). 1. Dusfour, I. et al. Deltamethrin resistance mechanisms in Aedes aegypti populations from three French overseas territories world- wide. PLoS Negl. Trop. Dis. 9, e0004226 (2015). wide. PLoS Negl. Trop. Dis. 9, e0004226 (2015). g p , ( ) 92. Faucon, F. et al. www.nature.com/scientificreports/ Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing. Genome Res. 25, 1347–1359 (2015). targeted sequencing. Genome Res. 25, 1347–1359 (2015). g q g ( ) 93. Lü, J., Yang, C., Zhang, Y. & Pan, H. Selection of reference genes for the normalization of RT-qPCR data in gene expression studies in insects: A systematic review. Front. Physiol. 9, 1560 (2018). 3. Lü, J., Yang, C., Zhang, Y. & Pan, H. Selection of reference genes for the normalization of RT-qPCR data in gene expression studies in insects: A systematic review. Front. Physiol. 9, 1560 (2018). 4 Matthe s B J et al Impro ed reference genome of Aedes aeg pti informs arbo ir s ector control Nature 563 501 507 (2018) B. J. et al. Improved reference genome of Aedes aegypti informs ar 94. Matthews, B. J. et al. Improved reference genome of Aedes aeg , J p g gyp , ( ) 5. Bariami, V., Jones, C. M., Poupardin, R., Vontas, J. & Ranson, H. Gene amplification, ABC transporters and cytochrome P450s Unraveling the molecular basis of pyrethroid resistance in the dengue vector Aedes aegypti. PLoS Negl. Trop. Dis. 6, e1692 (2012) 6. Kasai, S. et al. Mechanisms of pyrethroid resistance in the dengue mosquito vector, Aedes aegypti: Target site insensitivity, penetra- tion, and metabolism. PLoS Negl. Trop. Dis. 8, e2948 (2014). 95. Bariami, V., Jones, C. M., Poupardin, R., Vontas, J. & Ranson, H. Gene amplification, ABC transporters and cytochrome P450s: Unraveling the molecular basis of pyrethroid resistance in the dengue vector Aedes aegypti. PLoS Negl. Trop. Dis. 6, e1692 (2012). 96 Kasai S et al Mechanisms of p rethroid resistance in the deng e mosq ito ector Aedes aeg pti Target site insensiti it penetra Unraveling the molecular basis of pyrethroid resistance in the dengue vector Aedes aegypti. PLoS Negl. Trop. Dis. 6, e1692 (2012) 6. Kasai, S. et al. Mechanisms of pyrethroid resistance in the dengue mosquito vector, Aedes aegypti: Target site insensitivity, penetra- tion, and metabolism. PLoS Negl. Trop. Dis. 8, e2948 (2014).it 96. Kasai, S. et al. Mechanisms of pyrethroid resistance in the dengue mosquito vector, Aedes aegypti: T tion, and metabolism. PLoS Negl. Trop. Dis. 8, e2948 (2014).it 96. Kasai, S. et al. Mechanisms of pyrethroid resistance in the deng d b l l ( ) 97. Saavedra-Rodriguez, K. et al. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 64. Plernsub, S. et al. Temporal frequency of knockdown resistance mutations, F1534C and V1016G, in Aedes aegypti in Chiang Mai city, Thailand and the impact of the mutations on the efficiency of thermal fogging spray with pyrethroids. Acta Trop. 162, 125–132 (2016).i ( ) 65. Koou, S.-Y., Chong, C.-S., Vythilingam, I., Lee, C.-Y. & Ng, L.-C. Insecticide resistance and its underlying mechanisms in populations of Aedes aegypti adults (Diptera: Culicidae) in Singapore. Parasit. Vect. 7, 471 (2014). 6. Kawada, H. et al. Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypt populations in Myanmar. PLoS Negl. Trop. Dis. 8, 2 (2014). populations in Myanmar. PLoS Negl. Trop. Dis. 8, 2 (2014). p p y g p 7. Muthusamy, R. & Shivakumar, M. Involvement of metabolic resistance and F1534C kdr mutation in the pyrethroid resistance mechanisms of Aedes aegypti in India. Acta Trop. 148, 137–141 (2015). y g p 67. Muthusamy, R. & Shivakumar, M. Involvement of metabo mechanisms of Aedes aegypti in India. Acta Trop. 148, 137–14 gyp p ( ) 8. Fernando, S. D. et al. First report of V1016G and S989P knockdown resistant (kdr) mutations in pyrethroid-resistant Sri Lankan Aedes aegypti mosquitoes. Parasit. Vect. 11, 526 (2018).i Aedes aegypti mosquitoes. Parasit. Vect. 11, 526 (2018). gyp q 69. Zhou, X. et al. Knockdown resistance (kdr) mutations within seventeen field populations of Aedes albopictus from Beijing China: first report of a novel V1016G mutation and evolutionary origins of kdr haplotypes. Parasit. Vect. 12, 180 (2019). i p y g p yp ( ) 70. Chen, H. et al. First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid in d lb l f k l d h f ( ) i p y g p yp ( ) 0. Chen, H. et al. First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid insecticides in Aedes albopictus populations from Haikou City Hainan Island China Infect Dis Poverty 5 31 (2016) i p y g p yp ( 70. Chen, H. et al. First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyr in Aedes albopictus populations from Haikou City, Hainan Island China. Infect. Dis. Poverty 5, 31 (2016). 70. Chen, H. et al. Author contributions d f l d Ru.R. and A.J.M. formulated the original idea. Ru.R., I.U., M.A.M. and S.K. collected the samples, identified them, created F1 and helped in the logistics. Ru.R., L.P.B., L.C.S., B.S.S. and M.M.C. carried out biological and molecular assays. A.J.M. and Ru.R. performed the statistical analysis. Ru.R. and A.J.M. compiled the M.S. and M.A.M. and J.B.P.L. made the necessary editions. Acknowledgements g We would like to thank the World Academy of Sciences (TWAS), the Brazilian National Council for Scientific and Technological Research (CNPq), and the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES). We extend our gratitude to Professor Dr. Muhammad Tahir (Chairperson Zoology depart- ment, GC University Lahore) for his permission to use their insectary, Muhammad Asif, for his support during field visits and logistic support. Daniel Marland is thanked for the thorough review of the manuscript and for correcting all the grammatical and textual errors. This study was funded by Instituto Nacional em Ciência e Tecnologia em Entomologia Molecular (INCT-EM) (Grant No. 465678/2014-9) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Grant No. E-26/202.795/2019). https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \| www.nature.com/scientificreports/ Competing interests h p g The authors declare no competing interests. © The Author(s) 2021 Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1038/s41598-021-83465-w. Correspondence and requests for materials should be addressed to A.J.M. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-83465-w Scientific Reports \| (2021) 11:4555 \|
https://openalex.org/W2796633818	https://hal.sorbonne-universite.fr/hal-01708773/file/s41598-018-20281-9.pdf	English	null	New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia	Scientific reports	2,018	cc-by	9,050	To cite this version: Sandy Elbitar, Delia Susan-Resiga, Youmna Ghaleb, Petra El Khoury, Gina Peloso, et al.. New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholes- terolemia. Scientific Reports, 2018, 8, pp.1943. 10.1038/s41598-018-20281-9. hal-01708773 New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia Sandy Elbitar, Delia Susan-Resiga, Youmna Ghaleb, Petra El Khoury, Gina Peloso, Nathan Stitziel, Jean-Pierre Rabès, Valérie Carreau, Josée Hamelin, Ali Ben-Djoudi-Ouadda, et al. Distributed under a Creative Commons Attribution 4.0 International License New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia Received: 18 October 2017 Accepted: 15 January 2018 Published: xx xx xxxx Received: 18 October 2017 Accepted: 15 January 2018 Published: xx xx xxxx Sandy Elbitar 1,2,3, Delia Susan-Resiga 4, Youmna Ghaleb1,2,3, Petra El Khoury1,2, Gina Peloso5, Nathan Stitziel6, Jean-Pierre Rabès 1,7, Valérie Carreau8, Josée Hamelin4, Ali Ben- Djoudi-Ouadda4, Eric Bruckert8, Catherine Boileau1,3,9, Nabil G. Seidah4, Mathilde Varret 1,3 & Marianne Abifadel1,2 Autosomal dominant hypercholesterolemia (ADH) is characterized by elevated LDL-C levels leading to coronary heart disease. Four genes are implicated in ADH: LDLR, APOB, PCSK9 and APOE. Our aim was to identify new mutations in known genes, or in new genes implicated in ADH. Thirteen French families with ADH were recruited and studied by exome sequencing after exclusion, in their probands, of mutations in the LDLR, PCSK9 and APOE genes and fragments of exons 26 and 29 of APOB gene. We identified in one family a p.Arg50Gln mutation in the APOB gene, which occurs in a region not usually associated with ADH. Segregation and in-silico analysis suggested that this mutation is disease causing in the family. We identified in another family with the p.Ala3396Thr mutation of APOB, one patient with a severe phenotype carrying also a mutation in PCSK9: p.Arg96Cys. This is the first compound heterozygote reported with a mutation in APOB and PCSK9. Functional studies proved that the p.Arg96Cys mutation leads to increased LDL receptor degradation. This work shows that Next-Generation Sequencing (exome, genome or targeted sequencing) are powerful tools to find new mutations and identify compound heterozygotes, which will lead to better diagnosis and treatment of ADH. Hypercholesterolemia is a major risk factor for atherosclerosis and its premature cardiovascular complications. Autosomal dominant hypercholesterolemia (ADH) (MIM # 143890) is one of the most common monogenic dis- orders. It is caused by mutations in genes encoding key proteins involved in the LDL receptor (LDLR) endocytic and recycling pathways, causing decreased cellular uptake of LDL and increased plasma LDL-cholesterol (LDL-C) concentrations. Elevated plasma LDL-C levels give rise to tendon and skin xanthomas, arcus cornea, and vascular deposits, leading to progressive and premature atherosclerosis and coronary heart disease (CHD)1. HAL Id: hal-01708773 https://hal.sorbonne-universite.fr/hal-01708773v1 Submitted on 14 Feb 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License www.nature.com/scientificreports www.nature.com/scientificreports Received: 18 October 2017 Accepted: 15 January 2018 Published: xx xx xxxx New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia In-silico prediction analysis of both mutations using Polyphen, Sift, and Mutation taster tools. catalytic activity6; and APOE7. Mutations in LDL Receptor Adaptor protein 1 (LDLRAP1) gene are responsible of the recessive form of the disease8.h The prevalence of ADH is approximately estimated to 1/217 in Northern Europe9,10 but varies geographically from a region to another and is higher in some populations due to a founder effect11. Recent studies showed that ADH is under-diagnosed and undertreated in the general population12 and the probability of identifying a genetic component in individuals with hypercholesterolemia increases when LDL-C levels increased10.h The respective contribution of each known gene to ADH slightly varies from one country to another. A study in a French cohort, which included probands and families with hypercholesterolemia recruited through the French Research Network for ADH from several regions of France, showed that the LDLR gene is implicated in 73.9% of the cases, while mutations in APOB and PCSK9 are responsible of 6.6% and 0.7% of the cases respec- tively. Nevertheless, in 18.8% of the ADH probands studied, no mutation was found13. y p Genetic diagnosis of ADH was generally performed by direct DNA sequencing or a combination of direct sequencing with the multiplex ligation-dependent probe amplification (MLPA) to detect large insertion or dele- tion mutations in genes known to be implicated in the disease. Concerning the APOB gene, the p.Arg3527Gln, also known as APOB3527 or APOB3500, is the first and most common ADH-related mutation in APOB reported in late 1980s3. It is responsible alone for more than 95% of Familial Defective apolipoprotein B cases (FDB)14 (MIM# 144010). A few other mutations leading to hypercholesterolemia were described in the following years and were all located in a specific region of the APOB gene. Therefore, this gene was not classically entirely studied in ADH by Sanger sequencing and routinely only a fragment of exon 26 and another of exon 29 are analyzed, covering the regions where the functional mutations causing hypercholesterolemia have been described15,16. g g g yp Targeted next-generation sequencing (NGS) panel is now currently used to screen for ADH-causing muta- tions10,17, while exome sequencing (targeted sequencing of all protein-coding regions of the genome) is used to identify mutations in new genes. New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia Four genes are known to be implicated in the disease: LDLR, the gene encoding the low-density lipoprotein receptor2; APOB, which encodes the apolipoprotein B3, the ligand of the LDL receptor; PCSK9 (Proprotein convertase subtilisin kexin 9)4, which encodes a serine protease5 that plays a role in the degradation of the LDLR independently of its 1INSERM LVTS U1148, hôpital Bichat-Claude Bernard, Paris, France. 2Laboratory of Biochemistry and Molecular Therapeutics, Faculty of Pharmacy, Pôle Technologie- Santé, Saint-Joseph University, Beirut, Lebanon. 3Paris Diderot University, Paris, France. 4Laboratory of Biochemical Neuroendocrinology, Institut de Recherches Cliniques de Montréal, Affiliated to the Université de Montréal, Montréal, Québec, H2W1R7, Canada. 5Department of Biostatistics, School of Public Health, Boston University, Boston, MA, 02216, USA. 6Division of Cardiology, Department of Medicine; Department of Genetics, McDonnell Genome Institute, Washington University School of Medicine, Saint Louis, MO, USA. 7Assistance Publique-Hôpitaux de Paris, HUPIFO, hôpital Ambroise-Paré, Laboratoire de Biochimie et de Génétique Moléculaire, Boulogne-Billancourt et UVSQ, UFR des Sciences de la Santé Simone Veil, Montigny-le-Bretonneux, France. 8Assistance Publique-Hôpitaux de Paris, Endocrinology and Nutrition Department, Human Research Nutrition Center, Pitié-Salpêtrière Hospital, F-75013, Paris, France. 9Service de Génétique, hôpital Bichat-Claude Bernard, Paris, France. Correspondence and requests for materials should be addressed to M.A. (email: marianne.abifadel@usj.edu.lb) Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 1 www.nature.com/scientificreports/ Figure 1. Pedigree of family HC138 with status of the p.Ala3396Thr mutation of APOB and the p.Arg96Cys of PCSK9 for each patient. (A) Lipid levels are given when available in mg/dL with the age at clinical measurement. The −/− indicates the absence of the mutation while +/− indicates the heterozygous carriers. The asterisk shows the patients studied by exome sequencing. (B–C) Conservation of the alanine at position 3396 of APOB and the arginine at position 96 of PCSK9 between different species. In-silico prediction analysis of both mutations using Polyphen, Sift, and Mutation taster tools. Figure 1. Pedigree of family HC138 with status of the p.Ala3396Thr mutation of APOB and the p.Arg96Cys of PCSK9 for each patient. (A) Lipid levels are given when available in mg/dL with the age at clinical measurement. The −/− indicates the absence of the mutation while +/− indicates the heterozygous carriers. The asterisk shows the patients studied by exome sequencing. (B–C) Conservation of the alanine at position 3396 of APOB and the arginine at position 96 of PCSK9 between different species. New Sequencing technologies help revealing unexpected mutations in Autosomal Dominant Hypercholesterolemia This approach, now slowly substituted by whole genome sequencing, has emerged as an effective tool for gene discovery in families with suspected monogenic disorders18.ht gf g y p g The aim of our study was to investigate the genetic causes of ADH in French probands. After the exclusion in 127 ADH probands, of mutations in the LDLR, APOE and PCSK9 genes and fragments of exons 26 and 29 of APOB, thirteen ADH families were recruited. Two or three affected members of each family were studied by exome sequencing in order to identify new mutations in known genes or in new genes, which will eventually lead to a better understanding of the mechanism of this disease. Results The arginine at position 96 is highly conserved among species and its replacement by a cysteine is predicted to be pathologic by the in silico prediction tools (Fig. 1C). Characterization of the p.Arg96Cys PCSK9 mutation. Functional analyses were performed to study the effect of the p.Arg96Cys PCSK9 mutation on PCSK9 maturation and on PCSK9-mediated LDLR degrada- tion. PCSK9-WT and p.Ser127Arg (S127R), a well-characterized gain-of-function (GOF) mutation, were used as controls. Cell-based functional characterizations of wild-type (WT) PCSK9 and mutants R96C and S127R in HEK293 and HepG2 cells are shown in Fig. 2. Biosynthetic analysis (Fig. 2A), Western Blot (WB) (Fig. 2B) and Elisa (Fig. 2C) analyses for PCSK9 showed that the p.Arg96Cys mutation results in increased cellular levels of total PCSK9 (~60%), reduced processing of proPCSK9 zymogen to PCSK9 (by ~30%), and decreased PCSK9 secretion (by ~60%) relative to WT protein. Overexpression of PCSK9-R96C results in significantly increased LDLR degradation compared to PCSK9-WT. This was confirmed by WB and by Elisa assays for total overex- pressed LDLR after V5-tagged LDLR was co-transfected in HEK293 cells with V5-tagged PCSK9-WT (WT), PCSK9-R96C (R96C), PCSK9-S127R (S127R) or empty vector (V), as control (Fig. 2D), or in HepG2 cells (Fig. 2E). Similar results were obtained for endogenous LDLR in HepG2 cells after overexpression of WT and mutants PCSK9 (Fig. 2F). Measurement of Dil-LDL uptake under these conditions (Fig. 2F, right panel), showed that R96C PCSK9, like the GOF S127R, was significantly more potent than WT PCSK9 in reducing LDL uptake. However, when added extracellularly to HepG2 cells for short or long times, PCSK9-R96C degraded endogenous LDLR to the same extent as PCSK9-WT (Fig. 2G). A new mutation p.Arg50Gln in APOB, causing ADH. Furthermore, exome sequencing was performed in the HC706 family using DNA from II.6, II.7 and III.2 affected individuals (with asterisk in Fig. 3). The female proband (III.2) presented high levels of total cholesterol (235 mg/dL) and LDL-C (155 mg/dL) at the age of 44 even after treatment with rosuvastatin 20 mg and Ezetimibe. Family history indicated that her 65 years-old father (II.1) had a level of total cholesterol of 300 mg/dL before treatment, and kept high levels of total cholesterol (246 mg/dL) and LDL-C (149 mg/dL) at 65 years old under treatment. Results A double heterozygous patient with a mutation in APOB and PCSK9 in HC138 family. Exome sequencing was used to investigate the genetic cause of ADH in the HC138 family using DNA from patients I.1 and II.1 (Fig. 1). The proband II.1 had a history of arcus cornea with high levels of total-cholesterol (201 mg/dL) Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 2 www.nature.com/scientificreports/ and LDL-C (130 mg/dL) despite treatment by rosuvastatin 10 mg and Ezetimibe. His father I.1 suffered from myocardial infarction at the age of 50. He also presented arcus cornea and had a very high level of total-choles- terol reaching 440 mg/dL before he started statin treatment. The familial autosomal dominant transmission was confirmed by the recruitment of other members of the family. Details of their clinical measurements are given in Fig. 1A. and LDL-C (130 mg/dL) despite treatment by rosuvastatin 10 mg and Ezetimibe. His father I.1 suffered from myocardial infarction at the age of 50. He also presented arcus cornea and had a very high level of total-choles- terol reaching 440 mg/dL before he started statin treatment. The familial autosomal dominant transmission was confirmed by the recruitment of other members of the family. Details of their clinical measurements are given in Fig. 1A. g Exome sequencing of the two patients allowed the identification of the c.10186 G > A mutation in exon 26 of the APOB gene, which led to the substitution p.Ala3396Thr as previously described19. This mutation was then confirmed by Sanger sequencing. It segregated with the disease in the family with a penetrance of 100% and with no phenocopy. It was not reported in the different databases: Exome Variant Server (evs.gs.washington. edu/EVS/), dbSNP (ncbi.nlm.nih.gov/SNP/), and gnomAD browser (gnomad.broadinstitute.org/). The in-silico bioinformatics tools predicted that it is disease causing, and showed a high conservation of the alanine at position 3396 among different species (Fig. 1B). gf p ( g ) Furthermore, exome sequencing analysis showed in patient I.1 a variation in exon 2 of PCSK9: c.286 C > T leading to the p.Arg96Cys substitution. This variant was confirmed by Sanger sequencing. It was not carried by any other member of the family (Fig. 1A). The p.Arg96Cys (rs185392267) is reported with a very low frequency of 2.166e-5 (only 6 times over 277054 in gnomAD browser). Results Interestingly, a paternal aunt (II.6) also had a history of elevated level of total cholesterol (315 mg/dL before treatment) that reached 253 mg/dL on statin treatment with a LDL-C level of 148 mg/dL at the age of 68. Additionally, a paternal uncle (II.7) presented a level of total cholesterol of 410 mg/dL and LDL-C level of 319 mg/dL at the age of 37 before starting statin treatment. Recruitment of other family members and further investigations confirmed the autosomal dominant trait. Lipid measurements of the different members of the family before treatment (when available) or after are provided in Fig. 3A.i g A substitution c.149 G > A in exon 3 in the APOB gene causing a missense variant p.Arg50Gln was identified in the three affected members studied by exome sequencing. Sanger sequencing was used to validate the variation and showed that the variation segregated with the disease in the family with a penetrance of 91% and absence of phenocopy. Only one case of incomplete penetrance was detected over the eleven studied members of the family for whom DNA were available: individual III.8 who carries the variation but does not present high lipid levels as depicted in Fig. 3. This variant is not reported in the databases. The arginine residue at position 50 is a conserved amino acid in different species (Fig. 3B). The p.Arg50Gln variant is predicted to be damaging by bioinformatics tools such as Sift, mutation taster and Polyphen-2 as shown in Fig. 3C. t y g Subsequently, we analyzed by Sanger sequencing the DNA of 127 French probands with hypercholesterolemia in whom we excluded the known mutations in LDLR, APOE and PCSK9 genes and fragments of exons 26 and 29 of APOB, to look for the p.Arg50Gln variation in the APOB gene. None of those probands carried this variation. Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 Discussion Measured values are reported as % control. (G) HepG2 cells were incubated for 7 h or 18 h with conditioned media from HEK293 cells (produced, illustrated and quantified in B): no PCSK9 control-media (V) or PCSK9-media (330 ng/ml), WT, R96C or S127R, and analyzed by ELISA for total cellular LDLR. Data are representative of two independent experiments performed at least in duplicate, with the exception of (F), where LDLR-ELISA was completed for one experiment performed in triplicate, while Dil-LDL uptake was measured in one experiment performed in 16 independent replicates per condition. Quantifications are averages ± SD. p < 0.05; p < 0.01; *p < 0.001 (t-test). Full- length blots of Fig. 2A,B and D are presented in Supplementary Figure 2, where only the lanes selected in Fig. 2 are labeled. Figure 2. Cell-based functional characterization of PCSK9 wild-type (WT) and p.Arg96Cys (R96C). HEK293 cells were transiently transfected with V5-tagged PCSK9-WT (WT), PCSK9-R96C (R96C) or empty vector (V) (A), or gain of function PCSK9-S127R (S127R) (positive control) (B and C) and analyzed for PCSK9 cellular expression, zymogen processing and secretion of mature protein. (A) Biosynthetic analysis; 48 h post- transfection cells were pulsed-labeled with [35S]Met/Cys for 3 h, followed by anti-V5 immunoprecipitation, SDS-PAGE and autoradiography. (B) WB analysis using anti V5-HRP. Levels of total cellular and secreted PCSK9 were quantified by ELISA and the concentrations are listed. The bands corresponding to proPCSK9 and PCSK9 in (B) were quantified, their values normalized to β−Actin and ratios of the value of each form to the sum of the two forms were graphed in (C). V5-tagged LDLR was co-transfected with V5-tagged PCSK9-WT, PCSK9-R96C, PCSK9-S127R or empty vector, as control, in HEK293 cells (D) or HepG2 cells (E). Transfected HEK293 cells were analyzed by WB using anti V5-HRP (D). Total cellular levels of LDLR were quantified from the WB and by ELISA for HEK293 cells (D) or by ELISA only for HepG2 cells (E) and were normalized to values of the control. (F) HepG2 cells were transiently transfected with V5-tagged PCSK9-WT, PCSK9-R96C, PCSK9-S127R or empty vector, as control, and the total cellular endogenous levels of LDLR were quantified by ELISA (left). Dil-LDL uptake (right) was measured over 2 h. Measured values are reported as % control. Discussion In this study, we show that new sequencing technologies like exome sequencing are powerful tools to find new mutations in genes already known to be implicated in ADH, APOB in particular, especially because the Sanger sequencing strategies classically performed in ADH genetic diagnosis do not target the entire region of APOB. Indeed, we identified by exome sequencing in 2 unrelated French ADH families, 2 new mutations in the APOB gene, p.Arg50Gln and p.Ala3396Thr which are not located in the regions of exons 26 and 29 routinely investigated using Sanger sequencing when searching for APOB mutations implicated in ADH. In fact, direct Sanger sequenc- ing has been classically targeting only these particular regions due to the size of the apoB (4536 amino acids) and because the previously described functional ADH mutations are located in these specific regions of the gene15,16 (Fig. 4). Although numerous other mutations of APOB were reported in its entire coding-region, these mutations Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 3 www.nature.com/scientificreports/ Figure 2. Cell-based functional characterization of PCSK9 wild-type (WT) and p.Arg96Cys (R96C). HEK293 cells were transiently transfected with V5-tagged PCSK9-WT (WT), PCSK9-R96C (R96C) or empty vector (V) (A), or gain of function PCSK9-S127R (S127R) (positive control) (B and C) and analyzed for PCSK9 cellular expression, zymogen processing and secretion of mature protein. (A) Biosynthetic analysis; 48 h post- transfection cells were pulsed-labeled with [35S]Met/Cys for 3 h, followed by anti-V5 immunoprecipitation, SDS-PAGE and autoradiography. (B) WB analysis using anti V5-HRP. Levels of total cellular and secreted PCSK9 were quantified by ELISA and the concentrations are listed. The bands corresponding to proPCSK9 and PCSK9 in (B) were quantified, their values normalized to β−Actin and ratios of the value of each form to the sum of the two forms were graphed in (C). V5-tagged LDLR was co-transfected with V5-tagged PCSK9-WT, PCSK9-R96C, PCSK9-S127R or empty vector, as control, in HEK293 cells (D) or HepG2 cells (E). Transfected HEK293 cells were analyzed by WB using anti V5-HRP (D). Total cellular levels of LDLR were quantified from the WB and by ELISA for HEK293 cells (D) or by ELISA only for HepG2 cells (E) and were normalized to values of the control. (F) HepG2 cells were transiently transfected with V5-tagged PCSK9-WT, PCSK9-R96C, PCSK9-S127R or empty vector, as control, and the total cellular endogenous levels of LDLR were quantified by ELISA (left). Dil-LDL uptake (right) was measured over 2 h. Discussion (G) HepG2 cells were incubated for 7 h or 18 h with conditioned media from HEK293 cells (produced, illustrated and quantified in B): no PCSK9 control-media (V) or PCSK9-media (330 ng/ml), WT, R96C or S127R, and analyzed by ELISA for total cellular LDLR. Data are representative of two independent experiments performed at least in duplicate, with the exception of (F), where LDLR-ELISA was completed for one experiment performed in triplicate, while Dil-LDL uptake was measured in one experiment performed in 16 independent replicates per condition. Quantifications are averages ± SD. p < 0.05; p < 0.01; *p < 0.001 (t-test). Full- length blots of Fig. 2A,B and D are presented in Supplementary Figure 2, where only the lanes selected in Fig. 2 are labeled. are known to cause familial hypocholesterolemia (hypobetalipoproteinemia or FHBL) and are mostly nonsense, frameshift, or splicing variants that lead to various C-terminally truncated apoB species20 (Fig. 4). As for FDB, the most common mutation associated with an ADH phenotype is the p.Arg3527Gln carried by approximately 0.1% of Northern Europeans and US Caucasians. In addition, the p.Arg3527Trp variant is known to make a significant contribution to familial hypercholesterolemia among East Asians21. Few APOB mutations were proven to be pathogenic in ADH: p.Arg3507Trp, p.Arg3527Gln, and p.Trp4396Tyr15,22. Despite the fact that these mutations are not directly involved in the binding to the LDLR segment that concerns residues 3386 to 339623 (Fig. 4), they might destabilize the apoB–LDL receptor interaction by altering some critical residues that are crucial for apoB– LDL receptor affinity24, which result in a defective receptor-binding23,25. Interestingly, the p.Ala3396Thr mutation that we identified recently19 occurs in exon 26 of APOB and is located in the LDLR binding site B of apoB (Fig. 4). Consequently, this mutation might lead to a defective apoB-LDLR binding and an abnormal LDL internalization. Its good segregation with the disease in the family along with its localization in an important region for the nor- mal apoB function are strong evidences for its pathogenic effect. p g p gf Add to that, the p.Arg50Gln mutation is new and occurs in exon 3 of APOB, a region not routinely analyzed in ADH. Discussion Several arguments are in favor of a mutation responsible of the ADH phenotype in this family: (1) it showed good segregation with the disease in the family, (2) in-silico prediction tools are in favor of its deleterious Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 4 www.nature.com/scientificreports/ Figure 3. Segregation analysis of the p.Arg50Gln mutation of APOB in family HC706: (A) Lipid levels are given when available in mg/dL with the age of the patients at clinical measurement. The −/− indicates absence of the mutation while +/− indicates heterozygous carriers. The asterisk shows the patients studied by exome sequencing. (B) The arginine at position 50 of APOB is a conserved amino acid between different species. (C) This mutation is predicted to be probably damaging by Polyphen, tolerated by Sift, and disease causing by mutation taster when studied in-silico. Figure 3. Segregation analysis of the p.Arg50Gln mutation of APOB in family HC706: (A) Lipid levels are given when available in mg/dL with the age of the patients at clinical measurement. The −/− indicates absence of the mutation while +/− indicates heterozygous carriers. The asterisk shows the patients studied by exome sequencing. (B) The arginine at position 50 of APOB is a conserved amino acid between different species. (C) This mutation is predicted to be probably damaging by Polyphen, tolerated by Sift, and disease causing by mutation taster when studied in-silico. Figure 4. Different mutations reported in APOB, causing hypercholesterolemia (FDB) and hypocholesterolemia (FHBL). The APOB gene is constituted of 29 exons. The binding site for the LDL receptor originally described as site B is formed primarily by residues 3386–3396 (anciently known by 3359–3369). Regions 3475–3635 and 4363–4460 are the ones we classically sequence when looking for APOB mutations in ADH. Mutations causing familial hypocholesterolemia (FHBL) are distributed on the entire coding-region, and they are mostly nonsense, frameshift, or splicing variants. Few mutations causing hypercholesterolemia (FDB) are described in a particular region of APOB, the p.Arg3527Gln mutation being the most frequent one. Some others have been recently reported to cause ADH outside the classical regions of APOB, shown in italic; the p.Arg50Gln and p.Ala3396Thr are detailed in this article and highlighted in bold. Figure 4. Different mutations reported in APOB, causing hypercholesterolemia (FDB) and hh Figure 4. Different mutations reported in APOB, causing hypercholesterolemia (FDB) and hypocholesterolemia (FHBL). The APOB gene is constituted of 29 exons. Discussion The arginine at position 50 seems to be crucial for a normal cholesterol metabolism since 2 different mutations causing ADH have been identified at this same position. Interestingly, a recent study suggested that PCSK9 could bind to amino acid sequences within the N-terminal region of apoB28. Whether or not APOB mutations in this region might affect its interaction with other proteins such as PCSK9 for instance are still to be uncovered.hi Besides the p.Ala3396Thr identified in the HC138 family, the analyses of the variants observed in one of the members of this family in whom exome sequencing has been performed, allowed the identification of a new mutation in exon 2 of PCSK9: p.Arg96Cys. This patient carrying the 2 mutations in APOB and PCSK9 showed a severe phenotype with a maximal level of 440 mg/dL of total cholesterol before statin treatment. He also suffered from a myocardial infarction at the age of 50. The p.Arg96Cys mutation of PCSK9 has been recently reported to be responsible alone for the ADH phenotype in 3 patients from Denmark14, with mean untreated values of total cholesterol of 271.5 ± 46.0 mg/dL and LDL-C of 191.4 ± 34.4 mg/dL. Interestingly, 2 of those 3 patients presented coronary artery disease (CAD)14. Our functional studies performed herein demonstrated that the p.Arg96Cys mutation is a new GOF mutation of PCSK9, which alone may be responsible for the ADH phenotype. Although synthesized in cells at a higher level than the WT protein, the p.Arg96Cys is less secreted compared to WT. When overexpressed in cells, this GOF mutant degrades the LDLR to a higher extent than PCSK9-WT. The latter activity of PCSK9-R96C correlated with a decreased Dil-LDL uptake in HepG2 cells, an effect similar to that observed with the PCSK9-S127R GOF mutant. Interestingly, when added extracellularly to HepG2 cells, PCSK9-R96C degraded endogenous LDLR to the same extent as PCSK9-WT, whereas the GOF PCSK9-S127R displayed a sig- nificantly higher activity toward the LDLR. Collectively, our cell-based data suggest that PCSK9-R96C is a GOF mutant that better interacts with LDLR intracellularly (intracellular pathway)29, but similarly at the cell surface (extracellular pathway). In the liver, the extracellular pathway is predominant, as wild type PCSK9 acts mostly extracellularly on hepatocytes. However, the presence of a mutation on one allele of the PCSK9 gene could result in a shift of the prevalence of one pathway over the other, as was also reported for the LDLR-R410S mutation30. Discussion The binding site for the LDL receptor originally described as site B is formed primarily by residues 3386–3396 (anciently known by 3359–3369). Regions 3475–3635 and 4363–4460 are the ones we classically sequence when looking for APOB mutations in ADH. Mutations causing familial hypocholesterolemia (FHBL) are distributed on the entire coding-region, and they are mostly nonsense, frameshift, or splicing variants. Few mutations causing hypercholesterolemia (FDB) are described in a particular region of APOB, the p.Arg3527Gln mutation being the most frequent one. Some others have been recently reported to cause ADH outside the classical regions of APOB, shown in italic; the p.Arg50Gln and p.Ala3396Thr are detailed in this article and highlighted in bold. Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 5 www.nature.com/scientificreports/ effect at the protein level, (3) this variant was never reported before, and thus its rare incidence is in favor of a mutation rather than a polymorphism and (4) exome sequencing revealed no other interesting genetic event that could be responsible for the ADH phenotype in this family. Furthermore, another ADH causing mutation at the same position, the p.Arg50Trp, has been reported in another ADH family26 and was confirmed by exome sequencing in the UK10K project27. Thomas et al. showed that the p.Arg50Trp variant of APOB accumulates in the circulation of affected carriers, which suggest its defective hepatic uptake. In fact, exon 3 forms a small part of the first domain (βα1), which is predicted to direct hepatic assembly of lipoprotein molecules, as well as to affect the interaction of LDL particles with lipases and macrophage scavenger receptors. Nevertheless, detailed infor- mation on the specific role of exon 3 in these processes is not available26. pi p Furthermore, a recent study identified 2 novel APOB mutations, p.Arg3059Cys and p.Lys3394Asn, both asso- ciated with a significant decrease in binding to the LDL receptor, despite having a low penetrance when study- ing the co-segregation in the families16. Another study proved that p.Arg1164Thr and p.Gln4494del of APOB presented a 40% decrease in internalization in lymphocytes and HepG2 cells, very similar to APOB352715 even though they didn’t show complete penetrance. These results suggest that APOB can carry more ADH causing mutations outside of the classically studied regions, which means that all regions of the APOB should be investi- gated when diagnosing ADH. Discussion Thus, genetic evidence together with the cell-based functional characterization of PCSK9-R96C compared to WT and S127R are strong enough to conclude that the p.Arg96Cys is a GOF mutation of PCSK9, which alone could cause ADH, and aggravate the phenotype when carried together with another ADH causing mutation. gg p yp g g Consequently, exome sequencing helped us identify, in a patient with severe ADH, a mutation in APOB together with a new mutation in PCSK9. To the best of our knowledge, this is the first report of an ADH patient carrying a mutation in both APOB and PCSK9 genes concomitantly. Unfortunately, parents or other family mem- bers were not available for further investigations to rule out the possibility of a de-novo mutation that might have occurred in the PCSK9 gene in this patient, and that it was not transmitted to his children. These findings demonstrate that exome sequencing can help in the diagnosis and the identification of compound heterozygotes in ADH. In fact, some studies described patients and families with both LDLR/APOB31–36, or LDLR/PCSK9 muta- tions37,38. However, the clinical characteristics of double-heterozygous ADH patients are underreported and the diagnosis of double-heterozygous ADH can be easily missed39. Thus other APOB/PCSK9 double heterozygous might exist. It is noteworthy that the molecular identification of double heterozygosity is very important for fam- ily screening and adequate ADH diagnosis and treatment of all affected carriers of the family. Furthermore, the other genetic event occurring in another gene might explain differences in phenotypes and would be important for revealing the cause of phenocopies when studying familial segregation. This underscores the necessity of fully screening the LDLR, APOB, PCSK9 and APOE genes in all patients. g g p In summary, our work shows the importance of next generation sequencing technologies such as exome sequencing in identifying new mutations in the genes known to be implicated in ADH, and in revealing double heterozygous mutations, which improves familial screening and genetic counseling, as well as understanding the transmission of the disease and its severity in different members of the same family. The investigation for ADH mutations should include the entire coding regions of LDLR, PCSK9, APOE and APOB, with a particular attention to the region of the arginine at position 50 of the APOB. This will improve diagnosis and treatment of the disease and prevent its cardiovascular complications. Methods Starting with the total number of variants shared by individuals from the family, we excluded variants conflicting with the ADH inheritance pattern and common variants that have a frequency of ≥ 1% in the general population, by examining different databases such as dbSNP, 1000Genome, Exac and gnomAD browsers. Then, we excluded silent and non-genic variants which do not alter protein sequence since most Mendelian syndromes are caused by coding or splice site mutations that alter the protein sequence. The remaining single nucleotide variants and short insertions or dele- tions were considered candidates. The variants were subsequently analyzed separately by exploring those whose genes have physical protein-protein interaction with the 4 known genes of ADH, or share the same biological pathways. When an interesting variation is found, familial segregation was studied to demonstrate its possible co-segregation with the phenotype in the family. In silico Analyses. The frequency of the variations found by sequencing was estimated using different data- bases: Exome Variant Server (evs.gs.washington.edu/EVS/), dbSNP (ncbi.nlm.nih.gov/SNP/), and gnomAD browser (gnomad.broadinstitute.org/).hf The causal effect of each new molecular event was estimated with in silico prediction of protein function tools: Polyphen (genetics.bwh.harvard.edu/pph), SIFT (sift.jcvi.org), MutationTaster (mutationtaster.org) using Alamut Visual version 2.7.1. cDNAs, cell culture and transfections. The cDNAs encoding human LDLR30 and human PCSK9 and its mutants44 were cloned in pIRES2-EGFP (Clontech Labs), a bicistronic plasmid allowing the independent expres- sion of a fluorescent EGFP from an internal ribosome entry site (IRES) and C-terminally V5-tagged wild-type (WT) human PCSK9 or its p.Arg96Cys (R96C) mutant (identity confirmed by DNA sequencing) under the con- trol of a CMV promoter. The WT PCSK9 and the GOF PCSK9-S127R44 served as control. HEK293 (human embryonic kidney-derived epithelial) and HepG2 (human hepatocellular carcinoma) cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle medium (DMEM) (HEK293 cells) or in Eagle minimal essential medium (EMEM) (HepG2 cells) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen) and were maintained at 37 °C under 5% CO2. HEK293 cells were seeded in poly-L-lysine (50 µg/mL) coated 12-well plates (3.5 × 105 cells/well) or 10 cm plates (4 × 106 cells/well) for PCSK9 media pro- duction and the following day transfected using jetPRIME (PolyPlus) and a total of 0.5 µg of cDNA or 6 µg of cDNA, respectively. 24 h post-transfection, the culturing medium was changed to serum-free and the cells were treated according to each experiment. Methods Patients and Families. Probands and families from different cities in France were recruited by The French National Research Network on Hypercholesterolemia based on the inclusion criteria previously described13: LDL-C levels above the 95th percentile when compared with a sex and age-matched French population (STANISLAS cohort, B. Herbeth, G. Siest & S Visvikis-Siest41, personal communication), with normal levels of triglycerides and HDL-C, with an autosomal dominant transmission of hypercholesterolemia in the family. The study was performed in accordance with French bioethics regulations and all subjects gave informed con- sent. This study was conducted as part of trial # 05-07-06 approved by French Consultative Committee for the Protection of Person in Biomedical Research (CCPPRBs) Paris, Necker. Sanger Sequencing and MLPA. In all subjects, genes were studied sequentially: first, the p.Arg3527Gln mutation of the APOB (NM_000384.2) was looked for as previously described42 and then regions 3475–3635 and 4363–4460 of APOB were analyzed by Sanger sequencing. The promoters and the 18 exons of LDLR (NM_000527.4), as well as close flanking intronic sequences were amplified and sequenced. If no mutation was found, the search for a deletion/duplication of one or several LDLR’ exon(s) was performed with SALSA MLPA kit (P062) and data were analyzed with Coffalyser software (MRC-Holland). Finally, if no deletion/duplication was discovered, the 12 exons of PCSK9 (NM_174936.3) and the 4 exons of APOE (NM_000041.3) as well as the flanking intronic regions were sequenced. Primer sequences and annealing temperatures are available upon request. Electrophoregrams were analyzed using Gensearch®, or CodonCode Alligner®. Exome Sequencing. The family members analyzed by exome sequencing where chosen based on DNA availability for at least 2 affected members of the family and presence of informed consent allowing for genetic studies with prioritization of phenotypic extremes when possible. These selected samples underwent exome sequencing at the Broad Institute after that the institutional review board and all participating sites approved the study protocols and all individuals who were selected for sequencing provided informed consent as previously described19. Analysis of Exome Sequencing Data. In order to find the causal mutation out of thousands of differ- ent variations obtained by the exome sequencing, we used a comprehensive framework for prioritizing vari- ants, which is commonly used in the analysis of exome sequencing studies43. Discussion Physicians, clinical biochemists and health profession- als worldwide should join their effort to fight against this disease by offering the most adequate screening and diagnosis of individuals and families with ADH. Thus, genetic studies are of great importance in these extremely high-risk individuals and families. They can help implementing the most effective strategies to prevent and treat ADH, and might lead to new therapeutic class of lipid lowering drugs like it was the case with the discovery of PCSK940. Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 6 www.nature.com/scientificreports/ Methods Alternatively, for media swap experiments, HepG2 cells (3.5 × 105 cells/ well) were seeded in 12-well cell culture plates. 24 h later cells were starved for 24 h in serum-free media and following were incubated for 7 h or 18 h with 24 h serum-free conditioned media of HEK293 cells overexpress- ing human PCSK9 (WT, S127R or R96C) (see above). The concentrations of the secreted PCSK9 into the media were measured using an in-house ELISA assay as previously described45. For Dil-LDL uptake experiments and LDLR ELISA measurements, HepG2 cells were plated in 96-well plates (0.2 × 105 cells/well) (CellBind black plate with clear bottom, Corning; Cat # 3340) or 12-well plates (2 × 105 cells/well), respectively. Transfections were performed at the time of seeding with 0.125 µg of cDNA (96-well plate) or 1 µg of cDNA (12-well plate) and using FuGENE® HD (Promega). 24 h post-seeding and -transfection, the cells were starved for 24 h in serum-free media and treated according to each experiment. Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 7 www.nature.com/scientificreports/ Biosynthetic Analyses. 48 h post-transfection, HEK293 cells were washed and pulse-labeled for 3 h with 250 µCi/ml [35S]Met/Cys (PerkinElmer Life Sciences)44. Following 2 washes with ice-cold PBS, the cells were lysed in modified radioimmune precipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS) and protease inhibitor mixture (Roche Applied Science). Cell lysates and media were immunoprecipitated with monoclonal V5-Ab (1:500; Invitrogen) and immunoprecipitates were resolved by SDS-PAGE (8% Tris-Tricine gels) followed by autoradiography (2 h at −80 °C). Human LDLR ELISA in cell lysates. Following the treatments specific to each experiment, HEK293 or HepG2 cells were washed twice with ice-cold PBS and lysed on ice with ice-cold, non-denaturing cell lysis buffer (20 mM Tris-HCl, pH 8, 137 mM NaCl, 2 mM Na2EDTA, 1% NP-40, 10% glycerol, 4% protease inhibitor cocktail without EDTA) for 40 min, with gentle rotation. Cell lysates were cleared by centrifuging for 12 min at 15,000xg at 4 °C. The supernatants corresponding to the non-denatured cell lysates were saved and subjected to measurement of total human LDLR protein levels (human LDLR DuoSet ELISA Development kit, DY218; R&D Systems) and of total protein (Bio-Rad DC Protein assay), following the manufacturers’ protocol. The optical densities of the colored products were determined using a SpectraMax i3 plate reader (Molecular Devices). Methods All measured LDLR concentrations (pg/mL) were corrected for total protein concentration (mg/mL). Corrected LDLR content (pg LDLR/ml total protein) is reported as % vector (pIRES-EGFP) control. Western Blotting. Following the incubation times and treatments specific to each experiment, cultured cells were washed and lysed and the lysates cleared, as described above. Thirty to fifty micrograms of protein were sep- arated on 8% Tris glycine SDS-PAGE gels and transferred to a PVDF membrane. Western blotting was performed for human LDLR-V5 and human PCSK9-V5 (anti-V5-HRP, 1:5000; R96125; Invitrogen) and for β-actin (rabbit anti-β-actin, 1:5000; A2066; Sigma). After incubation with the appropriate secondary antibodies, if required, the membranes were revealed using Clarity Western ECL Substrate (Bio-Rad), imaged with a GelDoc XR+ instru- ment (Bio-Rad) and the bands of interest quantified using ImageLab 5.2.1 software (Bio-Rad). Dil-LDL uptake. 48 h post-seeding and -transfection and after a 24 h starvation in serum-free medium, HepG2 cells were incubated for 2 h at 37 °C with 6 μg/ml Dil-LDL (Alfa Aesar; Cat # J675330) (20 μl of 36 μg/ml Dil-LDL were added to each well containing 100 μl of conditioned media). Each condition was prepared in 16 replicates. At the end of the 2 h incubation, the media was removed and the cells washed 3 times (200 μl/well) with ice-cold D-PBS no ions (Wisent). After the final wash was removed, 100 μl of PBS was added to each well and the plate was scanned (bottom read) on a SpectraMax i3 plate reader (Molecular Devices). For each well, raw Dil-LDL uptake was measured as the average fluorescence intensity (RFU) (ex: 534 nm/em: 572 nm) of 21 points equally distributed in a fill pattern. Dil-LDL uptake in each well was corrected for the total number of transfected cells (average EGFP fluorescence intensity; ex: 488 nm/ em: 513 nm) and reported as the fluorescence ratio of Dil-LDL/ EGFP. Corrected Dil-LDL uptake is reported as % vector (pIRES-EGFP) control and was obtained from 16 wells. References 1. Abifadel, M. et al. Living the PCSK9 adventure: from the identification of a new gene in familial hypercholesterolemia towards a potential new class of anticholesterol drugs. Curr. Atheroscler. Rep. 16, 439 (2014). 1. Abifadel, M. et al. Living the PCSK9 adventure: from the identification of a new gene in familial hypercholesterolemia towards a potential new class of anticholesterol drugs. Curr. Atheroscler. Rep. 16, 439 (2014). p g p ( ) 2. Goldstein, J. L. & Brown, M. S. Familial hypercholesterolemia: pathogenesis of a receptor disease. Johns Hopkins Med. J. 143, 8–16 (1978). 3. Innerarity, T. L. et al. Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding. Proc. Natl Acad. Sci. USA 84, 6919–6923 (1987). ( ) 4. Abifadel, M. et al. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat. Genet. 34, 154–156 (2003).h 5. Seidah, N. G. et al. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Proc. Natl. Acad. Sci. USA 100, 928–933 (2003). f 6. McNutt, M. C., Lagace, T. A. & Horton, J. D. Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells. J. Biol. Chem. 282, 20799–20803 (2007). p p 7. Marduel, M. et al. Description of a large family with autosomal dominant hypercholesterolemia associated with the APOE p.Leu167del mutation. Hum. Mutat. 34, 83–87 (2013). p 8. Garcia, C. K. et al. Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein Science 292, 1394–1398 (2001). , ( ) 9. Benn, M., Watts, G. F., Tybjærg-Hansen, A. & Nordestgaard, B. G. Mutations causative of familial hypercholesterolaemia: screening of 98 098 individuals from the Copenhagen General Population Study estimated a prevalence of 1 in 217. Eur. Heart J. 37, 1384–1394 (2016).h 0. Wang, J. et al. Polygenic Versus Monogenic Causes of Hypercholesterolemia Ascertained Clinically. Arterioscler. Thromb. Vasc. Biol 36, 2439–2445 (2016). 1. Sjouke, B. et al. Homozygous autosomal dominant hypercholesterolaemia in the Netherlands: prevalence, genotype-phenotype relationship, and clinical outcome. Eur. Heart J. 36, 560–565 (2015). 12. Nordestgaard, B. G. et al. Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease: consensus statement of the European Atherosclerosis Society. Eur. Heart J. 34, 3478–3490a (2013). 13. Marduel, M. et al. Molecular spectrum of autosomal dominant hypercholesterolemia in France. Hum. Mutat. 31, E1811–1824 (2010). 14. Hopkins, P. N. et al. Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 e e e ces 1. Abifadel, M. et al. Living the PCSK9 adventure: from the identification of a new gene in familial hypercholesterolemia towards a potential new class of anticholesterol drugs. Curr. Atheroscler. Rep. 16, 439 (2014). 1. Abifadel, M. et al. Living the PCSK9 adventure: from the identification of a new gene in familial potential new class of anticholesterol drugs. Curr. Atheroscler. Rep. 16, 439 (2014). www.nature.com/scientificreports/ Chem. 279, 48865–48875 (2004). p ( ) 45. Dubuc, G. et al. A new method for measurement of total plasma PCSK9: clinical applications. J. Lipid Res. 51, 140–149 (2010) Acknowledgements We would like to thank Pr Sekar Kathiresan (Broad Institute of MIT and Harvard University, Cambridge, MA, USA; Cardiology Division, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA USA) for his help in the exome sequencing experiments and analysis. S.K. was supported by NIH R01 HL107816. This work was supported by a grant from Leducq Foundation [FLQ # 13 CVD 03] through the Transatlantic Networks of Excellence in Cardiovascular Research program (‘The function and regulation of PCSK9: a novel modulator of LDLR activity’); and Institut National de la Santé et de la Recherche Médicale (INSERM); and Conseil de la Recherche de l’Université Saint-Joseph, Beirut, Lebanon; and Lebanese National Council for Scientific Research (CNRS-L); and Canadian Institutes of Health Research (CIHR) Foundation grant (NGS; # 148363); and Canada Research Chair (NGS; # 950–231335); This work was also supported by the national project CHOPIN (CHolesterol Personalized Innovation) granted by the Agence Nationale de la Recherche (ANR-16-RHUS-0007) and coordinated by the Centre Hospitalo-Univesitaire (CHU) de Nantes. S Elbitar and Y Ghaleb are supported by grants from Lebanese National Council for Scientific Research (CNRS-L); and Council of Research of Saint-Joseph University of Beirut, Lebanon and Institut Français du Liban. www.nature.com/scientificreports/ yp 4. Rubinsztein, D. C. et al. Characterization of six patients who are double heterozygotes for familial hypercholesterolemia and familia defective apo B-100. Arterioscler. Thromb. J. Vasc. Biol. 13, 1076–1081 (1993). ph 5. Tai, E. S. et al. Compound heterozygous familial hypercholesterolemia and familial defective apolipoprotein B-100 produce exaggerated hypercholesterolemia. Clin. Chem. 47, 438–443 (2001). h 35. Tai, E. S. et al. Compound heterozygous familial hypercholesterolemia and fam exaggerated hypercholesterolemia. Clin. Chem. 47, 438–443 (2001). h 35. Tai, E. S. et al. Compound heterozygous familial hyperchole 36. Taylor, A. et al. A double heterozygote for familial hypercholesterolaemia and familial defective apolipoprotein B-100. Ann. Clin. Biochem. 47, 487–490 (2010).f 7. Pisciotta, L. et al. Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia Atherosclerosis 186, 433–440 (2006). 8. Bertolini, S. et al. Spectrum of mutations and phenotypic expression in patients with autosomal dominant hypercholesterolemia identified in Italy. Atherosclerosis 227, 342–348 (2013). i y 9. Sjouke, B. et al. Double-heterozygous autosomal dominant hypercholesterolemia: Clinical characterization of an underreported disease. J. Clin. Lipidol. 10, 1462–1469 (2016). p 0. Elbitar, S. et al. Proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors and the future of dyslipidemia therapy: an updated patent review (2011–2015). Expert Opin. Ther. Pat 26, 1377–1392 (2016). p ( ) p ph ( ) 41. Siest, G. et al. Objectives, design and recruitment of a familial and longitudinal cohort for studying gene-environment interactions in the field of cardiovascular risk: the Stanislas cohort. Clin. Chem. Lab. Med. 36, 35–42 (1998). i 2. Rabès, J. P. et al. Familial ligand-defective apolipoprotein B-100: simultaneous detection of the ARG3500– > GLN and ARG3531– > CYS mutations in a French population. Hum. Mutat. 10, 160–163 (1997). i 42. Rabès, J. P. et al. Familial ligand-defective apolipoprotein B-100: simultaneous detec ARG3531– > CYS mutations in a French population. Hum. Mutat. 10, 160–163 (1997). 3. Li, M.-X., Gui, H.-S., Kwan, J. S. H., Bao, S.-Y. & Sham, P. C. A comprehensive framework for prioritizing variants in exome sequencing studies of Mendelian diseases. Nucleic Acids Res. 40, e53 (2012).f 43. Li, M.-X., Gui, H.-S., Kwan, J. S. H., Bao, S.-Y. & Sham, P. C. A comprehensive framewor sequencing studies of Mendelian diseases. Nucleic Acids Res. 40, e53 (2012).f q g 4. Benjannet, S. et al. NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL receptor and LDL cholesterol. J. Biol. www.nature.com/scientificreports/ www.nature.com/scientificreports/ y p 21. Andersen, L. H., Miserez, A. R., Ahmad, Z. & Andersen, R. L. Familial defective apolipoprotein B-100: A review. J. Clin. Lipidol. 10, 1297–1302 (2016).fih 2. Rabès, J. P. et al. R3531C mutation in the apolipoprotein B gene is not sufficient to cause hypercholesterolemia. Arterioscler. Thromb Vasc. Biol. 20, E76–82 (2000).i ( ) 23. Boren, J. et al. Identification of the low density lipoprotein receptor-binding site in apolipoprotein B100 and the modulation of its binding activity by the carboxyl terminus in familial defective apo-B100. J. Clin. Invest. 101, 1084–1093 (1998).f 24. Miserez, A. R. & Keller, U. Differences in the phenotypic characteristics of subjects with familial defective apolipoprotein B-100 and familial hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol. 15, 1719–1729 (1995).h yph 5. Borén, J., Ekström, U., Agren, B., Nilsson-Ehle, P. & Innerarity, T. L. The molecular mechanism for the genetic disorder familia defective apolipoprotein B100. J. Biol. Chem. 276, 9214–9218 (2001). 26. Thomas, E. R. A. et al. Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia. Mol. Genet. Genomic Med. 1, 155–161 (2013). hypercholesterolemia. Mol. Genet. Genomic Med. 1, 155–161 (2 yp 7. Futema, M. et al. Whole exome sequencing of familial hypercholesterolaemia patients negative for LDLR/APOB/PCSK9 mutations J. Med. Genet. 51, 537–544 (2014). J ( ) 8. Sun, H. et al. Proprotein convertase subtilisin/kexin type 9 interacts with apolipoprotein B and prevents its intracellular degradation irrespective of the low-density lipoprotein receptor. Arterioscler. Thromb. Vasc. Biol. 32, 1585–1595 (2012). yh 29. Poirier, S. et al. Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route. J. Biol. Chem. 284, 28856–28864 (2009).h 30. Susan-Resiga, D. et al. The Proprotein Convertase Subtilisin/Kexin Type 9-resistant R410S Low Density Lipoprotein Receptor Mutation: A Novel Mechanism Causing Familial Hypercholesterolemia. J. Biol. Chem. 292, 1573–1590 (2017).i g yp 1. Rauh, G. et al. Identification of a heterozygous compound individual with familial hypercholesterolemia and familial defective apolipoprotein B-100. Klin. Wochenschr. 69, 320–324 (1991). i yg apolipoprotein B-100. Klin. Wochenschr. 69, 320–324 (1991). p p p 32. Benlian, P. et al. Phenotypic expression in double heterozygotes for familial hypercholesterolemia and familial defective apolipoprotein B-100. Hum. Mutat. 7, 340–345 (1996). 33. deCampo, A., Schallmoser, K., Schmidt, H., Toplak, H. & Kostner, G. M. A novel splice-site mutation in intron 7 causes more severe hypercholesterolemia than a combined FH-FDB defect. Atherosclerosis 157, 524–525 (2001). Author Contributions C.B. and M.AF. conceived and designed the study; S.EB., JP.R., M.AF. performed the molecular analyses; G.P., N.S. performed the exome sequencing; M.V., P.EK. and Y.G. participated in the validation of the molecular results; V.C. and E.B. performed the clinical assessments; N.S. conceived and designed biosynthetic analyses; D.SR., J.H. and A.BDO. performed the Western blot and in vitro analysis; S.EB., M.AF., C.B. and N.S. analyzed the data and wrote the article. All authors reviewed and approved the present article. References Characterization of Autosomal Dominant Hypercholesterolemia Caused by PCSK9 Gain of Function Mutations and Its Specific Treatment With Alirocumab, a PCSK9 Monoclonal Antibody. Circ. Cardiovasc. Genet. 8, 823–831 (2015). pi y 15. Alves, A. C., Etxebarria, A., Soutar, A. K. & Martin, C. & Bourbon, M. Novel functional APOB mutations outside LDL-bin i i f ili l h h l t l i H M l G t 23 1817 1828 (2014) i 5. Alves, A. C., Etxebarria, A., Soutar, A. K. & Martin, C. & Bourbon, M. Novel functional APOB mutations outside LDL-binding region causing familial hypercholesterolaemia. Hum. Mol. Genet. 23, 1817–1828 (2014). 16. Motazacker, M. M. et al. Advances in genetics show the need for extending screening strategies for autosomal dominant hypercholesterolaemia. Eur. Heart J. 33, 1360–1366 (2012). 17. Maglio, C. et al. Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing. J. Intern. Med. 276, 396–403 (2014). 18. Bamshad, M. J. et al. Exome sequencing as a tool for Mendelian disease gene discovery. Nat. Rev. Genet. 12, 745–755 (2011). 19. Stitziel, N. O. et al. Exome sequencing in suspected monogenic dyslipidemias. Circ. Cardiovasc. Genet. 8, 343–350 (2015). Scientific REpOrtS \| (2018) 8:1943 \| DOI:10.1038/s41598-018-20281-9 8 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 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https://openalex.org/W4319927915	https://digital.csic.es/bitstream/10261/335165/1/rolemice.pdf	English	null	Role of CB2 cannabinoid receptor in the development of food addiction in male mice	Neurobiology of disease	2,023	cc-by	15,091	Role of CB2 cannabinoid receptor in the development of food addiction in male mice a Laboratory of Neuropharmacology-Neurophar, Department of Medicine and Life Sciences, Universitat Pompeu Fabra (UPF), Barcelona, Spain b Neurosciences Institute, University Miguel Hern´andez-CSIC, Avda de Ram´on y Cajal s/n, San Juan de Alicante, Alicante 03550, Spain c Research Network in Primary Care of Addictions, Health Institute Carlos III, MICINN and FEDER, Madrid 28029, Spain d Instituto de Investigaci´on Sanitaria y Biom´edica de Alicante (ISABIAL), Alicante, Spain e Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain f ( ), , p ment de Psicobiologia i Metodologia de les Ci`encies de la Salut, Universitat Aut`onoma de Barcelona (UAB), Cerdanyola del Vall`es, Barcelona, Spain A R T I C L E I N F O Keywords: Food addiction CB2R Depressive-like disorder Compulsivity Impulsivity Chocolate Keywords: Food addiction CB2R Depressive-like disorder Compulsivity Impulsivity Chocolate The endocannabinoid system plays an important role in multiple behavioral responses due to its wide distri bution in the central nervous system. The cannabinoid CB1 receptor was associated to the loss of behavioral control over food intake occurring during food addiction. The cannabinoid CB2 receptor (CB2R) is expressed in brain areas canonically associated with addictive-like behavior and was linked to drug-addictive properties. In this study, we evaluated for the first time the specific role of the CB2R in food addiction by using a well-validated operant mouse model of long-term training to obtain highly palatable food. We have compared in this model the behavioral responses of wild-type mice, mutant mice constitutively lacking CB2R, and transgenic mice over expressing CB2R. The lack of CB2R constitutes a protective factor for the development of food addiction and the impulsive and depressive-like behavior associated. In contrast, the overexpression of CB2R induces a vulnerable phenotype toward food addiction after long-term exposure to highly palatable chocolate pellets. Relevant transcriptomic changes were associated to resilience and vulnerability to food addiction depending on the ge notype, which provides a mechanistic explanation for these behavioral changes. Therefore, CB2R may constitute a potential therapeutic target for the loss of eating control and the comorbid emotional effects associated to food addiction. Neurobiology of Disease 179 (2023) 106034 Neurobiology of Disease 179 (2023) 106034 Available online 10 February 2023 0969-9961/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Spain. E-mail addresses: elena.martin@upf.edu (E. Martín-García), rafael.maldonado@upf.edu (R. Maldonado). 1 These authors contributed equally. 2 These authors equally supervised this work. https://doi.org/10.1016/j.nbd.2023.106034 Received 23 December 2022; Received in revised form 5 February 2023; Accepted 6 February 2023 2 These authors equally supervised this work. * Corresponding authors at: Laboratory of Neuropharmacology-Neurophar, Department of Medicine and Life Sciences, Universitat Pompeu Fabra (UPF), Barcelona, Spain. E-mail addresses: elena.martin@upf.edu (E. Martín-García), rafael.maldonado@upf.edu (R. Maldonado). 1 Th th t ib t d ll 1. Introduction western society are leading to an increase of the socio-economic burden associated with food addiction, impaired by the lack of effective treat ments. Therefore, there is a need to understand the neurobiological mechanisms underlying food addiction in order to identify novel possible therapeutic approaches. Food addiction is a multifactorial disorder characterized by loss of control over food intake (Randolph, 1956). The concept of food addic tion is still controversial, and according to a well-accepted diagnosis tool, the Yale Food Addiction Scale 2.0 (Gearhardt et al., 2016), food addiction affects from 2% to 12% of healthy body mass index in dividuals. The prevalence rises among people suffering from obesity (18–24%), and eating disorders (50%), reaching the highest value in bulimia nervosa (85%) (Fernandez-Aranda et al., 2018). The exposure and accessibility to food with high palatability and caloric content in Food intake is regulated by homeostatic mechanisms that control energy intake and expenditure to maintain a metabolic balance (Onao lapo and Onaolapo, 2018). In parallel, allostatic mechanisms control food intake, regardless of energetic requirements, through the reward circuits under the influence of food palatability and environmental factors, among others (Caron and Richard, 2017). Both the homeostatic 2 These authors equally supervised this work. Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. female mice (Domingo-Rodriguez et al., 2020; García-Blanco et al., 2022; Mancino et al., 2015; Martín-García et al., 2020). All the behav ioral experiments were conducted in the animal facility at Universitat Pompeu Fabra-Barcelona Biomedical Research Park (UPF-PRBB; Bar celona, Spain). Mice were housed individually and maintained in a controlled temperature (21 ± 1 ◦C) and humidity (55 ± 10%) with food and water available ad libitum. All the experiments were performed during the dark phase of a reverse light/dark cycle (light on at 8:00 pm, light off at 8:00 am). All behavioral experiments were approved by the local ethical committee (Comit`e `Etic d'Experimentaci´o Animal-Parc de Recerca Biom`edica de Barcelona) and were performed in accordance with the European Communities Council Directive (2010/63/EU). All the experiments were performed under blind and randomized conditions. and the allostatic systems regulate food intake, although in pathological conditions, the hedonic system can override the homeostatic control (Caron and Richard, 2017). 1. Introduction The hypothalamus is the main brain area involved in the homeostatic control, whereas the allostatic mechanisms are mainly under the control of the mesocorticolimbic system, which includes the ventral tegmental area (VTA) and its projections to the nucleus accumbens (NAc) and cortical areas including the medial pre frontal cortex (mPFC). Misbalances in this brain reward system are commonly shared across food and drug addiction (Maldonado et al., 2021). Common neuroanatomical and neurophysiological alterations have been proposed to underlie the neurobiological substrate of both disorders that share multiple behavioral alterations including loss of control over intake, enhanced compulsivity and motivation, and altered reward sensitivity. i CB2 receptor overexpression in CB2xP mice was previously described in detail (García-Guti´errez et al., 2010). These studies showed the comparative CB2R relative gene expression profile between WT and CB2xP mice in several brain regions by real-time PCR. CB2R was significantly overexpressed in the caudate putamen (CPu), nucleus accumbens (NAcc), cingulate cortex (Cg Ctx), amygdala (Amy), hippo campus (Hipp; CA2, CA3, and DG subregions), ventromedial nucleus (VMN), arcuate nucleus (Arc), substantia nigra (SN), ventral tegmental area (VTA), and dorsal and medial raphe (DR and MnR, respectively). In addition, the cell type distribution of CB2 receptor in CB2xP was also previously described (Racz et al., 2008a, 2008b; Aracil-Fern´andez et al., 2012). The expression of transgenic CB2R was pronounced in both microglial cells and neurons of CB2xP (Racz et al., 2008a). Finally, (Aracil-Fern´andez et al., 2012) immunohistochemistry studies revealed that CB2R is overexpressed in neurons and astrocytes of CB2xP mice in the NAcc and VTA (Aracil-Fern´andez et al., 2012). Genetically modified mouse models lacking (CB2KO) or over expressing CB2R (CB2xP) have allowed important advances in under standing the physiological role of this receptor (García-Guti´errez et al., 2010; García-Guti´errez and Manzanares, 2011; Navarro et al., 2022). Previous studies reported reduced cocaine-reinforcing effects in CB2xP mice (Aracil-Fern´andez et al., 2012) and in WT mice receiving a CB2R agonist (Xi et al., 2011), although CB2R antagonists also decreased cocaine-reinforcing effects (Adamczyk et al., 2012). Alcohol-reinforcing effects were enhanced in CB2KO mice (Ortega-´Alvaro et al., 2015), although alcohol consumption was also increased by CB2R agonists (Onaivi et al., 2008). CB2KO mice showed protection against nicotine- reinforcing effects and withdrawal (Ignatowska-Jankowska et al., 2013; Navarrete et al., 2013) and the same effects were obtained by CB2R antagonist (Ignatowska-Jankowska et al., 2013; Navarrete et al., 2013) linking CB2R with increased vulnerability to nicotine abuse. 1. Introduction Therefore, the modulation of CB2R produces differential effects on the addictive properties of drugs depending on the model and the substance of abuse under study. Targeting CB2R for addictive processes represents an interesting approach considering the absence of several centrally mediated side effects that have limited the use of CB1R ligands, such as psychoactive, motor, and cognitive side effects (Caba˜nero et al., 2021a). 2.2. Operant behavior apparatus Operant responding maintained by chocolate-flavored pellets was performed in mouse operant chambers (Model ENV-307A-CT, Med As sociates, Georgia, VT, USA). The chambers were equipped with two retractable levers, one randomly assigned as the active lever and the other as the inactive for the entire experimental protocol. Pressing on the active lever resulted in a food pellet delivery paired with a stimulus- light (cue-light) located above the active lever, whereas pressing on the inactive lever had no consequences. A food dispenser equidistant be tween the two levers allows the delivery of food pellets when pertinent. The operant chambers were made of aluminum and acrylic and were housed inside soundproof boxes equipped with fans to provide ventila tion and white noise. The chambers' floor consists of a metal sheet with holes. The floor was changed in the shock test sessions by a grid floor made of metal bars able to conduct electrical current, which was also used as a contextual cue for the aversive cue reactivity test the day after the shock test allowing mice to discriminate between different contexts. This study aims to elucidate the involvement of CB2R in food addiction. We hypothesize that the modification in the expression of CB2 receptors will impact the development of food addiction. For this purpose, we used genetically modified mice either lacking or over expressing CB2R trained in a well-established operant food addiction model that evaluates 3 addiction criteria and 4 addiction-related phenotypic traits. At the end of the operant protocol, mice were evalu ated for anxiety and depression-like behavior. Several genes of interest were also investigated in different brain areas involved in food intake control and reward. The results obtained support the relevance of the endocannabinoid system in food addiction and highlight the role of CB2R as a novel potential therapeutic target for this disorder. 2.4. Experimental design As previously described (Martín-García et al., 2011), the operant response was acquired when all the following conditions were achieved: (1) mice maintained a stable response with <20% deviation from the mean of the total number of reinforcers earned in 3 consecutive sessions (80% of stability); (2) at least 70% responding on the active lever; and (3) a minimum of 10 reinforcers per session. 2.4.1. Operant training A total of 95 mice were trained for 118 days to obtain chocolate- flavored pellets. In the operant conditioning sessions, mice were under an FR1 schedule of reinforcement for 6 days (1 lever-press resulted in 1 pellet delivery) followed by 112 days of FR5 (5 lever-presses resulted in 1 pellet delivery) (Fig. 1a). The beginning of each session was signaled by turning on a house light placed on the chamber's ceiling during the first 3 s. Daily operant training sessions maintained by chocolate- flavored pellets lasted 1 h and were composed of 2 pellet periods (25 min each) separated by a pellet-free period (10 min). During the pellet periods, pellets were delivered contingently after an active response paired with a stimulus light (cue light). A time-out period of 10 s was established after each pellet delivery, where the cue light was off, and no reinforcer was provided after responding on the active lever. Responses on the active and inactive lever performed during the time-out periods were recorded. In contrast, the pellet-free period was signaled by the illumination of the entire operant box, and no pellet was delivered after responding on any lever. Mice were returned to their home cages after each session. 2.4.2. Three addiction criteria The food addiction criteria were evaluated at three different time points as previously described (García-Blanco et al., 2022): early (5–18 training days), middle (48–65 training days), and late (95–112 training days). The food addiction criteria gathered the main hallmarks of addiction based on DSM-IV (Deroche-Gamonet et al., 2004), DSM-5 and now included in the food addiction diagnosis through the YFAS 2.0 (Gearhardt et al., 2016) and therefore, are used to classify mice as nonaddicted (0–1 criteria) and addicted (2–3 criteria) (Fig. 1a). Persistence of response: persistent desire or unsuccessful efforts to cut down displayed by continuous food-seeking behavior even if the food reward is signaled as not available. It is measured by the number of non-reinforced active responses during the pellet-free period (10 min) on the 3 consecutive days before the progressive ratio (PR). Fig. 1. The reinforcement for chocolate-flavored pellets was reduced in CB2R-lacking mutants suggesting a resistant phenotype. a Experimental timeline of the protocol. b Schematic representation of the three mice genotypes used in this study. WT mice that express CB2R constitutively, mutant mice that do not express CB2R (CB2KO) and mutant mice that overexpress CB2R (CB2xP). c Number of chocolate-flavor pellets obtained in each daily operant training session (repeated-measures ANOVA; session effect p < 0.001 and interaction genotype/session p < 0.001). In the early period, both CB2xP (post-hoc LSD test ++p < 0.01) and CB2KO (post-hoc LSD test *p < 0.001) mice obtained less reinforcers than WT mice. CB2KO mice maintained this difference over the entire protocol (post-hoc LSD test WT vs CB2KO p < 0.001) while CB2xP mice reached the levels of WT mice in later stages of the protocol (post-hoc LSD test WT vs CB2xP NS) and therefore were different to CB2KO (post-hoc LSD test CB2KO vs CB2xP ###p < 0.001). Fig. 1. The reinforcement for chocolate-flavored pellets was reduced in CB2R-lacking mutants suggesting a resistant phenotype. a Experimental timeline of the protocol. b Schematic representation of the three mice genotypes used in this study. WT mice that express CB2R constitutively, mutant mice that do not express CB2R (CB2KO) and mutant mice that overexpress CB2R (CB2xP). c Number of chocolate-flavor pellets obtained in each daily operant training session (repeated-measures ANOVA; session effect p < 0.001 and interaction genotype/session p < 0.001). 2.3. Food pellets CD1 wild-type (WT) mice (n = 42) were purchased from Charles River (France). Transgenic mice overexpressing cannabinoid receptor 2 (CB2xP) (n = 28) and cannabinoid receptor 2 knockout (CB2KO) mice (n = 25) were kindly supplied by J. Manzanares laboratory (Instituto de Neurociencias, Universidad Miguel Hern´andez-CSIC Alicante, Spain). Homozygote CB2KO mice were initially generated on a C57BL/6 J congenic background (provided by Nancy E. Buckley, Cal State Poly technic University, Pomona, CA, USA), and the CB2KO founders were crossed with outbred CD1 (Charles River, France) background (Buckley et al., 2000) for eight generations. Mice overexpressing CB2R were on a CD1 congenic background (Racz et al., 2008b). All the mice were male, two months old and weighed 40 ± 3 g at the beginning of the experi ment. The male sex was chosen considering the previous literature that has validated the operant food addiction model only in male, but not in During the operant conditioning sessions, animals received after pressing the active lever a 20 mg chocolate-flavored pellet consisting in a highly palatable isocaloric food (TestDiet, Richmond, IN, USA). These pellets had a similar caloric value (3.44 kcal/g: 20.6% protein, 12.7% fat, 66.7% carbohydrate) to the standard maintenance diet provided to mice in their home cage (3.52 kcal/g: 17.5% protein, 7.5% fat, 75% carbohydrate) with some slight differences in their composition: choc olate flavor (2% pure unsweetened cocoa) and enhanced sucrose content (8.3% standard diet food vs 50.1% highly palatable pellets). These pellets were presented only during the operant behavior sessions, and animals were maintained on standard chow for their daily food intake. 2 A. García-Blanco et al. A. García-Blanco et al. Neurobiology of Disease 179 (2023) 106034 2.4. Experimental design 2.5. Anxiety-like behavior Anxiety-like behavior was evaluated by the elevated plus-maze test once the long-term operant training protocol was finished. A black Plexiglas apparatus consisting of 4 arms (29 cm long x 5 cm wide), 2 open and 2 closed, set in a cross from a neutral central square (5 × 5 cm) elevated 40 cm above the floor was used. Light intensity in the open and closed arms was 45 and 5 lx, respectively. Mice were placed in the central square facing one of the closed arms and tested for 5 min. The time spent in the open and closed arms of the maze was determined as a measure of anxiety-like behavior, whereas the total entries in the open and closed arms were considered a measure of locomotor activity, as previously reported (La Porta et al., 2015). Compulsivity: continued use despite negative consequences evalu ated as the resistance to punishment when chocolate-flavored pellets intake is coupled with an aversive stimulus. Mice were placed in an operant box without the metal sheet with holes and consequently with the grid floor exposed (contextual cue). During this session, mice un derwent an FR5 schedule in which they received an electric foot-shock (0.18 mA, 2 s) after 4 responses and received another electric foot- shock (0.18 mA, 2 s) and a pellet paired with the cue light after the 5th response. The schedule was reinitiated after time-out period (10 s after pellet delivery) and after the fourth response if mice did not perform the fifth response within 60 s. The total number of shocks performed in 50 min was used to evaluate compulsivity-like behavior, previously described as resistance to punishment (Deroche-Gamonet et al., 2004; Mancino et al., 2015). A. García-Blanco et al. Aversive cue-reactivity: To study the conditioning to an aversive stimulus (grid floor), non-reinforced active responses during the following session after the shock test were measured. Mice were placed in the operant box for 1 h with the same grid floor used during the shock test. However, during this session, pressing the active lever had no consequences: no shock, no chocolate-flavored pellets, and no cue light. Motivation: considerable effort and time spent in obtaining the reward measured by the PR schedule of reinforcement. The response required to earn one single pellet escalated according to the following series: 1, 5, 12, 21, 33, 51, 75, 90, 120, 155, 180, 225, 260, 300, 350, 410, 465, 540, 630, 730, 850, 1000, 1200, 1500, 1800, 2100, 2400, 2700, 3000, 3400, 3800, 4200, 4600, 5000, and 5500. The maximal number of responses that the animal is willing to perform to obtain one pellet is referred to as the breaking point. The maximum duration of the PR session was 5 h or until mice did not respond on any lever during 1 h. 2.7.1. Statistical analysis of behavioral data 2.7.1. Statistical analysis of behavioral data IBM SPSS 19 (SPSS Inc., Chicago, USA) was used to analyze all the data. Normality was determined by Kolmogorov-Smirnov's test. Para metric tests were performed if normality criteria were met. Repeated measures ANOVA was used to test the evolution over time. One-way ANOVA or two-way ANOVA were used when required for compari sons between groups followed by subsequent post hoc analysis (Fisher's Least Significant Difference “LSD” test) when required. Non-parametric tests were performed if normality criteria were not meet. Kruskal Wallis or Friedman test was applied followed by Mann-Whitney's U test when required. Chi-square analyses were performed to compare the percent age of addicted and nonaddicted mice, considering the observed fre quencies with those obtained in the control WT group. Results were expressed as individual values with the median and the interquartile range. A probability of 0.05 or less was considered statistically significant. 2.4.4. Behavioral tests to evaluate addiction-like phenotypic traits Four additional phenotypic traits were also evaluated as factors of vulnerability to addiction in each period (early, middle and late): i Impulsivity: Considered as motor disinhibition, is defined as the inability to stop a response once it is initiated (Dalley et al., 2011). Impulsivity was measured as the number of non-reinforced active re sponses during the time-out period (10 s) after each pellet delivery, as previously described (Domingo-Rodriguez et al., 2022; García-Blanco et al., 2022; Mancino et al., 2015; Martín-García et al., 2020). The three consecutive days before the progressive ratio test are considered for this criterion (Martín-García et al., 2020). In the cycle of addiction, there is a transition from impulsivity in the early stages to compulsivity in the later stages (Everitt et al., 2008). This transition has its neurobiological correlation in the shift from ventral to dorsal striatum control of this behavioral hallmark (Belin et al., 2008). li 2.6. Depressive-like behavior Depressive-like behavior was evaluated at the end of the long-term operant training protocol using the forced swimming test (Porsolt et al., 1978). Briefly, mice were individually placed into a glass cylinder (17.5 × 12.5 cm) filled 15 cm high with water (22 ± 1 ◦C). Mice were subjected to forced swimming for 6 min, and the total duration of immobility, disregarding small hind limb movements to keep the head above water, was measured during the last 4 min when mice showed a sufficiently stable level of immobility. 2.4.3. Establishment of mice subpopulations After performing the three behavioral tests to measure the food addiction behavior, mice were categorized as food addicted or non addicted depending on the number of positive criteria they achieved. A mouse was considered positive for a particular addiction criterion when the score of the specific behavioral test was above the 75th percentile of the normal distribution of the WT control group. Mice that achieved 2 or 3 addiction criteria were considered addicted animals, and mice that achieved 0 or 1 addiction criteria were considered nonaddicted animals, as previously reported (Domingo-Rodriguez et al., 2020; Mancino et al., 2015). 2.4.2. Three addiction criteria In the early period, both CB2xP (post-hoc LSD test ++p < 0.01) and CB2KO (post-hoc LSD test p < 0.001) mice obtained less reinforcers than WT mice. CB2KO mice maintained this difference over the entire protocol (post-hoc LSD test WT vs CB2KO p < 0.001) while CB2xP mice reached the levels of WT mice in later stages of the protocol (post-hoc LSD test WT vs CB2xP NS) and therefore were different to CB2KO (post-hoc LSD test CB2KO vs CB2xP ###p < 0.001). 3 Neurobiology of Disease 179 (2023) 106034 2.7. Statistics 2.7. Statistics 3.2. The impulsive-like behavior was decreased in CB2KO mice suggesting a resilient phenotype Relative gene expression analyses of cannabinoid receptor 1 (cnr1) in the NAc, prelimbic (PL), orbitofrontal (OFC) and infralimbic (IL) areas, opioid receptor (Oprm1) in the NAc, tyrosine hydroxylase (TH) in the VTA, corticotropin-releasing factor (Crf) in the paraventricular nucleus (PVN), dopamine receptor D1 (Drd1) and dopamine receptor D2 (Drd2) in PL, OFC and IL areas, were carried out. At the end of the behavioral procedure, mice were sacrificed by dislocation, and brains were removed from the skull and frozen over dry ice. Coronal sections (500 μm) containing the regions of interest were cut in a cryostat (−10 ◦C) according to Paxinos and Franklin atlas (Franklin and Paxinos, 2001), mounted onto slides, and stored at −80 ◦C. Sections were microdissected following the method described by Palkovits (Palkovits, 1983). Total RNA was obtained from brain micropunches using TRI extraction re agent (Applied Biosystems, Madrid, Spain). Reverse transcription to complementary DNA (cDNA) was carried out following the manufac turer's instructions (Applied Biosystems). The relative abundances of Cnr1 (Mm00432621_s1), Oprm1 (Mm01188089_m1), Crf (Mm01293920_s1), and Th (Mm00447546_m1) gene expressions were quantified in a StepOne Plus Sequence Detector System (Life Technol ogies, Madrid, Spain) and the relative abundances of Drd1 (5’-AGATT GACCAGGAAGAGGCC-3′ and 5’-GCAATCCAAGCCATACCAGG-3′) and Drd2 (5’-CCATCTCTTGCCCACTGCTCTTTGG-3′ and 5’-GGTGACGAT GAAGGGCACGTAGAAC-3′) were quantified in a QuantStudio 12 K System by Applied Biosystems. Each assay was undertaken in technical triplicate to ensure the reliability of single values and the average calculated for data analyses. All reagents were obtained from Life Technologies, and the manufacturer's protocols were followed. The reference genes used were 18S rRNA (Mm03928990_g1) and β-Actin (5’- CACAGCC1GGATGGCTACGT-3′ and 5’-CGTGAAAAGATGACCCA GATCA-3′). All primer-probe combinations were optimized and vali dated for relative quantification of gene expression. Data for each target gene was normalized to the endogenous reference gene, and the fold change in target gene expression was determined using the 2-ΔΔCt method (Livak and Schmittgen, 2001). As a measure of impulsive-like behavior, active lever presses during the 10 s of the time out period were recorded over each operant training session (repeated measures ANOVA, genotype F(2,92) = 3.69, p = 0.029; session F(111,10,212) = 16.78, p < 0.001; genotype/session interaction F(222,10,212) = 1.31, p = 0.0014). CB2KO mice showed a reduced number of non-reinforced active responses compared to WT mice among all FR5 sessions (post-hoc LSD test, p < 0.001 Fig. 2a), suggesting that CB2KO mice are less impulsive. 2.7.2. Principal component analysis Cognitive inflexibility: defined as the incapacity to shift responding to stimuli that have previously predicted the availability of reward. It is measured by the ability to modify the operant behavior when the active and the inactive levers were reversed in a single training session without previous learning. The errors are the number of active-reversed re sponses (previous inactive lever in a typical training session) performed in 1 h. The principal component analysis (PCA) technique was used to evaluate the multidimensional data obtained in mice chronically trained with chocolate-flavored pellets. PCA and orthogonal varimax rotation were conducted using the 3 addiction-like criteria and the 4 phenotypic traits considered as vulnerability factors of addiction and were dimen sionality reduced to the minimum number of components that best explain and maximize the variance present in the data set. An eigenvalue >1 was set as selecting components criterion according to the Kaiser criterion. PCA was used to explain the maximum total variance in a correlation matrix by transforming the original variables into linear components (Field, 2018). Factor loadings of the principal component 1 (PC1) and principal component 2 (PC2) in all variables were studied. Individual mice clustering according to addiction or non-addiction in the space yielded by the 2 PCA components, which accounted for the Appetitive cue-reactivity: The cue-induced food-seeking test, which consisted of a 90-min session, assessed the conditioning to an appetitive stimulus (cue-light). In the first 60 min, all active and inactive lever- presses were recorded but produced no consequences. In the next 30 min, the cue light associated with pellet delivery during a typical op erant training session was illuminated with no contingent pellet rein forcement. To signal the change in the schedule, the cue light was presented twice non-contingently and for 4 s. 4 Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. Fig. 1c). These results reveal that chocolate-flavored pellets were less reinforcing for CB2KO mice since the early period suggesting that the loss of CB2R may be a preexisting protective factor. maximum data variance, were represented. The order of factor loading of the different variables in PC1 and PC2 was considered, and loading >0.7 were considered as mainly contributing to the component. 3.3. Differences between genotypes in the classification of addicted and nonaddicted mice Considering the results of the 3 food addiction criteria in the late period, mice were individually categorized as nonaddicted (covering 0–1 criteria) or addicted (covering 2–3 criteria), as previously reported (Domingo-Rodriguez et al., 2020; García-Blanco et al., 2022; Mancino et al., 2015). Only 4.00% of CB2KO mice during the early training were classified as addicts, revealing a phenotype more resilient to develop food addiction compared to the WT control group (21.43% addicted mice) (Chi-square, χ2 = 4.51, p = 0.034, Fig. S1a). With regards to CB2xP, only 7.14% of mice were classified as addict in the early training (Chi-square, χ2 = 3.39, p = 0.065, Fig. S1a). During the middle training, the percentage of mice classified as addicts increased in all genotypes compared to the early period (WT = 28.57%, CB2KO = 20.00%, CB2xP = 17.86% animals covering 2–3 criteria). Neither CB2KO (Chi-square, χ2 = 0.90, p = 0.343) nor CB2xP (Chi-square, χ2 = 1.58, p = 0.209) differed from WT mice at this period (Fig. S2b). In the late period, similar percentage of CB2KO (28.00%) and WT mice (19.05%) were classified as addicts (Chi-square, χ2 = 1.30, p = 0.254, Fig. 3a). In contrast, CB2xP mice reached a higher percentage of addicts (35.71%) than WT mice (Chi-square, χ2 = 5.04, p = 0.025, Fig. 3a) suggesting that CB2R overexpression constitutes a risk factor to develop food addiction after long-term training. 3.1. The reinforcement for chocolate-flavored pellets was reduced in CB2KO mice suggesting a resistant phenotype WT (n = 42), CB2KO (n = 25), and CB2xP (n = 28) mice were trained in the operant chambers under FR1 schedule of reinforcement during 6 sessions followed by 112 sessions under FR5 to acquire an operant responding maintained by chocolate-flavored pellets (Fig. 1a, b). During FR1, all groups increased the number of reinforcements across sessions without significant differences between genotypes (repeated measures ANOVA, genotype F(2,92) = 0.24, p = 0.78; session F(5,460) = 37.30, p < 0.001; genotype/session interaction F(10,460) = 0.64, p = 0.65, Fig. 1c). When the effort was increased to 5 lever presses (FR5), all genotypes showed a progressive increase in the number of reinforcements across time (repeated measures ANOVA, genotype F(2,92) = 4.29, p = 0.17; session F(111,10,212) = 82.15, p < 0.001; genotype/session interaction F(222,10,212) = 2.89, p < 0.001 Fig. 1c). Interestingly, the number of re inforcements was significantly reduced in CB2KO compared to WT mice over the whole FR5 period (post-hoc LSD test p < 0.001). In contrast, CB2xP mice showed a reduced number of reinforcements compared to WT mice only in the early period, but no differences were found from the middle period onwards (post-hoc LSD test, NS, Fig. 1c). In the late period, CB2xP mice also showed a significantly higher number of re inforcements compared to CB2KO mice (post-hoc LSD test, p < 0.001, 3.2. The impulsive-like behavior was decreased in CB2KO mice suggesting a resilient phenotype In contrast, CB2xP mice exhibited a similar number of active lever presses in the time-out period compared to WT mice (post-hoc LSD test, NS, Fig. 2a). Most of the WT (91.30%), CB2KO (96.5%), and CB2xP (90.32%) mice achieved the acquisition criteria during the FR5 after an average of 17.36 ± 2.56, 26.72 ± 4.87, and 29.68 ± 5.40 sessions, respectively (Fig. 2b). No significant differences in the day of acquisition among genotypes were revealed indicating similar acquisition levels of the operant conditioning learning driven by chocolate-flavored pellets (Kruskal-Wallis, H = 2.76, p = 0.25, Fig. 2b). Therefore, the differences observed were not induced by a differential acquisition of the operant training. WT and CB2KO mice progressively gained weight over time (repeated measures ANOVA, genotype F(2,92) = 12.60, p = 0.029; week F(12,1104) = 197.51, p < 0.001; genotype/week interaction F(24,1104) = 15.94, p < 0.001; post-hoc LSD test, p = 0.63, Fig. 2c) without significant differences between them. In contrast, CB2xP mice showed significantly lower body weight than WT (post-hoc LSD test, p < 0.001) and CB2KO (post-hoc LSD test, p < 0.001) mice (Fig. 2c). 3.3. Differences between genotypes in the classification of addicted and nonaddicted mice 3.4. Evolution over time of training of the three-addiction criteria in the different genotypes The same was observed in CB2xP mice, which also showed decreased motivation than WT mice both in the early (Mann-Whitney's U, U = 404.5, p = 0.027) and middle (Mann-Whitney's U, U = 383.5, p = 0.013) periods (Fig. 3c). Regarding the compulsivity-like behavior, the three genotypes decreased their compulsivity across the protocol (WT mice Friedman's test, χ2 = 31.14, p < 0.001, CB2KO mice Friedman's test, χ2 = 15.52, p < 0.001, CB2xP mice Friedman's test, χ2 = 6.88, p = 0.032) (Fig. 3d). CB2KO mice did not showed different compulsivity compared to WT mice, while CB2xP showed increased compulsivity compared to WT mice in the middle (Mann-Whitney's U, U = 356, p = 0.004) and increased compulsivity compared to CB2KO mice in the middle (Mann-Whitney's U, U = 194.5, Regarding the nonaddicted mice subgroups, the most relevant results were the increased persistence of nonaddicted CB2xP mice compared to nonaddicted WT mice in the late period (Mann-Whitney's U, U = 203, p Regarding the nonaddicted mice subgroups, the most relevant results were the increased persistence of nonaddicted CB2xP mice compared to nonaddicted WT mice in the late period (Mann-Whitney's U, U = 203, p = 0.048) (Fig. S2a). Nonaddicted CB2KO (Mann-Whitney's U, U = 189, p = 0.023) and nonaddicted CB2xP (Mann-Whitney's U, U = 192, p = 0.027) mice showed a reduced motivation in the late period compared to nonaddicted WT mice (Fig. S2b). Nonaddicted CB2xP mice showed increased compulsivity in the middle period compared to nonaddicted WT (Mann-Whitney's U, U = 157, p = 0.003) and to nonaddicted CB2KO (Mann-Whitney's U, U = 77.5, p = 0.006) mice (Fig. S2c). = 0.048) (Fig. S2a). Nonaddicted CB2KO (Mann-Whitney's U, U = 189, p = 0.023) and nonaddicted CB2xP (Mann-Whitney's U, U = 192, p = 0.027) mice showed a reduced motivation in the late period compared to nonaddicted WT mice (Fig. S2b). Nonaddicted CB2xP mice showed increased compulsivity in the middle period compared to nonaddicted WT (Mann-Whitney's U, U = 157, p = 0.003) and to nonaddicted CB2KO (Mann-Whitney's U, U = 77.5, p = 0.006) mice (Fig. S2c). 3.4. Evolution over time of training of the three-addiction criteria in the different genotypes 3.4. Evolution over time of training of the three-addiction criteria in the different genotypes WT mice presented an stable persistence across the periods (Fried man's test, χ2 = 4.84, p = 0.089), while CB2KO (Friedman's test, χ2 = 11.76, p = 0.003) and CB2TG (Friedman's test, χ2 = 19.50, p < 0.001) mice increased their persistence over the periods (Fig. 3b). CB2KO did 5 Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. Fig. 2. The impulsive-like was decreased in CB2R-lacking mice suggesting a resilient phenotype. a Regarding the impulsivity across the protocol (repeated-measures ANOVA; session effect p < 0.001 and interaction genotype/session p < 0.01). CB2KO mice showed a reduced impulsivity compared to WT mice (post-hoc LSD test WT vs CB2KO p < 0.001), whereas CB2xP and WT mice exhibited similar responses (post-hoc LSD test WT vs CB2xP NS). b Day of acquisition of the operant con ditioning behavior during the FR5 period (Kruskal-Wallis, NS). c Weekly measurements of body weight in grams. CB2xP mice showed a reduced body weight compared to WT and CB2KO mice (repeated measures ANOVA, genotype effect, p < 0.001; post-hoc LSD test WT vs CB2xP +++p < 0.001, post-hoc LSD test CB2KO vs CB2xP ###p < 0.001). (Data are expressed as mean ± SEM, WT n = 42, CB2KO n = 25, CB2xP n = 28, Statistical details are included in Table S2). Fig. 2. The impulsive-like was decreased in CB2R-lacking mice suggesting a resilient phenotype. a Regarding the impulsivity across the protocol (repeated-measures ANOVA; session effect p < 0.001 and interaction genotype/session p < 0.01). CB2KO mice showed a reduced impulsivity compared to WT mice (post-hoc LSD test WT vs CB2KO p < 0.001), whereas CB2xP and WT mice exhibited similar responses (post-hoc LSD test WT vs CB2xP NS). b Day of acquisition of the operant con ditioning behavior during the FR5 period (Kruskal-Wallis, NS). c Weekly measurements of body weight in grams. CB2xP mice showed a reduced body weight compared to WT and CB2KO mice (repeated measures ANOVA, genotype effect, p < 0.001; post-hoc LSD test WT vs CB2xP +++p < 0.001, post-hoc LSD test CB2KO vs CB2xP ###p < 0.001). (Data are expressed as mean ± SEM, WT n = 42, CB2KO n = 25, CB2xP n = 28, Statistical details are included in Table S2). 3.4. Evolution over time of training of the three-addiction criteria in the different genotypes p = 0.005) and late (Mann-Whitney's U, U = 243, p = 0.049) periods (Fig. 3d). not differ from WT mice in any period, while CB2TG mice present lower persistence than WT mice in the early period (Mann-Whitney's U, U = 374.5, p = 0.010) and higher persistence than WT mice in the late period (Mann-Whitney's U, U = 382, p = 0.014) (Fig. 3b). The three genotypes increased their motivation across the protocol (WT mice Friedman's test, χ2 = 26.91, p < 0.001, CB2KO mice Friedman's test, χ2 = 19.49, p < 0.001, CB2xP mice Friedman's test, χ2 = 22.06, p < 0.001) (Fig. 3c). CB2KO mice showed decreased motivation than WT mice both in the early (Mann-Whitney's U, U = 372.5, p = 0.047) and middle (Mann- Whitney's U, U = 374, p = 0.048) periods. The same was observed in CB2xP mice, which also showed decreased motivation than WT mice both in the early (Mann-Whitney's U, U = 404.5, p = 0.027) and middle (Mann-Whitney's U, U = 383.5, p = 0.013) periods (Fig. 3c). Regarding the compulsivity-like behavior, the three genotypes decreased their compulsivity across the protocol (WT mice Friedman's test, χ2 = 31.14, p < 0.001, CB2KO mice Friedman's test, χ2 = 15.52, p < 0.001, CB2xP mice Friedman's test, χ2 = 6.88, p = 0.032) (Fig. 3d). CB2KO mice did not showed different compulsivity compared to WT mice, while CB2xP showed increased compulsivity compared to WT mice in the middle (Mann-Whitney's U, U = 356, p = 0.004) and increased compulsivity compared to CB2KO mice in the middle (Mann-Whitney's U, U = 194.5, not differ from WT mice in any period, while CB2TG mice present lower persistence than WT mice in the early period (Mann-Whitney's U, U = 374.5, p = 0.010) and higher persistence than WT mice in the late period (Mann-Whitney's U, U = 382, p = 0.014) (Fig. 3b). The three genotypes increased their motivation across the protocol (WT mice Friedman's test, χ2 = 26.91, p < 0.001, CB2KO mice Friedman's test, χ2 = 19.49, p < 0.001, CB2xP mice Friedman's test, χ2 = 22.06, p < 0.001) (Fig. 3c). CB2KO mice showed decreased motivation than WT mice both in the early (Mann-Whitney's U, U = 372.5, p = 0.047) and middle (Mann- Whitney's U, U = 374, p = 0.048) periods. 3.5. Evolution over time of training of the addiction-like phenotypic traits in the three genotypes 3.5. Evolution over time of training of the addiction-like phenotypic traits in the three genotypes The statistical analysis of evolution over the early, middle and late periods was performed in the four addiction-related phenotypic traits. The three genotypes increased their impulsivity over the periods (WT mice Friedman's test, χ2 = 36.16, p < 0.001, CB2KO mice Friedman's test, χ2 = 27.18, p < 0.001, CB2TG mice Friedman's test, χ2 = 34.83, p < 0.001) (Fig. S3a). CB2KO presented a decreased impulsivity compared to WT through the whole protocol (early period Mann-Whitney's U, U = 361.5, p = 0.033, middle period Mann-Whitney's U, U = 334, p = 0.013, late period Mann-Whitney's U, U = 312.5, p = 0.006), while the CB2xP mice showed a decreased impulsivity compared to WT mice only in the early period (Mann-Whitney's U, U = 408.5, p = 0.031). CB2xP showed an increased impulsivity compared to CB2KO mice in the late period (Mann-Whitney's U, U = 233, p = 0.037) (Fig. S3a). Regarding the nonaddicted mice subgroups the most relevant results were the decreased impulsivity of nonaddicted CB2KO mice compared to nonaddicted WT (Mann-Whitney's U, U = 136.5, p = 0.001) and nonaddicted CB2xP (Mann-Whitney's U, U = 88, p = 0.019) mice in the late period (Fig. S3e). No differences were found in the cognitive inflexibility trait between the subgroups of nonaddited mice of the three genotypes (Fig. S3f). Nonaddicted CB2KO mice showed a reduced appetitive cue reactivity in the early period compared to nonaddicted WT mice (Mann-Whitney's U, U = 198, p = 0.038) (Fig. S3g). Non addicted CB2xP mice showed decreased aversive cue reactivity in the middle period compared to nonaddicted WT mice (Mann-Whitney's U, U = 203, p = 0.049) (Fig. S3h). WT and CB2KO mice showed differences in the evolution of their cognitive inflexibity (WT mice Friedman's test, χ2 = 19.19, p < 0.001, CB2KO mice Friedman's test, χ2 = 7.48, p = 0.024) while CB2xP results in this phenotypic trait remained stable (CB2xP mice Friedman's test, χ2 = 0.87, p = 0.646) (Fig. S3b). Differences between genotypes were only found in the middle period. were CB2xP showed a decreased cognitive inflexibility compared to WT mice (Mann-Whitney's U, U = 387.5, p = 0.016) (Fig. S3b). 3.4. Evolution over time of training of the three-addiction criteria in the different genotypes Regarding the addicted mice subgroups the most interesting results were the decreased persistence of addicted CB2xP mice compared to addicted WT mice in the early period (Mann-Whitney's U, U = 0, p < 0.001) and the decreased persistence of addicted CB2KO mice compared to addicted WT mice in the middle period (Mann-Whitney's U, U = 11.5, p = 0.045) (Fig. S2d). Regarding the motivation criteria addicted WT mice presented an increased motivation in the middle period compared to CB2KO (Mann-Whitney's U, U = 8.5, p = 0.021) and CB2xP (Mann- Whitney's U, U = 16.5, p = 0.034) mice (Fig. S2e). Addicted CB2xP mice 6 Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. Fig. 3. Differences between genotypes in the classification of addicted and nonaddicted mice. a Percentage of mice classified as nonaddicted or addicted based on food addiction-like criteria scoring in the late period (Chi-square, WT vs CB2xP #p < 0.05) (WT n = 42, CB2KO n = 25, CB2xP n = 28, Statistical details are included in Table S3). b-d Behavioral evolution in the three addiction criteria tests over the early, middle, and late periods of all mice. Friedman test was used to evaluate evolution over time, statistical details are included in Table S4. Mann-Whitney's U test was used to compare groups in each period and significances between WT and CB2KO were represented as p < 0.05, p < 0.01 or p < 0.001, significances between WT and CB2xP mice were represented as +p < 0.05, ++p < 0.01 or +++p < 0.001 and significances between CB2KO and CB2xP mice were represented as #p < 0.05, ##p < 0.01, ###p < 0.001. (Data are expressed as mean ± SEM. WT n = 42, CB2KO n = 25, CB2xP n = 28. Statistical details are included in Table S4). Fig. 3. Differences between genotypes in the classification of addicted and nonaddicted mice. a Percentage of mice classified as nonaddicted or addicted based on food addiction-like criteria scoring in the late period (Chi-square, WT vs CB2xP #p < 0.05) (WT n = 42, CB2KO n = 25, CB2xP n = 28, Statistical details are included in Table S3). b-d Behavioral evolution in the three addiction criteria tests over the early, middle, and late periods of all mice. Friedman test was used to evaluate evolution over time, statistical details are included in Table S4. 3.4. Evolution over time of training of the three-addiction criteria in the different genotypes Mann-Whitney's U test was used to compare groups in each period and significances between WT and CB2KO were represented as p < 0.05, p < 0.01 or p < 0.001, significances between WT and CB2xP mice were represented as +p < 0.05, ++p < 0.01 or +++p < 0.001 and significances between CB2KO and CB2xP mice were represented as #p < 0.05, ##p < 0.01, ###p < 0.001. (Data are expressed as mean ± SEM. WT n = 42, CB2KO n = 25, CB2xP n = 28. Statistical details are included in Table S4). showed decreased compulsivity compared to addicted WT (Mann- Whitney's U, U = 157, p = 0.003) only in the early period (Fig. S2f). the early period compared to CB2KO (Mann-Whitney's U, U = 306.5, p = 0.005) and CB2xP (Mann-Whitney's U, U = 405, p = 0.028) mice (Fig. S3c). The aversive cue reactivity of WT and CB2KO mice showed an evo lution (WT mice Friedman's test, χ2 = 10.02, p = 0.007, CB2xP mice Friedman's test, χ2 = 11.84, p = 0.003) while CB2xP mice remained stable (Friedman's test, χ2 = 1.64, p = 0.441) (Fig. S3d). WT mice showed and increased aversive cue reactivity in the early period compared to CB2KO (Mann-Whitney's U, U = 297, p = 0.003) (Fig. S3d). 3.8. Alterations in gene expression after long-term exposure to palatable pellets The percentage of variance explained by the first principal compo nent (PC1) was 41.16% and by the second principal component (PC2) was 17.13%. The distribution of all mice according to these components is shown in Fig. 4a and Fig. 4b. Nonaddicted and addicted animals were distributed in two well-differentiated clusters (Fig. 4a). When repre senting mice by genotype and addictive phenotype, we can also observe a cluster organization of the different subgroups. The main addiction criterion loading in PC1 was motivation (Fig. 4c), whereas compulsivity was the predominant criterion in PC2 (Fig. 4d). Among the four phenotypic traits, impulsivity and appetitive and aversive cue- reactivities had the highest loading in PC1, whereas cognitive inflexi bility showed the lowest loading in both components. Thus, PC1 was the most useful component for discriminating between addicted and non addicted mice, as it accounts for more variability. Finally, the distribu tion of the different addiction criteria and phenotypic traits is represented in Fig. 4e, showing the motivation and impulsivity nearest. Molecular analysis was performed by RT-qPCR in two key areas of the mesolimbic system (NAc and VTA), the PVN and three cortical areas (PL, IL, and OFC), which are part of the medial prefrontal cortex, pre viously related to addiction (Fig. 6c, i) (Blakemore and Robbins, 2012; Diamond, 2013; Miller and Cohen, 2003; Radcliffe Hospital et al., 2005). In the NAc, the expression levels of the gene encoding CB1R (Cnr1) (Fig. 6a) were found to be different between the three genotypes (one- way ANOVA: F(2,45) = 5.59, p = 0.007). RT-qPCR showed decreased levels of Cnr1 gene expression in CB2xP compared to WT mice (post-hoc LSD test, I-J = 0.15, p = 0.048) and CB2KO (post-hoc LSD test, I-J = 0.25, p = 0.002) in the NAc. Regarding Oprm1 gene expression in the NAc (Fig. 6b), one-way ANOVA revealed differences between genotypes (F(2,45) = 3.74, p = 0.032). CB2xP mice showed lower levels of Oprm1 gene expression than CB2KO mice (post-hoc LSD test, I-J = 0.17, p = 0.012). Then, the three genotypes were evaluated by PCA separately. In the WT mice group, the percentage of variance explained by the two prin cipal components (PC) was 44.83% (PC1) and 15.21% (PC2). The dis tribution of WT mice according to these components is shown in Fig. S4a, and the distribution of the different addiction criteria and phenotypic traits in Fig. S4b. 3.8. Alterations in gene expression after long-term exposure to palatable pellets The main addiction criterion loading in PC1 for WT mice was motivation (Fig. S4c), whereas compulsivity was the predominant criterion in PC2 (Fig. S4d). Among the four phenotypic traits, impulsivity and both appetitive and aversive cue-reactivities had the highest loading in PC1, whereas cognitive inflexibility showed the lowest loading in PC1. Thus, PC1 was the most useful to discriminate between addicted and nonaddicted mice as it accounts for more variability. Concerning the expression of the Th gene in the VTA, one-way ANOVA revealed a genotype effect (F(2,40) = 19.22, p < 0.001), CB2xP displayed lower gene expression levels of Th than WT (post-hoc LSD test, I-J = 0.41, p < 0.001) and than CB2KO (post-hoc LSD test, I-J = 0.41, p < 0.001) (Fig. 6d). In the RT-qPCR of Crf gene in the PVN, one-way ANOVA revealed differences between genotypes (F(2,40) = 6.79, p = 0.003) (Fig. 6e). The Crf mRNA relative expression was significantly increased in CB2xP in comparison to WT (post-hoc LSD test, I-J = 0.23, p = 0.024) and CB2KO mice (post-hoc LSD test, I-J = 0.36, p < 0.001). Finally, one-way ANOVA analyses of the mRNA relative expression of the Drd1 gene in the IL, revealed differences between genotypes (F (2,29) = 3.41, p = 0.047), in which CB2KO mice showed a reduced mRNA expression of Drd1 gene compared to WT mice (post-hoc LSD test, I-J = 0.57, p = 0.022). In CB2KO mice, PC1 explained 47.99% of the variance and PC2 the 20.48% The distribution of CB2KO mice according to these components is represented in Fig. S5e, and the distribution of the addiction criteria and phenotypic traits in Fig. S5f. Similarly to WT mice, the main addiction criterion loading in PC1 of CB2KO mice was motivation, fol lowed by the persistence of response (Fig. S5g). The compulsivity cri terion did not meet the proposed loading criterion (0.40) in CB2R mutants (Field, 2018) (Fig. 3g). With regards to the phenotypic traits, impulsivity and appetitive cue-reactivity in CB2KO mice weighted more in PC1, whereas cognitive inflexibility weighted mainly in PC2 (Fig. S5h). PC1 was also the most useful to discriminate between addicted and nonaddicted mice. 3.7. Emotional alterations after long-term exposure to palatable pellets Depressive-like behavior was evaluated at the end of the 3.6. Principal component analysis revealed differential patterns of behavioral factor loadings in food addiction-like behavior in mice The links between the different behavioral addiction-like criteria and phenotypic traits were evaluated using PCA. i 3.5. Evolution over time of training of the addiction-like phenotypic traits in the three genotypes Regarding the addicted mice subgroups the most remarkable results were the decreased impulsivity of addicted CB2xP mice in the early period compared to addicted WT mice period (Mann-Whitney's U, U = 0, p < 0.001) and the decreased impulsivity of addicted CB2KO mice compared to addicted WT mice in the middle period (Mann-Whitney's U, U = 9, p = 0.029) (Fig. S3i). Cognitive inflexibility was decreased in addicted CB2xP mice compared to WT in the middle period (Mann- Whitney's U, U = 13, p = 0.016) (Fig. S3j). Regarding the appettive cue reactivity, addicted CB2KO mice presented a decreased response in the early period compared to WT mice (Mann-Whitney's U, U = 8.5, p = 0.021) (Fig. S3k). Addicted CB2KO mice showed decreased aversive cue The appetitive cue reactivity of CB2KO mice showed an evolution (Friedman's test, χ2 = 6.32, p = 0.042) while the appetitive cue reac tivity of WT and CB2xP mice remained stable (WT mice Friedman's test, χ2 = 0.95, p = 0.623, CB2xP mice Friedman's test, χ2 = 2.79, p = 0.248) (Fig. S3c). WT mice showed and increased appetitive cue reactivity in 7 A. García-Blanco et al. Neurobiology of Disease 179 (2023) 106034 reactivity compared to addicted WT mice in the early (Mann-Whitney's U, U = 0, p < 0.001) and middle (Mann-Whitney's U, U = 9, p = 0.029) period (Fig. S3l). reactivity compared to addicted WT mice in the early (Mann-Whitney's U, U = 0, p < 0.001) and middle (Mann-Whitney's U, U = 9, p = 0.029) period (Fig. S3l). experimental protocol by the forced swimming test. No significant ge notype differences were found in the immobility time shown (one-way ANOVA, NS) (Fig. 5a). Anxiety-like behavior was evaluated by the elevated plus maze test. Both CB2KO and CB2xP genotypes showed unaltered anxiety-like behavior when compared to the WT group (one-way ANOVA, NS) (Fig. 5c and Fig. 5d). 3.6. Principal component analysis revealed differential patterns of behavioral factor loadings in food addiction-like behavior in mice 4. Discussion In this study, we evaluate the role of CB2R in food addiction by dissecting the phenotype of CB2KO and CB2xP mice during the devel opment of this behavioral disorder. Different phenotypic signatures of resilience and vulnerability to develop food addiction were found in CB2KO and CB2xP. Thus, palatable food reinforcement was reduced in CB2KO mice and a low percentage of these mutants developed food addiction during the early training period suggesting a resistant phenotype in agreement with the decreased nicotine self-administration reported in CB2KO mice (Navarrete et al., 2013). In contrast, CB2xP mice showed decreased chocolate self-administration in the early period, but increased responding in the late period suggesting a switch in the reward sensitivity for these mutants. Thus, the reduction in food self-administration between genotypes only occurs in the early period. The length of the operant training seems crucial for these adaptive re sponses leading to differential behavioral responses. These effects could be related to the dopamine desensitization that has been only reported in long-termed substance abusers (Volkow et al., 2011). Decreased cocaine self-administration was previously exhibited by CB2xP mice during short-operant training, further supporting the relevance of the length of training for revealing this decreased seeking to different rewarding stimuli during short training periods (Aracil-Fern´andez et al., 2012). However, the percentage of mice reaching addiction criteria in In the CB2xP group, the percentage of variance explained by PC1 was 40.96% and 17.77% by PC2. The distribution of CB2xP mice according to the two components is represented in Fig. S6i, where the PC1 was again the most useful to discriminate between addicted and nonaddicted mice. Behavioral tests clustered according to the loading in two com ponents of the PCA are shown in Fig. S6j. The main addiction criterion loading in PC1 for CB2xP mice was the persistence of response, followed by motivation, whereas compulsivity showed the lowest loading in PC1 (Fig. S6k). Among the four phenotypic traits, aversive cue-reactivity and impulsivity had the highest loading in PC1. Appetitive cue-reactivity showed the main loading in PC2 (Fig. S6l). The cognitive inflexibility did not meet the proposed loading criterion (0.40). 3.7. Emotional alterations after long-term exposure to palatable pellets Depressive-like behavior was evaluated at the end of the 8 8 Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. Fig. 4. 4. Discussion Principal component analysis (PCA) revealed differential patterns of behavioral factor loadings in food addiction-like behavior in mice depending on their addictive phenotype and genotype. a PCA of the three addiction criteria and the four phenotypic traits performed in all mice with factor loadings of principal component 1 (PC1) (41.16%) and principal component 2 (PC2) (17.13%). Mice subjects clustered by addicted or nonaddicted on the space yielded two components of the PCA that account for the maximum variability (n = 25 addicted (A) mice represented in red, n = 70 nonaddicted (NA) mice represented in white). b PCA of the three addiction criteria and the four phenotypic traits performed in all mice. Mice subjects clustered by addiction categorization and by genotype on the space yielded two components of the PCA that account for the maximum variability (n = 34 nonaddicted WT (WT NA) mice represented as empty grey circles, n = 18 nonaddicted CB2KO (CB2KO NA) mice represented as empty blue circles, n = 18 nonaddicted CB2xP (CB2xP NA) mice represented as empty orange circles, n = 8 addicted WT (WT A) mice represented as filled grey circles, n = 7 addicted CB2KO (CB2KO A) mice represented as filled blue circles, n = 10 addicted CB2xP (CB2xP A) mice represented as filled orange circles). c-d Graphs with the order of factor loading of the different variables in the PC1 and PC2. The dashed horizontal line marked loadings >0.7 mainly contributing to the component. e Behavioral tests clustered according to the loading in two components of the PCA. Green colour represents PC1 and pink colour represents PC2. Results are summarized in Table S1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 4. Principal component analysis (PCA) revealed differential patterns of behavioral factor loadings in food addiction-like behavior in mice depending on their addictive phenotype and genotype. a PCA of the three addiction criteria and the four phenotypic traits performed in all mice with factor loadings of principal component 1 (PC1) (41.16%) and principal component 2 (PC2) (17.13%). Mice subjects clustered by addicted or nonaddicted on the space yielded two components of the PCA that account for the maximum variability (n = 25 addicted (A) mice represented in red, n = 70 nonaddicted (NA) mice represented in white). 4. Discussion b PCA of the three addiction criteria and the four phenotypic traits performed in all mice. Mice subjects clustered by addiction categorization and by genotype on the space yielded two components of the PCA that account for the maximum variability (n = 34 nonaddicted WT (WT NA) mice represented as empty grey circles, n = 18 nonaddicted CB2KO (CB2KO NA) mice represented as empty blue circles, n = 18 nonaddicted CB2xP (CB2xP NA) mice represented as empty orange circles, n = 8 addicted WT (WT A) mice represented as filled grey circles, n = 7 addicted CB2KO (CB2KO A) mice represented as filled blue circles, n = 10 addicted CB2xP (CB2xP A) mice represented as filled orange circles). c-d Graphs with the order of factor loading of the different variables in the PC1 and PC2. The dashed horizontal line marked loadings >0.7 mainly contributing to the component. e Behavioral tests clustered according to the loading in two components of the PCA. Green colour represents PC1 and pink colour represents PC2. Results are summarized in Table S1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 4. Principal component analysis (PCA) revealed differential patterns of behavioral factor loadings in food addiction-like behavior in mice depending on their addictive phenotype and genotype. a PCA of the three addiction criteria and the four phenotypic traits performed in all mice with factor loadings of principal component 1 (PC1) (41.16%) and principal component 2 (PC2) (17.13%). Mice subjects clustered by addicted or nonaddicted on the space yielded two components of the PCA that account for the maximum variability (n = 25 addicted (A) mice represented in red, n = 70 nonaddicted (NA) mice represented in white). b PCA of the three addiction criteria and the four phenotypic traits performed in all mice. 4. Discussion Other studies have reported that CB2KO mice show increased alcohol-drinking behavior and food intake (Pradier et al., 2015). Even though some of this evidence is dependent on the envi ronment, the diet, or the mouse strain (Ishiguro et al., 2010; Pradier et al., 2015), all these results together highlight the interest in targeting CB2R in obesity-associated pathologies in addictive processes closely related to hedonic control systems. In our previous study (Agudo et al., 2010), total food intake, mainly dependent on the homeostatic control of food intake, and several metabolic parameters including insulin sensitivity, adipose tissue inflammation and glucose uptake were eval uated in CB2KO mice. This parameter of food intake under the homeo static control was increased in CB2KO mice. In our current study, mice were fed ad libitum, fully covering their metabolic needs, and we evaluated the consumption of highly palatable isocaloric pellets during one hour daily operant sessions. Therefore, high palatable pellet seeking under these conditions is mainly dependent on the hedonic control of food intake rather than the homeostatic control (Onaolapo and Fig. 5. Addicted CB2KO mice present less depressive-like behavior after exposition to long-term operant training maintained by chocolate-favored pel lets. Characterization of anxiety-like behavior. a Time of immobility in seconds (s) in the forced swimming test as a measure of depressive-like behavior (one- way ANOVA, NS). b Percentage of time spent in the open arms as a measure of anxiety-like behavior (one-way ANOVA, NS). Data are expressed as mean ± SEM. WT n = 42, CB2KO n = 25, CB2xP n = 28. Statistical details are included in Table S5. both CB2KO and CB2xP mice was similar in the late period, pointing out the multifactorial nature of this disorder. i Here, the percentages of WT mice classified as vulnerable or resilient in the early and middle periods were equivalent across periods. In the late period, the 19.1% of WT mice were classified as addicted, similar to the prevalence reported in humans (19.9%) (Pursey et al., 2014) and to previous mice studies (22.2–25.5%) (Domingo-Rodriguez et al., 2020; García-Blanco et al., 2022; Mancino et al., 2015). The prolonged expo sure to highly palatable food enhanced the percentage of CB2KO mice reaching the addiction criteria in spite of the resilient phenotype observed in these mutants at the early training period. 4. Discussion CB2R expression in VTA dopaminergic neurons projecting to NAc was demonstrated to modulate the dopaminergic tone, which has a direct impact on dopamine-related behaviors such as the hedonic effect triggered by abuse of substances and palatable food (Zhang et al., 2014). Furthermore, neurons of the NAc and VTA expressed CB2R, and some of these neurons coexpressed D2R (Aracil-Fern´andez et al., 2012). Inter estingly, endocannabinoids have a role in the dopaminergic modulation of striatal MSNs (Andr´e and Gonthier, 2010). Conversely, endocanna binoids and exogenous cannabinoids may also produce aversive effects by activating CB1R on glutamatergic projections from the prefrontal cortex (PFC) to the VTA, whereas the activation of CB2R on dopami nergic neurons from the VTA to the NAc may decrease the rewarding effects (Caba˜nero et al., 2021b). CB2R localized on glial cells, such as astrocytes or microglia, could reduce dopamine levels by controlling the release of inflammatory cytokines from these cells. l y y The differences observed in palatable food reinforcement and impulsive-like behavior could not be explained by a learning impair ment since the three genotypes acquired the operant behavior in similar timing periods. The body weight of CB2KO mice did not differ from WT mice, and CB2xP mice exhibited reduced body weight gain, although this change did not affect the operant training acquisition. Pellet con sumption increased similarly in CB2xP and WT mice independently of the body weight, suggesting that a caloric need did not trigger the intake. In agreement, previous studies showed a lean phenotype of CB2xP and reduced body weight after chronic pharmacological CB2R activation (Ishiguro et al., 2010; Romero-Zerbo et al., 2012; Verty et al., 2015). Previous studies have revealed a selective decrease of food intake in CB2xP mice compared to WT only when measured during 12 h in fasted conditions and during 120 min after food presentation, but not when food intake was evaluated during longer periods (Romero-Zerbo et al., 2012). Furthermore, recent investigations have shown that the pharmacological activation of CB2 with JWH133 decreases sucrose self- administration (Bi et al., 2020), and the use of the CB2 agonist beta- caryophyllene decreases motivational salience and conditioning place preference for palatable food (Dos Santos Barbosa et al., 2023). Conversely, mice lacking CB2R present an age-related obese phenotype with increased food intake (Agudo et al., 2010; Pradier et al., 2015), and CB2R pharmacological blockade stimulates food consumption (Ishiguro et al., 2010). 4. Discussion Mice subjects clustered by addiction categorization and by genotype on the space yielded two components of the PCA that account for the maximum variability (n = 34 nonaddicted WT (WT NA) mice represented as empty grey circles, n = 18 nonaddicted CB2KO (CB2KO NA) mice represented as empty blue circles, n = 18 nonaddicted CB2xP (CB2xP NA) mice represented as empty orange circles, n = 8 addicted WT (WT A) mice represented as filled grey circles, n = 7 addicted CB2KO (CB2KO A) mice represented as filled blue circles, n = 10 addicted CB2xP (CB2xP A) mice represented as filled orange circles). c-d Graphs with the order of factor loading of the different variables in the PC1 and PC2. The dashed horizontal line marked loadings >0.7 mainly contributing to the component. e Behavioral tests clustered according to the loading in two components of the PCA. Green colour represents PC1 and pink colour represents PC2. Results are summarized in Table S1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. Fig. 5. Addicted CB2KO mice present less depressive-like behavior after exposition to long-term operant training maintained by chocolate-favored pel lets. Characterization of anxiety-like behavior. a Time of immobility in seconds (s) in the forced swimming test as a measure of depressive-like behavior (one- way ANOVA, NS). b Percentage of time spent in the open arms as a measure of anxiety-like behavior (one-way ANOVA, NS). Data are expressed as mean ± SEM. WT n = 42, CB2KO n = 25, CB2xP n = 28. Statistical details are included in Table S5. evaluated. The lack of CB2R decreased impulsive like-behavior, whereas CB2 overexpression did not modify this behavioral trait. In agreement, previous studies have demonstrated that CB2R blockade decreases impulsivity (Navarrete et al., 2012). Impulsivity has been widely asso ciated with the dopaminergic functioning, and increased impulsivity was correlated with diminished drd2 availability in the rat NAc (Dalley et al., 2007). Other studies showed that the pharmacological stimulation of CB2R in the VTA and NAc reduced dopaminergic neuron excitability and decreased dopamine extracellular levels in the NAc (Ma et al., 2019; Xi et al., 2011; Zhang et al., 2014). Therefore, CB2R might be involved in impulsive-seeking behavior through modulation of the dopaminergic system. 4. Discussion In our study, the decreased expression of th was found in CB2xP mice after a long protocol (4 months) of palatable food self-administration. This decrease in the limiting enzyme in dopamine synthesis in CB2xP mice could correlate with the dopamine desensitization that occurs in the later stages of addictive processes when the disease is fully consolidated (Nimitvilai et al., 2014; Volkow et al., 2011). i Neither lack nor overexpression of CB2R had an effect on depressive- nor anxiety-like behavior after the long-term operant training in our food addiction model. Indeed, both CB2KO, and CB2xP mice displayed equivalent despair-induced immobility time in the forced swimming test and exploration in the open arms of the elevated plus maze. Previous studies using the same genetic mouse models showed that CB2R deletion enhanced depressive-like behavior (Ortega-Alvaro et al., 2011) and vulnerability to stress and anxiety-like behavior (Ortega-´Alvaro et al., 2015), whereas CB2R overexpression protected from anxiogenic and depressive environmental conditions alteration (García-Guti´errez et al., 2010; García-Guti´errez and Manzanares, 2011). On the other hand, the CB2r antagonist AM630 led to anxiolytic and antidepressant effects in naïve mice, while pharmacological activation of CB2r by JWH133 pro duced anxiety and depressive-like behavior (García-Guti´errez et al., 2010; García-Guti´errez and Manzanares, 2011). Thus, genetic and pharmacological manipulations are not comparable in these pheno types, probably due to the complexity of the inherent neurobiological mechanism involved. In our study, long-term exposure to highly palat able food may interfere with the depressive- or anxiety-like behavior phenotypes of CB2KO and CB2xP mice, probably due to allostatic changes promoted by the repetitive activation of the reward circuits. In agreement with its resilient phenotype, addicted CB2KO mice displayed lower expression of the corticotropin-releasing factor (CRF) gene (crf) in the hypothalamus PVN, which is involved in the homeo static control of food intake (King, 2005). CRF is a stress hormone implicated in addictive processes and participates in drug-seeking and the manifestations of drug withdrawal (Blacktop et al., 2011). In agreement, we reported a lower expression in food addiction-resistant mice (addicted CB2KO) and increased expression in addiction- vulnerable mice (addicted CB2xP mice). This close relationship be tween crf expression and CB2R content in food addiction vulnerability suggests an important role of this hormone in the mechanisms involved in CB2R-mediated stress-induced vulnerability to food addiction. 4. Discussion Data are expressed as mean ± SEM (WT NA n = 8, CB2KO NA n = 8, CB2xP NA n = 8, WT A n = 8, CB2KO A n = 8, CB2xP A n = 8). Addicted WT mice are represented as grey filled circles, addicted CB2KO mice are represented as blue filled circles and addicted CB2xP mice are represented as orange filled circles, some animals were excluded due to technical limitations. Statistical details are included in Table S6. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Onaolapo, 2018), which was decreased in CB2KO mice. The meso corticolimbic circuit plays a crucial role in the hedonic system (Onao lapo and Onaolapo, 2018) that is altered in food-addicted individuals. Therefore, CB2R seems to play an opposite role in the homeostatic and hedonic control of food intake. with the resistant phenotype of these mutants. Previous studies have also reported increased Oprm1 in the NAc of CB2KO mice under naïve conditions and after acute ethanol administration (Ortega-´Alvaro et al., 2015). Furthermore, pharmacological CB2R blockade increased Oprm1 in the NAc (Navarrete et al., 2018). Therefore, the interaction between CB2R and Oprm1 seems crucial in food addiction development. Differential behavioral signatures with regard to the addiction criteria and the phenotypic traits were revealed in the two sub populations of addicted and nonaddicted mice in the three genotypes across the early, middle and late periods. Indeed, PCA results revealed an excellent clusterization of addicted and nonaddicted mice total mice and also in each genotype, suggesting that the model is highly predictive and that the addiction criteria and the phenotypic traits explain a suf ficient percentage of variance to distinguish the subpopulations. The expression of the gene encoding tyrosine hydroxylase (th), the rate-limiting enzyme in dopamine synthesis, was decreased in the VTA of CB2xP mice suggesting a hypodopaminergic state that was indepen dent of the criteria of addiction. Similar results were observed after the pharmacological activation of CB2R (Navarrete et al., 2018). Conversely, previous studies showed that th expression in the VTA was decreased in naïve CB2KO mice (Navarrete et al., 2018) and increased in naïve CB2xP mice (Aracil-Fern´andez et al., 2012), although others did not report differences in CB2KO (Ortega-´Alvaro et al., 2015). 4. Discussion Changes in the targeted gene expression were also revealed in the OFC of addicted mice where cnr1 expression was downregulated inde pendently of the genotype. This dysregulation can be associated with a compensatory mechanism that may appear to try to regulate gluta matergic and gabaergic release. The IL mPFC of CB2KO mice showed a reduced mRNA expression of the Drd1 gene compared to WT mice, in agreement with a protective factor since recent studies showed upre gulation of Drd1 mRNAs in addicted mice (Domingo-Rodriguez et al., 2022). The expression of the other targeted genes was not modified in the cortical areas in the different genotypes. Several limitations should be taken into account in the present study, such as the heterogeneity of the samples concerning individual differences in behavioral responses; the small fold change in gene fluctuations that could limit the detection of differences in lowly expressed genes. Changes in the expression of several genes closely related to reward activity were studied to disentangle the mechanisms involved in the behavioral changes promoted by the modification of CB2R content. CB2KO mice with a food addiction resilient phenotype showed the highest expression levels of the CB1R gene (cnr1) in the NAc compared to the other genotypes, and this change was more pronounced in addicted subgroup. In contrast, CB2xP, vulnerable to develop addiction, presented the lowest cnr1 gene expression level in the NAc. These results are in agreement with previous studies showing that the decrease of CB2R activity by CB2R pharmacological blockade increases CB1R in the NAc, whereas CB1R is decreased in NAc after CB2R agonist adminis tration (Navarrete et al., 2018). In agreement with the vulnerable phenotype of CB2xP mice, the expression of the mu opioid receptor (Oprm1) was lower in these mice. This finding correlates with previous studies reporting that Oprm1 is reduced in the NAc of CB2xPmice under naïve conditions or after cocaine pre-treatment (Aracil-Fern´andez et al., 2012) and in WT mice after CB2R pharmacological stimulation (Navarrete et al., 2018). High levels of Oprm1 were observed in addicted CB2KO mice in agreement 4. Discussion o Detection of Drd2 mRNA in OFC (one-way ANOVA, NS). Data are expressed as mean ± SEM (WT NA n = 8, CB2KO NA n = 8, CB2xP NA n = 8, WT A n = 8, CB2KO A n = 8, CB2xP A n = 8). Addicted WT mice are represented as grey filled circles, addicted CB2KO mice are represented as blue filled circles and addicted CB2xP mice are represented as orange filled circles, some animals were excluded due to technical limitations. Statistical details are included in Table S6. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) A. García-Blanco et al. Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. Fig. 6. Alterations in gene expression after long-term exposure to palatable pellets. a Detection of Cnr1 mRNA in NAc (one-way ANOVA, p < 0.001, post-hoc LSD test WT vs CB2xP + p < 0.05, CB2KO vs CB2xP ##p < 0.01). b Detection of Oprm1 in NAc (one-way ANOVA, p < 0.05, post-hoc LSD test CB2KO vs CB2xP #p < 0.05). c. NAc, PVN and VTA distribution in a sagittal view of the mice brain. d Detection of Th mRNA in VTA (one-way ANOVA, p < 0.001, post-hoc LSD test WT vs CB2xP +++p < 0.001, CB2KO vs CB2xP ###p < 0.001). e Detection of Crf mRNA in PVN (one-way ANOVA, p < 0.01, post-hoc LSD test WT vs CB2xP + p < 0.05, CB2KO vs CB2xP ###p < 0.001). f Detection of Cnr1 mRNA in PL (one-way ANOVA, NS). g Detection of Drd1 mRNA in PL (one-way ANOVA, NS). h Detection of Drd2 mRNA in PL (one-way ANOVA, NS). i. PL, IL and OFC distribution in a coronal view of the mice brain. j Detection of Cnr1 mRNA in IL (one-way ANOVA, NS). k Detection of Drd1 mRNA in IL (one-way ANOVA, p < 0.05, post-hoc LSD test WT vs CB2KO p < 0.05). l Detection of Drd2 mRNA in IL (one-way ANOVA, NS). m Detection of Cnr1 mRNA in OFC (one-way ANOVA, NS). n Detection of Drd1 mRNA in OFC (one-way ANOVA, NS). o Detection of Drd2 mRNA in OFC (one-way ANOVA, NS). 4. Discussion This result highlights that addiction is a multifactorial disease, and knocking out one single gene is not enough to fully revert this phenotype. The pro tective effect to develop food addiction in the absence of CB2R in a short-operant protocol is in accordance with the lower nicotine self- administration similarly shown by these mutant mice (Navarrete et al., 2013). CB2xP mice revealed an increased vulnerability to develop food addiction after long-term operant training. However, previous studies using these mutants (Aracil-Fern´andez et al., 2012) or pharma cological CB2R activation (Zhang et al., 2014) showed reduced cocaine self-administration in a short-operant training. These discrepancies can rely on the length of the training protocol or the different properties of the rewarding stimuli. Phenotypic traits related to vulnerability to food addiction were also 10 Neurobiology of Disease 179 (2023) a-Blanco et al. Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. (caption on next page) (caption on next page) 11 Neurobiology of Disease 179 (2023) 106034 Fig. 6. Alterations in gene expression after long-term exposure to palatable pellets. a Detection of Cnr1 mRNA in NAc (one-way ANOVA, p < 0.001, post-hoc LSD test WT vs CB2xP + p < 0.05, CB2KO vs CB2xP ##p < 0.01). b Detection of Oprm1 in NAc (one-way ANOVA, p < 0.05, post-hoc LSD test CB2KO vs CB2xP #p < 0.05). c. NAc, PVN and VTA distribution in a sagittal view of the mice brain. d Detection of Th mRNA in VTA (one-way ANOVA, p < 0.001, post-hoc LSD test WT vs CB2xP +++p < 0.001, CB2KO vs CB2xP ###p < 0.001). e Detection of Crf mRNA in PVN (one-way ANOVA, p < 0.01, post-hoc LSD test WT vs CB2xP + p < 0.05, CB2KO vs CB2xP ###p < 0.001). f Detection of Cnr1 mRNA in PL (one-way ANOVA, NS). g Detection of Drd1 mRNA in PL (one-way ANOVA, NS). h Detection of Drd2 mRNA in PL (one-way ANOVA, NS). i. PL, IL and OFC distribution in a coronal view of the mice brain. j Detection of Cnr1 mRNA in IL (one-way ANOVA, NS). k Detection of Drd1 mRNA in IL (one-way ANOVA, p < 0.05, post-hoc LSD test WT vs CB2KO *p < 0.05). l Detection of Drd2 mRNA in IL (one-way ANOVA, NS). m Detection of Cnr1 mRNA in OFC (one-way ANOVA, NS). n Detection of Drd1 mRNA in OFC (one-way ANOVA, NS). 5. Conclusions In summary, we characterized the role of CB2R in the development of food addiction by dissecting the phenotype of CB2KO and CB2xP mice. We have also identified differential gene expression signatures in 12 Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. org/10.1016/j.nbd.2023.106034. org/10.1016/j.nbd.2023.106034. the reward circuit depending on the CB2R content that may underlie the phenotypes of these mutants Different phenotypes with regards to reinforcement, impulsivity, compulsivity, and susceptibility to develop food addiction criteria were revealed in these mutants along our long- termed protocol. Our findings suggest that lack of CB2R activity de creases the reinforcement, protects against impulsivity- and compulsivity-like behavior, and therefore could be a protective factor to develop core features of food addiction in a time-dependent manner. However, it does not influence the final percentage of mice reaching addiction criteria. This phenotype of CB2KO mice was associated with increased cnr1 in the NAc, and decreased crf expression in the PVN. The overexpression of CB2R leads to a food addiction vulnerable phenotype associated with decreased Cnr1 and Oprm1 expression in the NAc, reduced levels of th in the VTA and increased crf expression in the PVN and decreased drd1 mRNA expression in the IL. These alterations in the reward circuit may underline substantial modifications in reward pro cessing due to CB2R overexpression that could explain the enhanced addictive vulnerability of these mice. In summary, our study unravels a new neurobiological mechanism involving CB2Rs in the substrate un derlying vulnerability to develop food addiction, which could pave the way toward novel therapeutic approaches for this disorder. Data will be made available on request. Data will be made available on request. Everitt, B.J., Belin, D., Economidou, D., Pelloux, Y., Dalley, J.W., Robbins, T.W., 2008. Neural mechanisms underlying the vulnerability to develop compulsive drug-seeking habits and addiction. Philos. Trans. R. Soc. B Biol. Sci. 363, 3125–3135. https://doi. org/10.1098/RSTB.2008.0089. Acknowledgements Fernandez-Aranda, F., Karwautz, A., Treasure, J., 2018. Food addiction: a transdiagnostic construct of increasing interest. Eur. Eat. Disord. Rev. 26, 536–540. https://doi.org/10.1002/ERV.2645. We thank M. Linares, R. Martín, D. Real, F. Porr´on, for their technical support. Field, A.P., 2018. Discovering Statistics Using IBM SPSS Statistics, 5th edition. SAGE Publ. Inc, p. 368. Declaration of Competing Interest Domingo-Rodriguez, L., Cabana-Domínguez, J., Fern`andez-Castillo, N., Cormand, B., Martín-García, E., Maldonado, R., 2022. Differential expression of miR-1249-3p and miR-34b-5p between vulnerable and resilient phenotypes of cocaine addiction. Addict. Biol. 27, e13201 https://doi.org/10.1111/ADB.13201. The authors declare no competing financial interests. Dos Santos Barbosa, L.A., Dutra, R.C., Moreira, E.L.G., de Carvalho, C.R., 2023. β-Caryophyllene, a cannabinoid receptor 2 agonist, decreases the motivational salience and conditioning place preference for palatable food in female mice. Addic Biol. 28, e13249 https://doi.org/10.1111/ADB.13249. References Adamczyk, P., Miszkiel, J., McCreary, A.C., Filip, M., Papp, M., Przegali´nski, E., 2012. The effects of cannabinoid CB1, CB2 and vanilloid TRPV1 receptor antagonists on cocaine addictive behavior in rats. Brain Res. 1444, 45–54. https://doi.org/ 10.1016/J.BRAINRES.2012.01.030. Agudo, J., Martin, M., Roca, C., Molas, M., Bura, A.S., Zimmer, A., Bosch, F., Maldonado, R., 2010. Deficiency of CB2 cannabinoid receptor in mice improves insulin sensitivity but increases food intake and obesity with age. Diabetologia 53, 2629–2640. https://doi.org/10.1007/S00125-010-1894-6. Andr´e, A., Gonthier, M.P., 2010. The endocannabinoid system: its roles in energy balance and potential as a target for obesity treatment. Int. J. Biochem. Cell Biol. 42, 1788–1801. https://doi.org/10.1016/j.biocel.2010.06.002. ´ Aracil-Fern´andez, A., Trigo, J.M., García-Guti´errez, M.S., Ortega-´Alvaro, A., Ternianov, A., Navarro, D., Robledo, P., Berbel, P., Maldonado, R., Manzanares, J., 2012. Decreased cocaine motor sensitization and self-Administration in Mice Overexpressing Cannabinoid CB2 receptors. Neuropsychopharmacol. 377 (37), 1749–1763. https://doi.org/10.1038/npp.2012.22. Belin, D., Mar, A.C., Dalley, J.W., Robbins, T.W., Everitt, B.J., 2008. High impulsivity predicts the switch to compulsive cocaine-taking. Science 320, 1352–1355. https:// doi.org/10.1126/SCIENCE.1158136. g Bi, G.H., Galaj, E., He, Y., Xi, Z.X., 2020. Cannabidiol inhibits sucrose self-administration by CB1 and CB2 receptor mechanisms in rodents. Addict. Biol. 25 https://doi.org/ 10.1111/ADB.12783. Blacktop, J.M., Seubert, C., Baker, D.A., Ferda, N., Lee, G., Graf, E.N., Mantsch, J.R., 2011. Augmented cocaine seeking in response to stress or CRF delivered into the ventral tegmental area following long-access self-administration is mediated by CRF receptor type 1 but not CRF receptor type 2. J. Neurosci. 31, 11396–11403. https:// doi.org/10.1523/JNEUROSCI.1393-11.2011. Funding Caron, A., Richard, D., 2017. Neuronal systems and circuits involved in the control of food intake and adaptive thermogenesis. Ann. N. Y. Acad. Sci. 1391 https://doi.org/ 10.1111/nyas.13263. This work was supported by the Spanish “Ministerio de Ciencia e Innovaci´on (MICIN), Agencia Estatal de Investigaci´on (AEI)” (PID2020- 120029GB-I00/MICIN/AEI/10.13039/501100011033, RD21/0009/ 0019, to RM; the “Generalitat de Catalunya, AGAUR” (2017 SGR-669, to RM; the “ICREA-Acad`emia” (2020) to RM; the “European Commission- DG Research” (PainFact, H2020-SC1–2019-2-RTD-848099, QSPain Re lief, H2020-SC1–2019-2-RTD-848068) to RM; the Spanish”la Caixa” Foundation under the project code LCF/PR/HR22/52420017 to R.M., the Spanish “Instituto de Salud Carlos III, RETICS-RTA” (RD16/0017/ 0020) to RM; the Spanish “Ministerio de Sanidad, Servicios Sociales e Igualdad, Plan Nacional Sobre Drogas” (PNSD- 2021I076, to RM; PNSD- 2019I006, to EMG; Ministerio de Ciencia e Innovaci´on (ERA-NET) PCI2021–122073-2A to EMG; and “Instituto de Salud Carlos III”, Spanish “Ministerio de Ciencia e Innovaci´on” (RD21/0009/0008, “Red de Investigaci´on en Atenci´on Primaria de Adicciones”) to JM. Dalley, J.W., Fryer, T.D., Brichard, L., Robinson, E.S.J., Theobald, D.E.H., L¨a¨ane, K., Pe˜na, Y., Murphy, E.R., Shah, Y., Probst, K., Abakumova, I., Aigbirhio, F.I., Richards, H.K., Hong, Y., Baron, J.C., Everitt, B.J., Robbins, T.W., 2007. Nucleus accumbens D2/3 receptors predict trait impulsivity and cocaine reinforcement. Science 315 (80), 1267–1270. https://doi.org/10.1126/science.1137073. p g Dalley, J.W., Everitt, B.J., Robbins, T.W., 2011. Impulsivity, compulsivity, and top-down cognitive control. Neuron 69, 680–694. https://doi.org/10.1016/J. NEURON.2011.01.020. Deroche-Gamonet, V., Belin, D., Piazza, P.V., 2004. Evidence for addiction-like behavior in the rat. Science 305 (80), 1014–1017. https://doi.org/10.1126/ SCIENCE.1099020/SUPPL_FILE/DEROCHEGAMONET.SOM.PDF. Diamond, A., 2013. Executive Functions, 64, pp. 135–168. https://doi.org/10.1146/ ANNUREV-PSYCH-113011-143750. Domingo-Rodriguez, L., Ruiz de Azua, I., Dominguez, E., Senabre, E., Serra, I., Kummer, S., Navandar, M., Baddenhausen, S., Hofmann, C., Andero, R., Gerber, S., Navarrete, M., Dierssen, M., Lutz, B., Martín-García, E., Maldonado, R., 2020. A specific prelimbic-nucleus accumbens pathway controls resilience versus vulnerability to food addiction. Nat. Commun. 111 (11), 1–16. https://doi.org/ 10.1038/s41467-020-14458-y. Authors contribution E.M-G., J.M. and R.M. conceived and designed the behavioral and gene expression studies, did the funding acquisition, project adminis tration and supervision; A.G.-B. and A.R.-L. performed the behavioral experiments and the statistical analyses and graphs with the supervision of E.M-G. and R.M.; A.G.-B., A.R.-L., F.N. and M.S.G-G: performed the qPCRs. E.M-G., A.G.-B. and R.M. wrote the original draft of the manu script with review and edits from all the other authors. Blakemore, S.J., Robbins, T.W., 2012. Decision-making in the adolescent brain. Nat. Neurosci. 159 (15), 1184–1191. https://doi.org/10.1038/nn.3177. ´ Buckley, N.E., McCoy, K.L., Mezey, ´E., Bonner, T., Zimmer, Anne, Felder, C.C., Glass, M., Zimmer, Andreas, 2000. Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2 receptor. Eur. J. Pharmacol. 396, 141–149. https://doi.org/10.1016/S0014-2999(00)00211-9. p g Caba˜nero, D., Martín-García, E., Maldonado, R., 2021a. The CB2 cannabinoid receptor as a therapeutic target in the central nervous system, 25, 659–676. https://doi.org/ 10.1080/14728222.2021.1971196. Caba˜nero, D., Martín-García, E., Maldonado, R., 2021b. The CB2 cannabinoid receptor as a therapeutic target in the central nervous system. Expert Opin. Ther. Targets 25, 659–676. https://doi.org/10.1080/14728222.2021.1971196. Appendix A. Supplementary data Role of cannabinoid CB2 receptor in the reinforcing actions of ethanol. Addict. Biol. 20, 43–55. https://doi.org/10.1111/ ADB.12076/SUPINFO. p g Ishiguro, Carpio, Horiuchi, Shu, Higuchi, Schanz, Benno, R., Arinami, T., Onaivi, E.S., 2010. A nonsynonymous polymorphism in cannabinoid CB2 receptor gene is associated with eating disorders in humans and food intake is modified in mice by its ligands. Synapse 64, 92–96. https://doi.org/10.1002/syn.20714. Palkovits, M., 1983. [23] punch sampling biopsy technique. Methods Enzymol. 103, 368–376. https://doi.org/10.1016/S0076-6879(83)03025-6. associated with eating disorders in humans and food intake is modified in mice by its ligands. Synapse 64, 92–96. https://doi.org/10.1002/syn.20714. King, P., 2005. The hypothalamus and obesity. Curr. Drug Targets 6, 225–240. https:// doi.org/10.2174/1389450053174587. Porsolt, R.D., Anton, G., Blavet, N., Jalfre, M., 1978. Behavioural despair in rats: a new model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47, 379–391. https://doi.org/10.1016/0014-2999(78)90118-8. La Porta, C., Andreea Bura, S., Llorente-Onaindia, J., Pastor, A., Navarrete, F., García- Gutierrez, M.S., De La Torre, R., Manzanares, J., Monfort, J., Maldonado, R., 2015. Role of the endocannabinoid system in the emotional manifestations of osteoarthritis pain. Pain 156, 2001–2012. https://doi.org/10.1097/J.PAIN.0000000000000260. Pradier, B., Erxlebe, E., Markert, A., R´acz, I., 2015. Interaction of cannabinoid receptor 2 and social environment modulates chronic alcohol consumption. Behav. Brain Res. 287, 163–171. https://doi.org/10.1016/J.BBR.2015.03.051. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real- time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408. https://doi. org/10.1006/meth.2001.1262. Pursey, K.M., Stanwell, P., Gearhardt, A.N., Collins, C.E., Burrows, T.L., 2014. The prevalence of food addiction as assessed by the Yale food addiction scale: a systematic review. Nutrients. https://doi.org/10.3390/nu6104552. g Ma, Z., Gao, F., Larsen, B., Gao, M., Luo, Z., Chen, D., Ma, X., Qiu, S., Zhou, Y., Xie, J., Xi, Z.X., Wu, J., 2019. Mechanisms of cannabinoid CB2 receptor-mediated reduction of dopamine neuronal excitability in mouse ventral tegmental area. EBioMedicine 42, 225. https://doi.org/10.1016/J.EBIOM.2019.03.040. Racz, I., Nadal, X., Alferink, J., Ba˜nos, J.E., Rehnelt, J., Martín, M., Pintado, B., Gutierrez- Adan, A., Sanguino, E., Bellora, N., Manzanares, J., Zimmer, A., Maldonado, R. Racz, Nadal, X., Alferink, J., Ba˜nos, J.E., Rehnelt, J., Martín, M., Pintado, B., Gutierrez- Adan, A., Sanguino, E., Bellora, N., Manzanares, J., Zimmer, A., Maldonado, R., 2008a. Interferon-gamma is a critical modulator of CB(2) cannabinoid receptor signaling during neuropathic pain. J. Neurosci. 28, 12136–12145. https://doi.org/ 10.1523/JNEUROSCI.3402-08.2008. Maldonado, R., Calv´e, P., García-Blanco, A., Domingo-Rodriguez, L., Senabre, E., Martín- García, E., 2021. Vulnerability to addiction. Neuropharmacology 186, 108466. https://doi.org/10.1016/j.neuropharm.2021.108466. Appendix A. Supplementary data Franklin, K., Paxinos, G., 2001. Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates. Supplementary data to this article can be found online at https://doi. 13 Neurobiology of Disease 179 (2023) 106034 A. García-Blanco et al. the endocannabinoid system in psychiatric disorders. Int. J. Mol. Sci. 23, 4764. https://doi.org/10.3390/IJMS23094764. García-Blanco, A., Domingo-Rodriguez, L., Cabana-Domínguez, J., Fern´andez- Castillo, N., Pineda-Cirera, L., Mayneris-Perxachs, J., Burokas, A., Espinosa- Carrasco, J., Arboleya, S., Latorre, J., Stanton, C., Cormand, B., Fern´andez-Real, J. M., Martín-García, E., Maldonado, R., 2022. miRNA signatures associated with vulnerability to food addiction in mice and humans. J. Clin. Invest. 132 https://doi. org/10.1172/JCI156281. the endocannabinoid system in psychiatric disorders. Int. J. Mol. Sci. 23, 4764. https://doi.org/10.3390/IJMS23094764. Nimitvilai, S., Herman, M., You, C., Arora, D.S., McElvain, M.A., Roberto, M., Brodie, M. S., 2014. Dopamine D2 receptor desensitization by dopamine or corticotropin releasing factor in ventral tegmental area neurons is associated with increased glutamate release. Neuropharmacology 82, 28–40. https://doi.org/10.1016/J. NEUROPHARM.2014.03.006. García-Guti´errez, M.S., Manzanares, J., 2011. Overexpression of CB2 cannabinoid receptors decreased vulnerability to anxiety and impaired anxiolytic action of alprazolam in mice. J. Psychopharmacol. 25, 111–120. https://doi.org/10.1177/ 0269881110379507/ASSET/IMAGES/LARGE/10.1177_0269881110379507-FIG 2. JPEG. Onaivi, E.S., Ishiguro, H., Gong, J.P., Patel, S., Meozzi, P.A., Myers, L., Perchuk, A., Mora, Z., Tagliaferro, P.A., Gardner, E., Brusco, A., Akinshola, B.E., Hope, B., Lujilde, J., Inada, T., Iwasaki, S., Macharia, D., Teasenfitz, L., Arinami, T., Uhl, G.R., 2008. Brain neuronal CB2 cannabinoid receptors in drug abuse and depression: from mice to human subjects. PLoS One 3, e1640. https://doi.org/10.1371/JOURNAL. PONE.0001640. García-Guti´errez, M.S., P´erez-Ortiz, J.M., Guti´errez-Ad´an, A., Manzanares, J., 2010. Depression-resistant endophenotype in mice overexpressing cannabinoid CB(2) receptors. Br. J. Pharmacol. 160, 1773–1784. https://doi.org/10.1111/j.1476- 5381.2010.00819.x. Onaolapo, A.Y., Onaolapo, O.J., 2018. Food additives, food and the concept of ‘food addiction’: is stimulation of the brain reward circuit by food sufficient to trigger addiction? Pathophysiology 25, 263–276. Gearhardt, A.N., Corbin, W.R., Brownell, K.D., 2016. Development of the Yale food addiction scale version 2.0. Psychol. Addict. Behav. 30, 113–121. https://doi.org/ 10.1037/ADB0000136. Ortega-Alvaro, A., Aracil-Fern´andez, A., García-Guti´errez, M.S., Navarrete, F., Manzanares, J., 2011. Deletion of CB2 Cannabinoid Receptor Induces Schizophrenia- Related Behaviors in Mice. Neuropsychopharmacol 367 (36), 1489–1504. https:// doi.org/10.1038/npp.2011.34. Ignatowska-Jankowska, B.M., Muldoon, P.P., Lichtman, A.H., Damaj, M.I., 2013. The cannabinoid CB2 receptor is necessary for nicotine-conditioned place preference, but not other behavioral effects of nicotine in mice. Psychopharmacology 229, 591–601. https://doi.org/10.1007/S00213-013-3117-6/FIGURES/6. Ortega-´Alvaro, A., Ternianov, A., Aracil-Fern´andez, A., Navarrete, F., García- Guti´errez, M.S., Manzanares, J., 2015. Appendix A. Supplementary data p g j p Mancino, S., Burokas, A., Guti´errez-Cuesta, J., Guti´errez-Martos, M., Martín-García, E., Pucci, M., Falconi, A., D’Addario, C., Maccarrone, M., Maldonado, R., 2015. Epigenetic and proteomic expression changes promoted by eating addictive-like behavior. Neuropsychopharmacology 40, 2788–2800. https://doi.org/10.1038/ npp.2015.129. Racz, I., Nadal, X., Alferink, J., Ba˜nos, J.E., Rehnelt, J., Martín, M., Pintado, B., Gutierrez- Adan, A., Sanguino, E., Manzanares, J., Zimmer, A., Maldonado, R., 2008b. Crucial role of CB2 cannabinoid receptor in the regulation of central immune responses during neuropathic pain. J. Neurosci. 28, 12125–12135. https://doi.org/10.1523/ JNEUROSCI.3400-08.2008. Radcliffe Hospital, J., Way, H., Kringelbach, M.L., 2005. The human orbitofrontal cortex: linking reward to hedonic experience. Nat. Rev. Neurosci. 69 (6), 691–702. https:// doi.org/10.1038/nrn1747. Martín-García, E., Burokas, A., Kostrzewa, E., Gieryk, A., Korostynski, M., Ziolkowska, B., Przewlocka, B., Przewlocki, R., Maldonado, R., 2011. New operant model of reinstatement of food-seeking behavior in mice. Psychopharmacology 215, 49–70. https://doi.org/10.1007/s00213-010-2110-6. Randolph, T.G., 1956. The descriptive features of food addiction; addictive eating and drinking. Q. J. Stud. Alcohol 17, 198–224. p g Martín-García, E., Domingo-Rodriguez, L., Maldonado, R., 2020. An operant conditioning model combined with a Chemogenetic approach to study the neurobiology of food addiction in mice. Bio-protocol 10. https://doi.org/10.21769/ BIOPROTOC.3777. Romero-Zerbo, S.Y., Garcia-Gutierrez, M.S., Su´arez, J., Rivera, P., Ruz-Maldonado, I., Vida, M., Rodriguez de Fonseca, F., Manzanares, J., Bermúdez-Silva, F.J., 2012. Overexpression of cannabinoid CB2 receptor in the brain induces hyperglycaemia and a lean phenotype in adult mice. J. Neuroendocrinol. 24, 1106–1119. https://doi. org/10.1111/j.1365-2826.2012.02325.x. i Miller, E.K., Cohen, J.D., 2003. An integrative theory of prefrontal cortex function, 24, 167–202. https://doi.org/10.1146/ANNUREV.NEURO.24.1.167. g j Verty, A.N.A., Stefanidis, A., McAinch, A.J., Hryciw, D.H., Oldfield, B., 2015. Anti- obesity effect of the CB2 receptor agonist JWH-015 in diet-induced obese mice. PLoS One 10, e0140592. https://doi.org/10.1371/journal.pone.0140592. Navarrete, F., P´erez-Ortiz, J.M., Manzanares, J., 2012. Cannabinoid CB2 receptor- mediated regulation of impulsive-like behaviour in DBA/2 mice. Br. J. Pharmacol. 165, 260–273. Navarrete, F., Rodríguez-Arias, M., Martín-García, E., Navarro, D., García-Guti´errez, M. S., Aguilar, M.A., Aracil-Fern´andez, A., Berbel, P., Mi˜narro, J., Maldonado, R., Manzanares, J., 2013. Role of CB2 cannabinoid receptors in the rewarding, reinforcing, and physical effects of nicotine. Neuropsychopharmacol. 3812 (38), 2515–2524. https://doi.org/10.1038/npp.2013.157. Volkow, N.D., Wang, G.J., Fowler, J.S., Tomasi, D., Telang, F., 2011. Addiction: beyond dopamine reward circuitry. Proc. Natl. Acad. Sci. U. S. A. 108, 15037–15042. https://doi.org/10.1073/PNAS.1010654108. Xi, Z.X., Peng, X.Q., Li, X., Song, R., Zhang, H.Y., Liu, Q.R., Yang, H.J., Bi, G.H., Li, J., Gardner, E.L., 2011. Appendix A. Supplementary data Brain cannabinoid CB2 receptors modulate cocaine’s actions in mice. Nat. Neurosci. 149 (14), 1160–1166. https://doi.org/10.1038/nn.2874. Navarrete, F., García-Guti´errez, M.S., Manzanares, J., 2018. Pharmacological regulation of cannabinoid CB2 receptor modulates the reinforcing and motivational actions of ethanol. Biochem. Pharmacol. 157, 227–234. https://doi.org/10.1016/J. BCP.2018.07.041. Zhang, H.Y., Gao, M., Liu, Q.R., Bi, G.H., Li, X., Yang, H.J., Gardner, E.L., Wu, J., Xi, Z.X., 2014. Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice. Proc. Natl. Acad. Sci. U. S. A. 111, E5007–E5015. https://doi.org/10.1073/pnas.1413210111. Navarro, D., Gasparyan, A., Navarrete, F., Torregrosa, A.B., Rubio, G., Marín-Mayor, M., Acosta, G.B., Garcia-Guti´errez, M.S., Manzanares, J., 2022. Molecular alterations of 14
https://openalex.org/W4207012990	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0262736&type=printable	English	null	Two-phase learning-based 3D deblurring method for digital breast tomosynthesis images	PloS one	2,022	cc-by	10,866	OPEN ACCESS Citation: Choi Y, Han M, Jang H, Shim H, Baek J (2022) Two-phase learning-based 3D deblurring method for digital breast tomosynthesis images. PLoS ONE 17(1): e0262736. https://doi.org/ 10.1371/journal.pone.0262736 Editor: Chi-Hua Chen, Fuzhou University, CHINA Received: June 18, 2021 Accepted: January 4, 2022 Published: January 24, 2022 Copyright: © 2022 Choi 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: Chi-Hua Chen, Fuzhou University, CHINA Received: June 18, 2021 Accepted: January 4, 2022 Published: January 24, 2022 Copyright: © 2022 Choi 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 data (Reference, DBT, deblurred volumes) used in the experiments can be downloaded from the link below: https://doi. org/10.6084/m9.figshare.18095639.v1. Funding: This research was supported by the Bio and Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science and ICT (NRF2019R1A2C2084936 and 2020R1A4A1016619) and the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Two-phase learning-based 3D deblurring method for digital breast tomosynthesis images Yunsu ChoiID, Minah HanID, Hanjoo JangID, Hyunjung Shim, Jongduk BaekID School of Integrated Technology and Yonsei Institute of Convergence Technology, Yonsei University, Incheon, South Korea Yunsu ChoiID, Minah HanID, Hanjoo JangID, Hyunjung Shim, Jongduk BaekID School of Integrated Technology and Yonsei Institute of Convergence Technology, Yonsei University, Incheon, South Korea * kateshim@yonsei.ac.kr (HS); jongdukbaek@yonsei.ac.kr (JB) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract In digital breast tomosynthesis (DBT) systems, projection data are acquired from a limited number of angles. Consequently, the reconstructed images contain severe blurring artifacts that might heavily degrade the DBT image quality and cause difficulties in detecting lesions. In this study, we propose a two-phase learning approach for artifact compensation in a coarse-to-fine manner to mitigate blurring artifacts effectively along all viewing directions of the DBT image volume (i.e., along the axial, coronal, and sagittal planes) to improve the detection performance of lesions. The proposed method employs a convolutional neural network model comprising two submodels/phases, with Phase 1 performing three-dimen- sional (3D) deblurring and Phase 2 performing additional 2D deblurring. To investigate the effects of loss functions on the proposed model’s deblurring performance, we evaluated several loss functions, such as the pixel-based loss function, adversarial-based loss func- tion, and perception-based loss function. Compared with the DBT image, the mean squared error of the image and the root mean squared errors of the gradient of the image decreased by 82.8% and 44.9%, respectively, and the contrast-to-noise ratio increased by 183.4% in the in-focus plane. We verified that the proposed method sequentially restored the missing frequency components as the DBT images were processed through the Phase 1 and Phase 2 steps. These results indicate that the proposed method performs effective 3D deblurring, significantly reducing the blurring artifacts in the in-focus plane and other planes of the DBT image, thus improving the detection performance of lesions. PLOS ONE RESEARCH ARTICLE PLOS ONE PLOS ONE PLOS ONE PLOS ONE Two-phase learning-based 3D deblurring Ministry of Health Welfare, Republic of Korea, the Ministry of Food and Drug Safety) (202011A03). Ministry of Health Welfare, Republic of Korea, the Ministry of Food and Drug Safety) (202011A03). As the DBT system obtains patient data scanned from a limited range of angles (e.g., 30˚ to 60˚ [9]), severe blurring artifacts occur when conventional filtered backprojection methods are used for image reconstruction (e.g., the Feldkamp–Davis–Kress (FDK) [10] algorithm). Although analysis-based methods, such as the gradient-projection Barzilai-Borwein algorithm (GP-BB) [11], have been developed to improve the image quality of DBT systems, they still have limitations in terms of reducing the blurring artifacts, especially in breast tissue images, as illustrated in Fig 1. Competing interests: The authors have declared that no competing interests exist. In recent studies, convolutional neural networks (CNNs) for mitigating blurring artifacts caused by camera motion [12] have been proposed. The camera motion is formulated as con- volutions between the reference images and motion kernels. Theoretically, considering that the FDK algorithm is a linear system, the DBT image reconstructed by FDK algorithm can be expressed as a convolution between the reference image and the point spread function (PSF) [13], similar to camera motion deblurring as follows: rðx; y; zÞ ¼ iðx; y; zÞ pðx; y; zÞ þ nðx; y; zÞ ð1Þ ð1Þ rðx; y; zÞ ¼ iðx; y; zÞ pðx; y; zÞ þ nðx; y; zÞ where i(x, y, z) is an ideal breast image, p(x, y, z) is the 3D PSF of DBT system, r(x, y, z) is the reconstructed DBT image, and n(x, y, z) is the reconstructed noise. The conventional deconvo- lution method such as Richardson-Lucy (RL) [14], which requires a manual control of the parameters in RL deconvolution, is not suitable for deblurring DBT image because accurate estimation of the PSF is difficult [13]. However, due to the robust characteristics of CNN and its wide receptive field, a more accurate deblurring kernel with spatially varying properties can be estimated. In our previous work [15], we proposed a method to deblur DBT images using a deep residual-block-based CNN (DRCNN), where the cone-beam computed tomography (CBCT) images reconstructed by the FDK algorithm were used as target images. As the CBCT data were acquired over a 360˚ range, the reconstructed image did not contain blurring arti- facts caused by insufficient view sampling. Introduction Digital breast tomosynthesis (DBT) imaging systems widely used for chest, wrist, head, neck, dental and breast for medical diagnostics [1–5]. Recent developments in high-quality digital receptors have allowed DBT systems to be used in detecting breast cancer [5, 6]. Unlike mam- mograms, DBT systems use multiple projection data from different viewing angles, resulting in a significant improvement in detection accuracies in reconstructed DBT images [7, 8]. 2020R1A4A1016619) and the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Our previously proposed CNN learned the local and global properties of the blurring artifacts; thus, it reduced these blurring artifacts effec- tively in the in-focus plane using the two-dimensional (2D) in-focus slice data for training the model. However, the blurring artifacts could not be effectively reduced in the images along the coronal and sagittal planes because the DBT system captures less sufficient data along the coro- nal and sagittal planes. To solve the afore-mentioned problem, a new method for 3D deblur- ring the DBT volume is required. In this study, we propose a two-phase learning-based 3D deblurring technique to reduce the blurring artifacts along all imaging planes of DBT images from anatomical backgrounds. Fig 1. Limitation of the conventional DBT reconstruction algorithm. (a) CBCT images, (b) DBT images reconstructed using FDK, and (c) DBT images reconstructed using GP-BB. The display window is [0.0456 0.0844] cm−1. https://doi.org/10.1371/journal.pone.0262736.g001 Fig 1. Limitation of the conventional DBT reconstruction algorithm. (a) CBCT images, (b) DBT images reconstructed using FDK, and (c) DBT images reconstructed using GP-BB. The display window is [0.0456 0.0844] cm−1. https://doi.org/10.1371/journal.pone.0262736.g001 2 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Two-phase learning-based 3D deblurring As the DBT system produces much blurrier images along the coronal plane, we designed our network to reflect this spatially varying property of DBT images for effective deblurring. Our proposed two-phase learning method involves two different network models with a sequential training scheme. In Phase 1, we perform an initial 3D deblurring on the 3D DBT volume, where the entire volume is restored at a coarse scale. In Phase 2, we increase the sharpness of the restored 3D volume obtained from Phase 1 by applying U-Net [16] along the coronal plane, where the blurring artifacts are observed to be most severe. To investigate the effects of loss functions on our model’s deblurring performance, we eval- uate various loss functions (i.e., pixel-based loss, adversarial-based loss [17], and perception- based loss [18]). Pixel-based evaluations are conducted using the mean squared error of the image (MSE) and the root mean squared errors of the gradient of the image (GRMSE) [19] between the CBCT and deblurred images. Contrast enhancement of the lesions is also evalu- ated using the contrast-to-noise ratio (CNR). The effectiveness of the proposed deblurring method is analyzed by comparing the restored frequency components between the CBCT and deblurred images. Data preparation In Phase 1 training, generated 100 CBCT and DBT volume pairs using the characteristics of clinical mammograms [20, 21] were divided with a ratio of 1:1:3 for training, validation, and testing set, respectively. The testing set used in Phase 1 was divided with a ratio of 1:1:1 for training, validation, and testing set, respectively, further being used to train Phase 2 CNN. Breast volumes were simulated using a randomly generated inverse power law noise model [22, 23]. A Gaussian noise volume of voxel size 899 × 899 × 899 pixels was generated and trans- formed into the frequency domain using the discrete Fourier transform (DFT). The trans- formed volume was multiplied with a filtering kernel (i.e., 1/f3/2, where f is the radial frequency in per millimeter) and transformed into the spatial domain via the inverse DFT [20] to obtain the actual breast statistics. Note that the zero frequency value of the filter was designated as twice the first non-zero frequency component to prevent an infinite value at zero frequency [24]. To avoid the wrap-around effect caused by DFT, a central spherical volume with a diame- ter of 450 voxels was extracted. Next, to implement a 30% volumetric glandular fraction (VGF), the voxel values were sorted in descending order. The upper (lower) 30% (70%) were assigned 0.0802 cm−1(0.0456 cm−1), corresponding to the attenuation coefficient of the glandu- lar (adipose) tissue at an energy of 20 keV [25]. A rectangular volume with a short z-axis direc- tion (i.e., 288 × 288 × 144) was extracted, reflecting a compressed breast volume. We generated projection data from the rectangular volume using Siddon’s algorithm [26]. The DBT image was reconstructed using 41 projection data (−20˚ to 20˚), and the CBCT image was reconstructed using 360 projection data (−180˚ to 180˚) based on the FDK algorithm with a Hanning-weighted ramp filter. We did not use a slice thickness (ST) filter [27] to maintain the high-frequency components [21] of the breast volume. Fig 2 illustrates the data acquisition geometry of the DBT system, and Table 1 summarizes the details of the simulation parameters. For noise simulation, quantum noise with Poisson statistics 2 × 105 incident photons per detector cell, which is equivalent to the dose level of 1.6 mGy for a 4 cm breast with 20 KeV energy, was added to the projection data. PLOS ONE Experiments with 3D breast volume datasets demonstrate that our proposed network achieves excellent deblurring compared to the network described in our previous study [15]. Data preparation The dose level is similar to the exposure level measured in the work of Zeng et al [28]. The total flux was matched for the DBT and CBCT data acquisition systems. Breast tissue near the volume center 3 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Two-phase learning-based 3D deblurring Fig 2. Data acquisition geometry of the DBT system. https://doi.org/10.1371/journal.pone.0262736.g002 Fig 2. Data acquisition geometry of the DBT system. https://doi.org/10.1371/journal.pone.0262736.g002 was replaced by a 2 mm or 4 mm diameter spherical lesion in 40 test volumes to examine the generalization performance of the trained CNN. The attenuation coefficient of the lesion was 0.0844 cm−1, corresponding to 20 keV [25] energy. To evaluate the generalized performance for different background structures, we applied the trained CNN model to deblur 15% VGF DBT images, as the 15% VGF represents the median value of women’s VGF statistics [29]. Two-phase CNN architecture Our proposed method was motivated by the model-stacking approach. Although the training time is relatively longer than that required for a single network training, previous studies [30, 31] have shown that model-stacking demonstrates better performance in terms of accuracy in medical image segmentation and classification. We focused on the fact that our target dataset is a 3D DBT volume dataset, which has similar visual patterns across the training dataset as PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 4 / 19 PLOS ONE Two-phase learning-based 3D deblurring Table 1. Simulation parameters. Parameters CBCT DBT Source to iso-center distance 545 mm Detector to iso-center distance 105 mm Data acquisition angle −180˚ * 180˚ −20˚ * 20˚ Number of views 360 41 Detector cell size 0.125 × 0.125mm2 Detector array size 450 × 450 Reconstructed volume size 47.2 × 47.2 × 47.2mm3 Reconstructed voxel size 0.105 × 0.105 × 0.105mm3 VGF 30% (Training and testset) 15% (Generalization testset) Number of incident X-ray photons 2 × 105/Number of views Reconstruction algorithm FDK https://doi.org/10.1371/journal.pone.0262736.t001 https://doi.org/10.1371/journal.pone.0262736.t001 other fine-grained datasets. Inspired by the success of the model-stacking approach in predic- tion tasks on fine-grained datasets, we employed it in our artifact compensation procedure on the 3D DBT volume. Various studies [32, 33] have confirmed the advantages of model-stack- ing for more accurate prediction and reliable estimation than the single network model for the same number of filters. Owing to these benefits, we adopted a two-phase learning-based approach. The proposed CNN architecture is presented in Fig 3. The depth of the CNN model must be increased to increase the modeling power of Phase 1. In this case, however, the training became more difficult because of the gradient vanishing problem [34, 35]. Therefore, the residual network was adopted to increase the model capacity to mitigate the gradient vanishing problem [36, 37]. Phase 1 has several residual network building blocks [38]. Each residual block comprises of two different steps. First, an input passes through the convolutional layer, rectified linear unit (ReLU) layer, and an additional convolu- tional layer. Second, the input and the output of the first step are added. Phase 2 was designed to render the output of Phase 1 more accurately by learning the tex- ture of the breast tissue and reducing any remaining blurring artifacts in the coronal plane. Two-phase CNN architecture As the PSF in the coronal plane is particularly wide, the most severe blurring artifacts are pro- duced here compared to other image planes. The U-Net was adopted to reflect this during the deblurring procedure because it is known to have a wide receptive field. As indicated in Phase 2 of Fig 3, the number of filters is doubled when the feature map passes through the max-pool- ing layer, whereas the number of filters is halved when the feature map passes through the upsampling layer. Loss function In Phase 1, we used the mean absolute error (MAE) as a loss function for the relatively good sharpness of the output image [39]. The MAE loss function is defined as follows: LMAE ¼ 1 w h k G1ðzÞ x k1; ð2Þ ð2Þ where, G1 is the Phase 1 CNN, x is the CBCT image, z is the input DBT image reconstructed using the FDK algorithm, and w and h are the width and height of the input DBT image, respectively. Algorithm 1 Optimization procedure of PL-MAE. Algorithm 1 Optimization procedure of PL-MAE. 5 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Two-phase learning-based 3D deblurring Fig 3. Architecture of the proposed CNN. The proposed CNN is composed of two phase. Phase 1 is composed of several residual blocks. The 2D slice of the coronal plane of the Phase 1 output volume is the input of Phase 2. Phase 2 has the structure of U-Net and it yields the final output of the proposed CNN. Fig 3. Architecture of the proposed CNN. The proposed CNN is composed of two phase. Phase 1 is composed of several residual blocks. The 2D slice of the coronal plane of the Phase 1 output volume is the input of Phase 2. Phase 2 has the structure of U-Net and it yields the final output of the proposed CNN. https://doi.org/10.1371/journal.pone.0262736.g003 https://doi.org/10.1371/journal.pone.0262736.g003 https://doi.org/10.1371/journal.pone.0262736.g003 Require: Set hyperparameters, α = 5 × 10−3, β1 = 0.9, β2 = 0.999, λ2 = 0.05, the number of total epochs, Nepoch = 100, the batch size n = 2, and patch size of 48 × 48 × 48. Require: Set hyperparameters, α = 5 × 10−3, β1 = 0.9, β2 = 0.999, λ2 = 0.05, the number of total epochs, Nepoch = 100, the batch size n = 2, and patch size of 48 × 48 × 48. p Require: Initial G1 (i.e., Phase 1) parameters φ0, initial G2 (i.e., Phase 2) parameters θ0 p Require: Initial G1 (i.e., Phase 1) parameters φ0, initial G2 (i.e., Phase 2) parameters θ0 Phase 2) parameters θ0 p 0 Require: Load pretrained VGG-16 network parameters 1: for epoch = 0, . . ., Nepoch do 2: Sample a batch of DBT image patches fzðiÞg n i¼1 and corresponding CBCT patches fxðiÞg n i¼1 patches fxðiÞg n i¼1 3: for i = 1, . . Loss function ., n do (i) (i) 3: for i = 1, . . ., n do (i) (i) 4: L(i)(G1) LMAE(z(i), x(i)) 4: L(i)(G1) LMAE(z(i), x(i)) 6: Update G1; Adam 5 1 n Pi¼1 n LðiÞðG1Þ; ; a; b1; b2 7: end for 8: Aggregate G1(z;φ) in the form of 3D volume ^z, aggregate G1(x;φ) in the form of 3D volume ^x 8: Aggregate G1(z;φ) in the form of 3D volume z, aggregate G1(x;φ) in the form of 3D volume ^x ^ ~ ^ ~ 9: Divide ^z into coronal slices ~z, divide ^x into coronal slices ~x 10: for epoch = 0, . . ., Nepoch do 10: for epoch = 0, . . ., Nepoch do 11: Sample a batch of f~zðiÞg n i¼1 and f~xðiÞg n i¼1 After deblurring the DBT images in Phase 1, further deblurring was performed in Phase 2. To investigate the effect of the loss function on breast tissue restoration, we used the pixel- based loss function (i.e., MAE), adversarial loss function with MAE (AL-MAE), and percep- tion-based loss function with MAE (PL-MAE). For the adversarial loss function, we used the Wasserstein generative adversarial network with a gradient penalty (WGAN-GP) [17] and a discriminator of 144 × 144 PatchGAN [40]. The adversarial-based loss function is defined as PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 6 / 19 PLOS ONE Two-phase learning-based 3D deblurring follows: LAL ¼ DðG2ðzÞÞ DðxÞ þ Zðk r^xDð^xÞ k2 2 1Þ 2 ; ð3Þ ð3Þ where, D is a discriminator, G2 is the Phase 2 CNN, r denotes the gradient, ^x ¼ x þ ð1 Þz, and has the standard uniform distribution. The weighting parameter η was set to 0.1 following the recommendations in previous work [17]. ^x ¼ x þ ð1 Þz, and has the standard uniform distribution. The weighting parameter η was set to 0.1 following the recommendations in previous work [17]. We used the first 13 layers of the VGG-16 network [41] for the perception-based loss func- tion, which was pretrained on the ImageNet dataset [42]. The CBCT and deblurred images of the proposed CNN model were passed through the VGG-16 network, and the outputs were used for loss calculation. Training and test dataset In Phase 1 training, a total of 20 CBCT and DBT volume pairs (i.e., 288 × 288 × 144) was used. Each of the volume pairs was divided into 108 non-overlapping patches of size 48 × 48 × 48; a total of 2,160 patch pairs was used during the training. After passing the DBT image patches through the trained Phase 1, the output patches were aggregated in the form of breast volume and separated into the 288 coronal plane slices of size 288 × 144. These output slices of Phase 1 and the corresponding CBCT slices were used for Phase 2 training. Since we used 20 volumes during the Phase 2 training, a total of 5,760 slice pairs was used. Loss function The perception-based loss function is defined as follows: LPL ¼ 1 W H C k ðG2ðzÞÞ ðxÞ k2 2; ð4Þ ð4Þ where, W, H, and C are the width, height, and the number of channels in the feature space, and ϕ is the feature extractor. When using the adversarial-based and perception-based loss functions, MAE loss was used together to render a deblurred image that is more similar to the CBCT image. We determined the weighting values (i.e., λ1 and λ2) to minimize the loss function on the validation dataset in a search range of [0.0001 0.1]. The optimal values were 0.001 and 0.05 for λ1 and λ2 respec- tively. The objective functions of Phase 2 (i.e., MAE, AL-MAE, and PL-MAE loss functions) are defined as follows. LALMAE ¼ LMAE þ l1LAL; ð5Þ LPLMAE ¼ LMAE þ l2LPL; ð6Þ ð5Þ LALMAE ¼ LMAE þ l1LAL; ð5Þ LPLMAE ¼ LMAE þ l2LPL; ð6Þ ð6Þ The definition of the MAE loss function is the same as in (2), except that G1 is replaced by G2. The definition of the MAE loss function is the same as in (2), except that G1 is replaced by G2. Performance evaluation Pixel-based evaluation. The means of the 2D MSE and 2D GRMSE were used to evaluate the similarities between the CBCT and deblurred images. The mean of the 2D MSE is calcu- lated as follows: MSEmeanðy; ~yÞ ¼ 1 mn X m i¼1 X n j¼1 ðyij ~yijÞ 2; ð7Þ ð7Þ where yij is the jth pixel of the central slice image of ith CBCT volume, ~yij is the jth pixel of the central slice image of the ith deblurred volume, m is the number of images, and n is the number of pixels in the image. For the subjective visual assessment [19], we used the mean of the 2D GRMSE, defined as follows: GRMSEmeanðy; ~yÞ ¼ 1 m X m i¼1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Pn j¼1 ðOðyijÞ Oð~yijÞÞ 2 n s ; ð8Þ ð8Þ where we used the intermediate operator O as a gradient function. The mean of the 2D MSE and 2D GRMSE between the CBCT and deblurred images were compared in the axial, coronal, and sagittal planes. Lesion contrast. The CNR was calculated for the images with 4 mm lesions inserted to evaluate the contrast improvement of lesions against the background in the deblurred image. The CNR is strongly associated with the reader preference score for lesion contrast [44]. We extracted the central slice of the breast volume to calculate the CNR. In the extracted slice, the circular-shaped lesion was set as the foreground, and the outer part of the lesion was set as the background. The mean of CNR is calculated as follows: CNRmean ¼ 1 m X m i¼1 jufðyiÞ ubðyiÞj ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðs2 f ðyiÞ þ s2 bðyiÞÞ=2 q ; ð9Þ ð9Þ fififififififififififififififififififififififififififififififififififififi where yi is the central slice image of ith CBCT volume, ~yi is the central slice image of the ith deblurred volume, ub(uf) is the mean CT number outside (inside) the mass lesion, and σb(σf) is the standard deviation outside (inside) the mass lesion. Frequency domain analysis. To evaluate the ability of the CNN to fill in the missing data of the DBT image in the frequency domain, we examined the frequency responses for the axial, coronal, and sagittal planes. We extracted the central slice in each direction from 20 independently generated breast volumes. Then, the extracted images were 2D Fourier trans- formed and its absolute values were averaged. We displayed them on the log scale. Model and implementation details In Phase 1, the network was composed of the residual network building blocks, and all convo- lutional layers have 40 filters of 3 × 3 × 3 size with a stride 1. The number of filters was selected experimentally to achieve the best performance without sacrificing training efficiency. We attached these results in the supplementary material. We evaluated different network depths by adjusting the number of residual network building blocks to 6, 8, 10, 12, and 14 blocks. The network with 10 residual network building blocks is superior to the others, as depicted in Fig 4. Furthermore, Fig 5 demonstrates the capability of the 10-block CNN on the validation data- set. Thus, we selected 10 residual network building blocks for the network design of Phase 1. In Phase 2, to restore the fine texture of breast tissue, we used U-Net structures with 32 fil- ters of size 3 × 3 with a stride of 1, which have a 140 × 140 wide receptive field size covering 7 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Two-phase learning-based 3D deblurring Fig 4. The influence of different residual network building blocks in Phase 1. https://doi.org/10.1371/journal.pone.0262736.g004 Fig 4. The influence of different residual network building blocks in Phase 1. Fig 4. The influence of different residual network building blocks in Phase 1. https://doi.org/10.1371/journal.pone.0262736.g004 https://doi.org/10.1371/journal.pone.0262736.g004 the PSF of the coronal plane. The PSF of the coronal plane has an elongated shape spanning a 60-pixel length. In Phase 1, the network was trained using the adaptive moment estimation (Adam) opti- mizer [43] with a batch size of 2 due to the memory issue. We excluded the batch normaliza- tion layer, as our network is stably trained without the batch normalization layer. The training efficiency with and without batch normalization is compared in the supplementary material. Fig 5. The training and validation loss of the ten-block CNN (i.e., Phase 1) with each training epoch. https://doi.org/10.1371/journal.pone.0262736.g005 Fig 5. The training and validation loss of the ten-block CNN (i.e., Phase 1) with each training epoch. https://doi.org/10.1371/journal.pone.0262736.g005 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 8 / 19 PLOS ONE Two-phase learning-based 3D deblurring We trained the network using 100 epochs by setting the Adam optimizer’s exponential decay rates for the first and second moment (i.e., β1 and β2) estimates to 0.9 and 0.999, respectively, as recommended in the previous study [43]. Model and implementation details The learning rate (i.e., α) was 5 × 10−3, which was found experimentally in the range [0.0001 0.01]. In Phase 2, we used the same Adam optimizer and hyperparameters as in Phase 1 and observed that the CNN with all proposed loss functions converged stably within 100 epochs in each phase. Convergence required about 8 h each using the Keras library on a system with an Nvidia Titan XP (Pascal) 12 GB GPU and Intel (R) Core (TM) i7–6700 3.40 GHz processor. Results We compared the proposed two-phase learning-based scheme with the FDK algorithm, total- variation iterative reconstruction with GP-BB (TV-IR), and DRCNN [15]. In TV-IR method, we applied the algorithm by setting the iteration number to 100 and regularization parameter (i.e., λ) to 5 × 10−4. In DRCNN method, we trained the network using 100 epochs by setting the β1 and β2 estimates to 0.9 and 0.999, respectively. The learning rate was 1 × 10−3, which was found experimentally in the range [0.0001 0.01]. Fig 6 illustrates the DBT images recon- structed using the FDK algorithm and TV-IR, DRCNN, CNN-based deblurred images with different loss functions, and CBCT images. In Fig 6(a)–6(c), we observe that severe blurring artifacts in the DBT image are reduced in all planes using the proposed method. In particular, the coronal and sagittal planes of the DBT image contain very severe blurring artifacts due to the limited range of data acquisition angles, and it is difficult to recognize the original struc- tures compared to the axial plane. We also observed that GP-BB has a low lesion contrast com- pared with the FDK algorithm, although it could enhance the edge. In the image deblurred using DRCNN, the deblurring is not properly performed in the coronal and sagittal planes. However, the proposed deblurring method recovers the original structures of these planes reli- ably with notably improved image quality. We also observed that different loss functions produce different textures in the deblurred images. Compared with the CBCT images, the deblurred images with MAE loss functions introduce slight blurs, reflected as reduced image noise. In addition, we observed that the pro- posed method using AL-MAE loss overestimated the original structures and amplified the noise. Using the proposed method with WGAN-GP loss to restore extensive missing data in DBT images would not be appropriate due to the amplification of high-frequency components. However, the deblurred images with PL-MAE loss functions exhibit textures more similar to the CBCT images due to their ability to preserve feature information via the perception-based loss functions. Overall, the image sharpness is preserved well in the deblurred images with MAE and PL-MAE loss functions. Table 2 summarizes the means of the 2D MSE and 2D GRMSE between the CBCT and deblurred images for different loss functions. A smaller value indicates better performance in the MSE and GRMSE. Performance evaluation The MSE values between the 2D FFTs of the CBCT and deblurred images were compared. The central PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 9 / 19 PLOS ONE Two-phase learning-based 3D deblurring vertical profiles of each 2D frequency response were also compared, as this area contains the most missing data in the DBT images. vertical profiles of each 2D frequency response were also compared, as this area contains the most missing data in the DBT images. PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 Results In the DBT image, we only used the axial plane for pixel-based evalua- tions because the other planes do not contain any useful information due to the severe blur- ring. The quantitative results confirm our observation in Fig 6, implying that the proposed method achieves excellent deblurring performance comparable to or better than that of the DRCNN. Compared with other loss functions, the deblurred image by MAE provides slightly better scores in terms of the MSE. This result may be attributable to the generated anatomical background image being relatively piecewise linear, thus rendering the MAE loss function more appropriate for these metrics. As GRMSE reflects perceptual characteristics, the deblurred image using PL-MAE provides better results in the GRMSE evaluation. Fig 6(d)–6(i) displays the DBT images reconstructed by FDK algorithm and TV-IR, deblurred images, and CBCT images with the presence of 4 mm and 2 mm diameter lesions. Three lesions were included along the x-direction to examine how well the proposed method can recover spatially varying blurring artifacts in DBT images. It is challenging to identify the lesions in the coronal and sagittal planes in the DBT image due to the severe blurring artifacts. In the image deblurred by the DRCNN, the shapes of lesions are distorted. However, the pro- posed method restores the original lesion shapes more effectively. In particular, the lesion PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 10 / 19 PLOS ONE Two-phase learning-based 3D deblurring Fig 6. Results using 30% VGF DBT images. DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE loss functions, and CBCT images (from left to right). Images without mass lesion for (a) axial, (b) coronal, and (c) sagittal planes; images containing 4 mm lesions for (d) axial, (e) coronal, and (f) sagittal planes, and images containing 2 mm lesions for (g) axial, (h) coronal, and (i) sagittal planes. The display window is [0.0456 0.0844] in cm−1. https://doi.org/10.1371/journal.pone.0262736.g006 Fig 6. Results using 30% VGF DBT images. DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE loss functions, and CBCT images (from left to right). https://doi.org/10.1371/journal.pone.0262736.t002 Results Images without mass lesion for (a) axial, (b) coronal, and (c) sagittal planes; images containing 4 mm lesions for (d) axial, (e) coronal, and (f) sagittal planes, and images containing 2 mm lesions for (g) axial, (h) coronal, and (i) sagittal planes. The display window is [0.0456 0.0844] in cm−1. https://doi.org/10.1371/journal.pone.0262736.g006 https://doi.org/10.1371/journal.pone.0262736.g006 detectability in the coronal and sagittal planes is superior to the FDK algorithm, TV-IR, and DRCNN. Despite the powerful deblurring performance, we observed that the boundaries of 4 mm and 2 mm lesions are not recovered well in the coronal and sagittal planes compared to the axial plane. It appears that the proposed CNN experiences difficulties in filling extensive missing data in the coronal and sagittal planes compared to the axial plane. Table 3 summarizes the CNR of each plane for the 4 mm lesions. In the 4 mm lesions, the deblurred axial plane image achieves significantly improved CNR performance, which is 2.84 times higher than that of the original DBT image reconstructed using the FDK algorithm. Even coronal and sagittal plane images exhibit a much higher CNR than DRCNN images. Table 2. Pixel-based evaluation with 30% VGF DBT images. MSE and GRMSE results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE. (mean±standard deviation). MSE (×10−5) GRMSE (×10−2) Method Axial Coronal Sagittal Axial Coronal Sagittal DRCNN 9.67 ± 1.01 10.51 ± 2.20 10.21 ± 2.06 0.92 ± 0.03 0.87 ± 0.07 0.77 ± 0.06 MAE 7.09 ± 0.78 8.28 ± 1.32 7.56 ± 1.20 0.59 ± 0.01 0.59 ± 0.01 0.64 ± 0.01 AL-MAE 8.72 ± 3.40 8.51 ± 1.82 11.48 ± 0.12 0.65 ± 0.15 0.56 ± 0.01 0.73 ± 0.20 PL-MAE 8.90 ± 1.30 10.02 ± 2.15 9.62 ± 2.06 0.59 ± 0.01 0.55 ± 0.01 0.64 ± 0.02 FDK 41.09 ± 5.56 1.07 ± 0.06 TV-IR 11.04 ± 2.39 0.61 ± 0.01 DBT images. MSE and GRMSE results of the DBT images reconstructed with FDK and TV-IR, deblurred images by od with MAE, AL-MAE, and PL-MAE. (mean±standard deviation). Table 2. Pixel-based evaluation with 30% VGF DBT images. MSE and GRMSE results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE. (mean±standard deviation). el-based evaluation with 30% VGF DBT images. PLOS ONE Contrast-to-Noise Ratio Method Axial Coronal Sagittal DRCNN 2.25 ± 0.54 2.01 ± 0.57 1.98 ± 0.65 MAE 2.91 ± 0.79 2.42 ± 0.76 2.67 ± 0.73 AL-MAE 2.17 ± 0.81 2.06 ± 0.66 2.04 ± 0.82 PL-MAE 3.24 ± 0.75 2.98 ±1.05 3.19 ± 1.00 FDK 1.14 ± 0.57 TV-IR 0.65 ± 0.42 https://doi.org/10.1371/journal.pone.0262736.t003 Fig 7. Frequency domain analysis. Frequency responses of DBT images using FDK, deblurred images, and CBCT images for fx-fy plane (Top), fx-fz plane (middle), and fy-fz plane (bottom). (a) DBT images with FDK reconstruction, (b) deblurred images after Phase 1 and deblurred images after Phase 2 with (c) MAE, (d) PL-MAE, and (e) CBCT images The display window is [1 4] The red arrows indicate the missing data regions in the DBT images Table 3. Lesion contrast evaluation with 30% VGF DBT images. CNR results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE. (mean±standard deviation). Contrast-to-Noise Ratio Method Axial Coronal Sagittal DRCNN 2.25 ± 0.54 2.01 ± 0.57 1.98 ± 0.65 MAE 2.91 ± 0.79 2.42 ± 0.76 2.67 ± 0.73 AL-MAE 2.17 ± 0.81 2.06 ± 0.66 2.04 ± 0.82 PL-MAE 3.24 ± 0.75 2.98 ±1.05 3.19 ± 1.00 FDK 1.14 ± 0.57 TV-IR 0.65 ± 0.42 https://doi org/10 1371/journal pone 0262736 t003 While all loss functions provide similar improvements in the CNR, the PL-MAE achieves a rel- atively higher CNR over all planes than other loss functions because the PL-MAE compro- mises the sharpness and textures of the original image more effectively for this task. Through the CNR results, the CNR of the axial plane has a relatively high value compared with the other planes. As the missing data in the axial plane is smaller than in the other planes, the lesion con- trast improvement of the proposed method seems better. The 2D frequency responses of the deblurred images from Phases 1 and 2 were calculated, as listed in Fig 7, to analyze the restoring power of the proposed method in each phase. We selected the MAE (PL-MAE) loss for this comparison because it yielded the highest perfor- mance in the pixel-based evaluation (lesion contrast) with the CBCT images. As expected, the DBT image contains many missing data points due to the limited data acquisition angle, as depicted in Fig 7(a). PLOS ONE PLOS ONE Two-phase learning-based 3D deblurring While all loss functions provide similar improvements in the CNR, the PL-MAE achieves a rel- atively higher CNR over all planes than other loss functions because the PL-MAE compro- mises the sharpness and textures of the original image more effectively for this task. Through the CNR results, the CNR of the axial plane has a relatively high value compared with the other planes. As the missing data in the axial plane is smaller than in the other planes, the lesion con- trast improvement of the proposed method seems better. The 2D frequency responses of the deblurred images from Phases 1 and 2 were calculated, as listed in Fig 7, to analyze the restoring power of the proposed method in each phase. We selected the MAE (PL-MAE) loss for this comparison because it yielded the highest perfor- mance in the pixel-based evaluation (lesion contrast) with the CBCT images. As expected, the DBT image contains many missing data points due to the limited data acquisition angle, as depicted in Fig 7(a). However, most of the missing data are appropriately filled in by the pro- posed method. Note that the high-frequency components are observed in the DBT image because the ST filter is not applied. When we used the ST filter, the high-frequency compo- nents are reduced, which is common in many breast tomosynthesis imaging cases. The DBT images with a ST filter are included in the supplementary material. Table 4 summarizes the MSE between the 2D FFTs of the CBCT and deblurred images. The PL-MAE achieves a rela- tively low MSE value in all planes, demonstrating the effectiveness of using the perception- based loss function to fill in the missing data in the frequency domain. Fig 8 compares the cen- tral vertical profiles in Fig 7 for the CBCT, DBT, and deblurred images with MAE and Table 3. Lesion contrast evaluation with 30% VGF DBT images. CNR results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE. (mean±standard deviation). Results MSE and GRMSE results of the DBT images reconstructed with FDK and T blurred images by the proposed method with MAE, AL-MAE, and PL-MAE. (mean±standard deviation). PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 11 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE However, most of the missing data are appropriately filled in by the pro- posed method. Note that the high-frequency components are observed in the DBT image because the ST filter is not applied. When we used the ST filter, the high-frequency compo- nents are reduced, which is common in many breast tomosynthesis imaging cases. The DBT images with a ST filter are included in the supplementary material. Table 4 summarizes the MSE between the 2D FFTs of the CBCT and deblurred images. The PL-MAE achieves a rela- tively low MSE value in all planes, demonstrating the effectiveness of using the perception- based loss function to fill in the missing data in the frequency domain. Fig 8 compares the cen- tral vertical profiles in Fig 7 for the CBCT, DBT, and deblurred images with MAE and Fig 7. Frequency domain analysis. Frequency responses of DBT images using FDK, deblurred images, and CBCT images for fx-fy plane (Top), fx-fz plane (middle), and fy-fz plane (bottom). (a) DBT images with FDK reconstruction, (b) deblurred images after Phase 1 and deblurred images after Phase 2 with (c) MAE, (d) PL-MAE, and (e) CBCT images. The display window is [1 4]. The red arrows indicate the missing data regions in the DBT images. https://doi.org/10.1371/journal.pone.0262736.g007 Fig 7. Frequency domain analysis. Frequency responses of DBT images using FDK, deblurred images, and CBCT images for fx-fy plane (Top), fx-fz plane (middle), and fy-fz plane (bottom). (a) DBT images with FDK reconstruction, (b) deblurred images after Phase 1 and deblurred images after Phase 2 with (c) MAE, (d) PL-MAE, and (e) CBCT images. The display window is [1 4]. The red arrows indicate the missing data regions in the DBT images. https://doi.org/10.1371/journal.pone.0262736.g007 12 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Two-phase learning-based 3D deblurring Table 4. MSE between the 2D FFTs of the CBCT and deblurred images by the proposed method with MAE and PL-MAE. MSE in the frequency domain Method Axial Coronal Sagittal Phase 1 0.82 1.31 1.12 Phase 2 (MAE) 0.51 0.59 0.34 Phase 2 (PL-MAE) 0.42 0.57 0.29 FDK 12.21 11.91 6.19 https://doi.org/10.1371/journal.pone.0262736.t004 Table 4. MSE between the 2D FFTs of the CBCT and deblurred images by the proposed method with MAE and PL-MAE. between the 2D FFTs of the CBCT and deblurred images by the proposed method with MAE and https://doi.org/10.1371/journal.pone.0262736.t004 PL-MAE loss functions. PLOS ONE The proposed method sequentially restores the missing frequency components as the DBT image is processed through Phases 1 and 2. As we intended, Phase 1 performs the initial deblurring to fill in the missing data of the DBT image, as presented in Fig 7(b), but small differences in the CBCT image are still observed, as presented in Fig 8. The image sharpness is restored further by Phase 2, producing improved similarity between the CBCT and deblurred images, as indicated in Fig 8. The proposed method’s generalization performance is tested using 15% VGF data, and the corresponding deblurred images are illustrated in Fig 9. The results demonstrate that the pro- posed CNN is still effective for reducing blurring artifacts and exhibits robust characteristics, even for unseen data. The results of the quantitative evaluation are summarized in Tables 5 and 6. The proposed CNN using different loss functions demonstrated better results than DRCNN over all planes, even for unseen data through these results. In particular, the CNN using AL-MAE exhibits good performance for generalization tests represented by the MSE results. In contrast, the CNN using PL-MAE still produces the best score using the GRMSE. g p g Based on the different values of VGF, we compared the relative improvements from the MSE, GRMSE, and CNR based on the data from the axial plane of the DBT image recon- structed using the FDK algorithm. For the 15% (30%) VGF dataset, the MSE and GRMSE decreased by 81.0% (82.8%) and 34.1% (44.9%), respectively, compared with the DBT image, and the CNR increased by 191.2% (183.4%) compared with the DBT image. Discussion and conclusion In this study, we reduced the blurring artifacts in the DBT images using a two-phase learning- based CNN and evaluated the image quality using the MSE, GRMSE, and CNR. Although the simulated lesions included in the DBT image were slightly distorted, images deblurred by the proposed method achieved a higher CNR compared with the conventional method. We also demonstrated that the proposed method could reduce the blurring artifacts for unseen data, which was tested using data obtained based on different VGF values. Given the limited access to actual breast CT volumes, we validated the proposed method using 3D volume data generated using a computer simulation. Further validation of the pro- posed method using clinically available DBT image datasets could be an interesting future research topic. We generated the training data pair by generating the DBT and CBCT volume using a computer simulation in this work. In actual clinical situations, acquiring such paired data would not be feasible. In this case, the DBT image can be generated by conducting a for- ward projection of the CBCT volume by reflecting the data acquisition geometry of the DBT system. We specifically aimed to achieve digital tomosynthesis image deblurring in anatomical backgrounds, but the proposed two-phase CNN structure could also be applied to deblur other digital tomosynthesis images, such as chest images. The publicly available clinical CBCT chest PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 13 / 19 PLOS ONE Two-phase learning-based 3D deblurring Fig 8. Central vertical profiles of the frequency domain. Central vertical profiles of Fig 7 for (a) fx-fy, (b) fx-fz, and (c) fy-fz planes. Note that f is the pixel frequency in mm−1. https://doi.org/10.1371/journal.pone.0262736.g008 Fig 8. Central vertical profiles of the frequency domain. Central vertical profiles of Fig 7 for (a) fx-fy, (b) fx-fz, and (c) fy-fz planes. Note that f is the pixel frequency in mm−1. Fig 8. Central vertical profiles of the frequency domain. Central vertical profiles of Fig 7 for (a) fx-fy, (b) fx-fz, and (c) fy-fz planes. Note that f is the pixel frequency in mm−1. https://doi.org/10.1371/journal.pone.0262736.g008 https://doi.org/10.1371/journal.pone.0262736.g008 data provided by the NIH clinical center was used to verify that the proposed CNN is effective for other types of clinical data as well. We observed that the MSE of the axial plane with the proposed method was reduced by 60% compared with the digital tomosynthesis image. PLOS ONE Two-phase learning-based 3D deblurring Table 5. Pixel-based evaluation with 15% VGF DBT images. MSE and GRMSE results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE in generalization testset. (mean±standard deviation). MSE (×10−5) GRMSE (×10−2) Method Axial Coronal Sagittal Axial Coronal Sagittal DRCNN 6.33 ± 1.41 8.89 ± 2.50 7.60 ± 1.89 0.81 ± 0.04 0.69 ± 0.06 0.66 ± 0.04 MAE 4.62 ± 1.23 5.06 ± 1.23 4.93 ± 1.45 0.57 ± 0.01 0.56 ± 0.02 0.62 ± 0.02 AL-MAE 4.47 ± 1.32 4.63 ± 1.35 6.16 ± 4.91 0.60 ± 0.02 0.56 ± 0.06 0.69 ± 0.01 PL-MAE 5.10 ± 1.80 5.26 ± 1.89 5.63 ± 2.10 0.56 ± 0.02 0.51 ± 0.02 0.60 ± 0.02 FDK 23.47 ± 7.98 0.85 ± 0.10 TV-IR 7.10 ± 2.67 0.56 ± 0.02 https://doi.org/10.1371/journal.pone.0262736.t005 conditions in which deblurring may be difficult. To examine the generalization performance of the proposed algorithm, we generated a 30% VGF DBT volume acquired over the ranges of −40˚ to 40˚ and −10˚ to 10˚ for the same breast volume. These two volumes were deblurred using the CNN, pretrained with the DBT volume acquired over the range of −20˚ to 20˚ and the corresponding CBCT volume pair. The generalization performance is much better for a larger data acquisition angle (i.e., −40˚ to 40˚). Because the primary role of the proposed method is to fill in the missing data of DBT volume in frequency space (or equivalently, deblurring in image space), the generalization performance of the CNN for the DBT volumes acquired over a −10˚ to 10˚ data acquisition angle is worse because it contains much more missing data in frequency space. We attached these results in the supplementary material. conditions in which deblurring may be difficult. To examine the generalization performance of the proposed algorithm, we generated a 30% VGF DBT volume acquired over the ranges of −40˚ to 40˚ and −10˚ to 10˚ for the same breast volume. These two volumes were deblurred using the CNN, pretrained with the DBT volume acquired over the range of −20˚ to 20˚ and the corresponding CBCT volume pair. The generalization performance is much better for a larger data acquisition angle (i.e., −40˚ to 40˚). Table 6. Lesion contrast evaluation with 15% VGF DBT images. CNR results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE in generalization testset. (mean±standard deviation). PLOS ONE Because the primary role of the proposed method is to fill in the missing data of DBT volume in frequency space (or equivalently, deblurring in image space), the generalization performance of the CNN for the DBT volumes acquired over a −10˚ to 10˚ data acquisition angle is worse because it contains much more missing data in frequency space. We attached these results in the supplementary material. We adopted U-Net in phase 2, because it contains a large receptive field size to cover the length of PSF in the DBT system, which is a key aspect of the proposed method. When we used REDCNN [45] or ResNet [38] in phase 2, the performance of the deblurring was not effective compared to the case of U-Net. Detailed results are included in the supplementary material. For further validation of the proposed method, the PSF deblurring method based on itera- tive blind deconvolution (i.e., PSF deblur) [13] was compared with the proposed method (i.e., PL-MAE). The image deblurred by the PSF deblur method slightly increased the CNR of 4 mm lesions compared to the image reconstructed by FDK, similar to the result of the previous study [13]. However, the MSE and GRMSE between the PSF deblurred image and the refer- ence image were increased compared to the image reconstructed by the FDK due to the increased noise level. These results demonstrate that our proposed method showed a higher performance than the PSF deblur method. The following results are shown in the supplemen- tary material. Table 6. Lesion contrast evaluation with 15% VGF DBT images. CNR results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE in generalization testset. (mean±standard deviation). CNR Method Axial Coronal Sagittal DRCNN 3.22 ± 0.49 2.98 ± 0.61 3.22 ± 0.86 MAE 4.27 ± 0.85 3.64 ± 0.84 3.32 ± 0.77 AL-MAE 1.94 ± 1.12 2.97 ± 1.12 1.96 ± 1.08 PL-MAE 4.67 ± 0.92 4.25 ± 0.80 3.84 ± 0.83 FDK 1.61 ± 0.40 TV-IR 1.07 ± 0.40 https://doi.org/10.1371/journal.pone.0262736.t006 Table 6. Lesion contrast evaluation with 15% VGF DBT images. CNR results of the DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE in generalization testset. (mean±standard deviation). Discussion and conclusion We believe further improvements can be achieved using an optimized network structure and training strategy for the chest dataset, which is a topic for future research. We attached these results in the supplementary material. We used breast volumes with 30% VGF for training and testing the model. In the previous study [29], it was reported that 80% of women have a VGF lower than 27%, and 95% have a VGF below 45%. Although the VGF is a little higher, breast volumes with a 30% VGF were generated to verify that the proposed CNN could reduce blurring artifacts under harsh Fig 9. Results using 15% VGF DBT images. VGF 15% DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE loss functions, and CBCT images (from left to right). Images without mass lesion for (a) axial, (b) coronal, and (c) sagittal planes; images containing 4 mm lesions for (d) axial, (e) coronal, and (f) sagittal planes, and images containing 2 mm lesions for (g) axial, (h) coronal, and (i) sagittal planes. The display window is [0.0456 0.0844] in cm−1. https://doi org/10 1371/journal pone 0262736 g009 Fig 9. Results using 15% VGF DBT images. VGF 15% DBT images reconstructed with FDK and TV-IR, deblurred images by DRCNN, deblurred images by the proposed method with MAE, AL-MAE, and PL-MAE loss functions, and CBCT images (from left to right). Images without mass lesion for (a) axial, (b) coronal, and (c) sagittal planes; images containing 4 mm lesions for (d) axial, (e) coronal, and (f) sagittal planes, and images containing 2 mm lesions for (g) axial, (h) coronal, and (i) sagittal planes. The display window is [0.0456 0.0844] in cm−1. https://doi.org/10.1371/journal.pone.0262736.g009 14 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE PLOS ONE PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 1 15 / 19 PLOS ONE Two-phase learning-based 3D deblurring In this study, we trained the proposed CNN using the MAE, AL-MAE, and PL-MAE loss functions. All these loss functions exhibited verifiable image quality improvement compared to the DRCNN and the FDK algorithm reconstruction methods. In particular, the PL-MAE loss function exhibited the best deblurring performance in the results for the GRMSE, CNR, and frequency domain analysis. Because previous works [46–50] have reported that adver- sarial loss effectively recovers missing data, we also used the WGAN-GP as a loss function to determine the performance of the proposed method. However, the deblurring performance of WGAN-GP was worse than that of the MAE and PL-MAE. We conjecture that WGAN-GP is not effective for filling in extensive missing data, as is the case for the DBT system. We used ±20˚ data acquisition, which falls within the range of data acquisition angle (i.e., [±7.5˚ ±25˚]) of the commercialized DBT systems [51]. Depending on the imaging applica- tions, digital tomosynthesis systems use different data acquisition angles (e.g., ±25˚ for scaph- oid [52], ±45˚ for head and neck [3], and ±102.5˚ for dental [4]), producing fewer blurring artifacts compared to the current work. Extending the proposed method to different angle dig- ital tomosynthesis imaging systems and different background structures would be interesting for future research. In this study, the proposed deblurring method was tested only on FDK reconstructed DBT images. However, the proposed method can also be used for any practical reconstruction as long as the training data pair can be acquired; when DBT images are reconstructed with differ- ent apodization filters, the network can be separately trained for each apodization filters. Moreover, using transfer learning [53] could be an additional solution when only a limited amount of training dataset can be acquired. In conclusion, we proposed the two-phase learning-based 3D deblurring technique consid- ering the wide PSF of the DBT system. We quantitatively analyzed the deblurring results using quantitative evaluation (i.e., MSE, GRMSE, and CNR). The results reveal that the proposed method performs effective 3D deblurring and reduces the blurring artifacts effectively from the in-focus plane and other planes of the DBT image. Combining the proposed method with the DBT system would be extremely useful for computer-assisted diagnosis. Supporting information S1 File. Supplementary material includes additional implementation details and further clarification. (PDF) S1 File. Supplementary material includes additional implementation details and further clarification. PLOS ONE External valida- tion through experimental results will be performed in future work, as all datasets used in the experiment were generated using a computer simulation. References 1. Dobbins JT III, McAdams HP. Chest tomosynthesis: technical principles and clinical update. European journal of radiology. 2009; 72(2):244–51. https://doi.org/10.1016/j.ejrad.2009.05.054 2. Duryea J, Dobbins J III, Lynch J. Digital tomosynthesis of hand joints for arthritis assessment. Medical physics. 2003; 30(3):325–33. https://doi.org/10.1118/1.1543573 PMID: 12674232 3. Bachar G, Siewerdsen J, Daly M, Jaffray D, Irish J. Image quality and localization accuracy in C-arm tomosynthesis-guided head and neck surgery. Medical physics. 2007; 34(12):4664–77. https://doi.org/ 10.1118/1.2799492 PMID: 18196794 4. Ogawa K, Langlais R, McDavid W, Noujeim M, Seki K, Okano T, et al. Development of a new dental panoramic radiographic system based on a tomosynthesis method. Dentomaxillofacial Radiology. 2010; 39(1):47–53. https://doi.org/10.1259/dmfr/12999660 PMID: 20089744 5. Bonafede MM, Kalra VB, Miller JD, Fajardo LL. Value analysis of digital breast tomosynthesis for breast cancer screening in a commercially-insured US population. ClinicoEconomics and outcomes research: CEOR. 2015; 7:53. 6. Gao Y, Babb JS, Toth HK, Moy L, Heller SL. Digital breast tomosynthesis practice patterns following 2011 FDA approval: a survey of breast imaging radiologists. Academic radiology. 2017; 24(8):947–53. https://doi.org/10.1016/j.acra.2016.12.011 PMID: 28188043 7. Niklason LT, Christian BT, Niklason LE, Kopans DB, Castleberry DE, Opsahl-Ong B, et al. Digital tomo- synthesis in breast imaging. Radiology. 1997; 205(2):399–406. https://doi.org/10.1148/radiology.205.2. 9356620 PMID: 9356620 8. Gennaro G, Toledano A, Di Maggio C, Baldan E, Bezzon E, La Grassa M, et al. Digital breast tomo- synthesis versus digital mammography: a clinical performance study. European radiology. 2010; 20 (7):1545–53. https://doi.org/10.1007/s00330-009-1699-5 PMID: 20033175 9. Sechopoulos I. A review of breast tomosynthesis. Part I. The image acquisition process. Medical phys- ics. 2013; 40(1):014301. https://doi.org/10.1118/1.4770279 PMID: 23298126 10. Feldkamp LA, Davis LC, Kress JW. Practical cone-beam algorithm. Josa a. 1984; 1(6):612–9. https:// doi.org/10.1364/JOSAA.1.000612 11. Park JC, Song B, Kim JS, Park SH, Kim HK, Liu Z, et al. Fast compressed sensing-based CBCT recon- struction using Barzilai-Borwein formulation for application to on-line IGRT. Medical physics. 2012; 39 (3):1207–17. https://doi.org/10.1118/1.3679865 PMID: 22380351 12. Nah S, Hyun Kim T, Mu Lee K. Deep multi-scale convolutional neural network for dynamic scene deblurring. Proceedings of the IEEE conference on computer vision and pattern recognition; 2017. 13. Mota AM, Clarkson MJ, Almeida P, Matela N. An Enhanced Visualization of DBT Imaging Using Blind Deconvolution and Total Variation Minimization Regularization. IEEE Transactions on Medical Imaging. 2020; 39(12):4094–101. https://doi.org/10.1109/TMI.2020.3013107 PMID: 32746152 14. Fish D, Brinicombe A, Pike E, Walker J. Blind deconvolution by means of the Richardson–Lucy algo- rithm. JOSA A. 1995; 12(1):58–65. https://doi.org/10.1364/JOSAA.12.000058 15. Author Contributions Conceptualization: Yunsu Choi, Hanjoo Jang, Hyunjung Shim, Jongduk Baek. Data curation: Yunsu Choi. Formal analysis: Yunsu Choi, Hanjoo Jang, Jongduk Baek. Funding acquisition: Hyunjung Shim, Jongduk Baek. Investigation: Yunsu Choi, Minah Han, Jongduk Baek. Methodology: Yunsu Choi, Minah Han, Hanjoo Jang, Jongduk Baek. Project administration: Yunsu Choi, Minah Han, Hyunjung Shim, Jongduk Baek. Conceptualization: Yunsu Choi, Hanjoo Jang, Hyunjung Shim, Jongduk Baek. Formal analysis: Yunsu Choi, Hanjoo Jang, Jongduk Baek. Formal analysis: Yunsu Choi, Hanjoo Jang, Jongduk Baek. Funding acquisition: Hyunjung Shim, Jongduk Baek. Investigation: Yunsu Choi, Minah Han, Jongduk Baek. Methodology: Yunsu Choi, Minah Han, Hanjoo Jang, Jongduk Baek. Project administration: Yunsu Choi, Minah Han, Hyunjung Shim, Jongduk Baek. Project administration: Yunsu Choi, Minah Han, Hyunjung Shim, Jongduk Baek. 16 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Two-phase learning-based 3D deblurring Resources: Yunsu Choi. Software: Yunsu Choi. Supervision: Yunsu Choi, Minah Han, Jongduk Baek. Validation: Yunsu Choi, Minah Han, Hanjoo Jang. Visualization: Yunsu Choi, Minah Han, Hanjoo Jang. Resources: Yunsu Choi. Software: Yunsu Choi. Supervision: Yunsu Choi, Minah Han, Jongduk Baek. Validation: Yunsu Choi, Minah Han, Hanjoo Jang. Visualization: Yunsu Choi, Minah Han, Hanjoo Jang. Writing – original draft: Yunsu Choi. Writing – review & editing: Yunsu Choi, Minah Han, Hanjoo Jang, Hyunjung Shim, Jongduk Baek. Writing – original draft: Yunsu Choi. Writing – review & editing: Yunsu Choi, Minah Han, Hanjoo Jang, Hyunjung Shim, Jongduk Baek. Writing – review & editing: Yunsu Choi, Minah Han, Hanjoo Jang, Hyunjung Shim, Jongduk Baek. References Choi Y, Shim H, Baek J. Image Quality Enhancement of Digital Breast Tomosynthesis Images by Deblurring with Deep Residual Convolutional Neural Network. 2018 IEEE Nuclear Science Symposium and Medical Imaging Conference Proceedings (NSS/MIC); 2018: IEEE. 16. Ronneberger O, Fischer P, Brox T. U-net: Convolutional networks for biomedical image segmentation. International Conference on Medical image computing and computer-assisted intervention; 2015: Springer. PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 17 / 19 PLOS ONE Two-phase learning-based 3D deblurring 17. Gulrajani I, Ahmed F, Arjovsky M, Dumoulin V, Courville A. Improved training of wasserstein gans. arXiv preprint arXiv:170400028. 2017. 18. Gatys LA, Ecker AS, Bethge M. Image style transfer using convolutional neural networks. Proceedings of the IEEE conference on computer vision and pattern recognition; 2016. 19. Rose SD, Sanchez AA, Sidky EY, Pan X. Investigating simulation-based metrics for characterizing lin- ear iterative reconstruction in digital breast tomosynthesis. Medical physics. 2017; 44(9):e279–e96. https://doi.org/10.1002/mp.12445 PMID: 28901614 20. Gong X, Glick SJ, Liu B, Vedula AA, Thacker S. A computer simulation study comparing lesion detec- tion accuracy with digital mammography, breast tomosynthesis, and cone-beam CT breast imaging. Medical physics. 2006; 33(4):1041–52. https://doi.org/10.1118/1.2174127 PMID: 16696481 21. Richard S, Samei E. Quantitative imaging in breast tomosynthesis and CT: Comparison of detection and estimation task performance. Medical physics. 2010; 37(6Part1):2627–37. https://doi.org/10.1118/ 1.3429025 PMID: 20632574 22. Burgess AE, Jacobson FL, Judy PF. Human observer detection experiments with mammograms and power-law noise. Medical physics. 2001; 28(4):419–37. https://doi.org/10.1118/1.1355308 PMID: 11339738 23. Reiser I, Nishikawa RM. Task-based assessment of breast tomosynthesis: Effect of acquisition param- eters and quantum noise a. Medical physics. 2010; 37(4):1591–1600. https://doi.org/10.1118/1. 3357288 PMID: 20443480 24. Burgess AE, Judy PF. Signal detection in power-law noise: effect of spectrum exponents. JOSA A. 2007; 24(12):B52–60. https://doi.org/10.1364/JOSAA.24.000B52 PMID: 18059914 25. Johns PC, Yaffe MJ. X-ray characterisation of normal and neoplastic breast tissues. Physics in Medi- cine & Biology. 1987; 32(6):675. https://doi.org/10.1088/0031-9155/32/6/002 PMID: 3039542 26. Siddon RL. Fast calculation of the exact radiological path for a three-dimensional CT array. Medical physics. 1985; 12(2):252–5. https://doi.org/10.1118/1.595715 PMID: 4000088 27. Zhou J, Zhao B, Zhao W. A computer simulation platform for the optimization of a breast tomosynthesis system. Medical physics. 2007; 34(3):1098–109. https://doi.org/10.1118/1.2558160 PMID: 17441255 28. Zeng R, Park S, Kakic P, Myers KJ. Evaluating the sensitivity of the optimization of acquisition geometry to the choice of reconstruction algorithm in digital breast tomosynthesis through a simulation study. Physics in Medicine & Biology. 2015; 60(3):1259. https://doi.org/10.1088/0031-9155/60/3/1259 29. References Yaffe M, Boone JM, Packard N, Alonzo-Proulx O, Huang SY, Peressotti C, et al. The myth of the 50–50 breast. Medical physics. 2009; 36(12):5437–43. https://doi.org/10.1118/1.3250863 PMID: 20095256 30. Kim M, Yun J, Cho Y, Shin K, Jang R, Bae H-j, et al. Deep learning in medical imaging. Neurospine. 2019; 16(4):657. https://doi.org/10.14245/ns.1938396.198 PMID: 31905454 31. Nguyen TT, Liew AW-C, Pham XC, Nguyen MP. A novel 2-stage combining classifier model with stack- ing and genetic algorithm based feature selection. International Conference on Intelligent Computing; 2014: Springer. 32. Jarrett K, Kavukcuoglu K, Ranzato MA, LeCun Y. What is the best multi-stage architecture for object recognition?. 2009 IEEE 12th international conference on computer vision; 2009: IEEE. 33. Graczyk M, Lasota T, Trawiński B, Trawiński K. Comparison of bagging, boosting and stacking ensem- bles applied to real estate appraisal. Asian conference on intelligent information and database systems; 2010: Springer. 34. Tong T, Li G, Liu X, Gao Q. Image super-resolution using dense skip connections. Proceedings of the IEEE international conference on computer vision; 2017. 35. Han X-H, Zheng Y, Chen Y-W. Multi-level and multi-scale spatial and spectral fusion CNN for hyper- spectral image super-resolution. Proceedings of the IEEE/CVF International Conference on Computer Vision Workshops; 2019. 36. Mao X-J, Shen C, Yang Y-B. Image restoration using very deep convolutional encoder-decoder net- works with symmetric skip connections. arXiv preprint arXiv:160309056. 2016. 37. Yue B, Fu J, Liang J. Residual recurrent neural networks for learning sequential representations. Infor- mation. 2018; 9(3):56. https://doi.org/10.3390/info9030056 38. He K, Zhang X, Ren S, Sun J. Deep residual learning for image recognition. Proceedings of the IEEE conference on computer vision and pattern recognition; 2016. 39. Zhao H, Gallo O, Frosio I, Kautz J. Loss functions for image restoration with neural networks. IEEE Transactions on computational imaging. 2016; 3(1):47–57. https://doi.org/10.1109/TCI.2016.2644865 40. Isola P, Zhu J-Y, Zhou T, Efros AA. Image-to-image translation with conditional adversarial networks. Proceedings of the IEEE conference on computer vision and pattern recognition; 2017. 18 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 PLOS ONE Two-phase learning-based 3D deblurring 41. Simonyan K, Zisserman A. Very deep convolutional networks for large-scale image recognition. arXiv preprint arXiv:14091556. 2014. 42. Russakovsky O, Deng J, Su H, Krause J, Satheesh S, Ma S, et al. Imagenet large scale visual recogni- tion challenge. International journal of computer vision. 2015; 115(3):211–52. https://doi.org/10.1007/ s11263-015-0816-y 43. Kingma DP, Ba J. Adam: A method for stochastic optimization. PLOS ONE \| https://doi.org/10.1371/journal.pone.0262736 January 24, 2022 References arXiv preprint arXiv:14126980. 2014. 44. Goodsitt MM, Chan H-P, Schmitz A, Zelakiewicz S, Telang S, Hadjiiski L, et al. Digital breast tomosynth- esis: studies of the effects of acquisition geometry on contrast-to-noise ratio and observer preference of low-contrast objects in breast phantom images. Physics in Medicine & Biology. 2014; 59(19):5883. https://doi.org/10.1088/0031-9155/59/19/5883 PMID: 25211509 45. Chen H, Zhang Y, Kalra MK, Lin F, Chen Y, Liao P, et al. Low-dose CT with a residual encoder-decoder convolutional neural network. IEEE transactions on medical imaging. 2017; 36(12):2524–35. https://doi. org/10.1109/TMI.2017.2715284 PMID: 28622671 46. Su S, Delbracio M, Wang J, Sapiro G, Heidrich W, Wang O. Deep video deblurring for hand-held cam- eras. Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition; 2017. 47. Pathak D, Krahenbuhl P, Donahue J, Darrell T, Efros AA. Context encoders: Feature learning by inpainting. Proceedings of the IEEE conference on computer vision and pattern recognition; 2016. 48. Yang C, Lu X, Lin Z, Shechtman E, Wang O, Li H. High-resolution image inpainting using multi-scale neural patch synthesis. Proceedings of the IEEE conference on computer vision and pattern recogni- tion; 2017. 49. Iizuka S, Simo-Serra E, Ishikawa H. Globally and locally consistent image completion. ACM Transac- tions on Graphics (ToG). 2017; 36(4):1–14. https://doi.org/10.1145/2897824.2925974 50. Li Y, Liu S, Yang J, Yang M-H. Generative face completion. Proceedings of the IEEE conference on computer vision and pattern recognition; 2017. 51. Tirada N, Li G, Dreizin D, Robinson L, Khorjekar G, Dromi S, Ernst T. Digital breast tomosynthesis: physics, artifacts, and quality control considerations. Radiographics. 2019; 32(2):413–26. https://doi. org/10.1148/rg.2019180046 PMID: 30768362 52. Mermuys K, Vanslambrouck K, Goubau J, Steyaert L, Casselman JW. Use of digital tomosynthesis: case report of a suspected scaphoid fracture and technique. Skeletal Radiology. 2008; 37(6):569–72. https://doi.org/10.1007/s00256-008-0470-3 PMID: 18343919 53. Pan SJ, Yang Q. A survey on transfer learning. IEEE Transactions on knowledge and data engineering. 2009; 22(10):1345–59. https://doi.org/10.1109/TKDE.2009.191 19 / 19
https://openalex.org/W2160232795	https://ebmh.bmj.com/content/ebmental/7/4/98.full.pdf	English	null	The worm turns: publication bias and trial registers revisited	Evidence-based mental health	2,004	public-domain	4,258	Evidence-based medicine: ethically obligatory or ethically suspect? Similarly, knowingly using less accurate information is less likely to optimise patients’ health and is therefore inherently un- ethical. I This ethical argument relies on an epistemological assump- tion—that EBM provides us with a more reliable way of knowing than pre-EBM medicine. To date, there is no body of evidence, although there are case examples, demonstrating that EBM is more able to generate accurate medical information than pre-EBM medicine. Furthermore, it is also uncertain whether EBM achieves its ultimate goal of improving patient health compared with previous modes of practice. In the absence of such evidence, EBM relies on this ethical argument to support—and indeed demand—its use. Because EBM’s ethical rationale depends upon the correctness of its epistemological assumption, the assump- tion must be capable of withstanding scrutiny before one is ethically obliged to practice EBM. In EBM, as the name suggests, it is evidence upon which medical practice is based and it is evidence that will improve the accuracy and reliability of our knowledge. Thus, in order to test EBM’s epistemological assumption, it is essential to understand how EBM defines ‘‘evidence’’. Source of funding bias and technical bias affect the generation of research data. Publication bias, in which certain types of data are knowingly kept from publication, distorts the dissemination of research data.7 Because of publication bias, the total pool of research data from which we draw conclusions about medical interventions cannot be relied upon because it is incomplete. The recent controversy over the use of SSRIs in childhood major depression demonstrates this bias.8 In this case, clinical trials which demonstrated no advantage of the active drugs as compared with placebo were not published and not released for professional scrutiny. Conclusions drawn about these drugs by the psychiatric community were made without being aware of these unpublished data. In the authoritative accounts of EBM, ‘‘evidence’’ is never formally defined, but is implied to be quantitative data obtained from research studies—preferably randomised controlled trials or meta-analyses of these trials. Data generated by other research methods are less preferred or located lower down on the ‘‘evidence hierarchy’’. Some consideration is given to non-quantitative research data, such as qualitative data, although the status and weight of such data remains unclear. EBM’s proponents also allege EBM’s increased reliability by portraying it as inherently neutral, a corrective measure against the biases inherent in any other form of reasoning in clinical practice. Evidence-based medicine: ethically obligatory or ethically suspect? whereas there is no equivalent body to favour the funding of other types of psychiatric interventions such as psycho- social treatments. While fewer data do not mean that psychosocial interventions are ineffective, over time the gradual accumulation of research data concerning pharma- ceuticals suggests greater evidence of their effectiveness compared with other types of interventions. The greater availability of research data concerning medications as compared to psychosocial interventions is also fostered by ‘‘technical bias’’, which is actually built into EBM’s structure. Technical bias favours research that we already know how to do. The ethos of technical bias is aptly captured in step one of EBM’s five steps of EBM practice: converting the need for information into an ‘‘answerable’’ question.4 As Miettinen points out, there may well be intellectual and clinical value in considering unanswerable questions, or at least, difficult to answer questions.5 Furthermore, there may be a gap between what one needs to know, and the answers that the medical research literature can provide. Because EBM prefers certain research methods and certain types of data in its evidence hierarchy, EBM favours those interventions that can best be studied using EBM rules.6 For example, research studies of pharmaceuticals are more likely to be conducted according to EBM preferred methods and these methods will generate data that is ranked more highly by EBM (quantitative data from RCTs). Psychotherapy research, on the other hand, is difficult to conduct according to EBM rules—it can never even meet the basic EBM requirement of double blinding. Psychotherapy research methods are also fraught with methodological problems, particularly when compared with studies of pharmaceutical agents. As a result, psychotherapy research data will necessarily be considered tentative and less definitive than data generated by RCTs and meta-analyses of pharmaceuticals. I n the pages of Evidence-Based Mental Health and elsewhere, authors have argued that it is ethically obligatory to practice evidence-based medicine (EBM).1–3 The argument in favour of this position begins with the assumption that EBM is more able than other strategies to provide us with accurate information about the effectiveness of medical interventions. Accurate information is essential to making clinical recommendations that will be effective in optimising our patients’ health. Optimising patients’ health is a basic ethical duty for medical practitioners. Therefore, the most accurate information—delivered by EBM—is required for practitioners to fulfil a primary ethical obligation. EBMH Notebook............................................................................... Evidence-based medicine: ethically obligatory or ethically suspect? EBMH Notebook.. 96 96 www.ebmentalhealth.com EBMH Notebook.. Evidence-based medicine: ethically obligatory or ethically suspect? Evidence-based medicine: ethically obligatory or ethically suspect? There is no mention of the social, political, and economic contexts in which EBM is practiced or any acknowledgement of how these contexts could influence the generation or dissemination of research data. A few examples suggest that this view of EBM as neutral may be unfounded. In light of these examples, it is reasonable to conclude that EBM is not bias free. The social context in which EBM operates cannot be ignored, for this context influences the production of data. But in addition to ignoring potential sources of bias in producing data, EBM also promulgates a view that data alone will tell us which interventions are or are not effective. This view obscures the process by which data becomes evidence. Data do not support conclusions by themselves—they must first be interpreted. Interpretation is ‘‘Source of funding bias’’ is apparent in psychiatric research when we consider the far more rapidly growing body of research on pharmaceuticals compared with psycho- social interventions. Large, private corporations with a com- mercial interest in pharmaceuticals are a source of funding for researching one type of intervention—medications— 97 a process in which judgement is used to evaluate the relevance and weight of data. It is through this process that data are thought to support certain conclusions. However, interpretation is a human process that is necessarily influenced by a variety of factors including power relations, and commercial and other vested interests.9 The debate concerning SSRI use in childhood depression again illustrates this point. Now that some leading experts have had the opportunity to examine what we think is the total pool of data on this question, various interpretations of the data have arisen. Garland claims that the total data do not support the view that SSRIs are effective in childhood depression whereas Korenblum disagrees.10 This dispute highlights the fact that the same data may be interpreted differently depending on who is examining them, and what knowledge and background they bring to this exercise. Interpreters’ judgement may also be affected, consciously or uncon- sciously, by other factors such as economic concerns, peer pressure, and personal issues of ego and status. optimise patients’ health remains to be seen. In the absence of epistemological justification, there is no ethical obligation to practice EBM. REFERENCES 1 Szatmari P. The art of evidence-based child psychiatry. Evidence-Based Mental Health 2003;6:1–2. 1 Szatmari P. The art of evidence-based child psychiatry. Evidence-Based Mental Health 2003;6:1–2. ; 2 Paris J. Canadian psychiatry across 5 decades: from clinical inference to evidence-based practice. Can J Psychiatry 2000;45:34–8. 2 Paris J. Canadian psychiatry across 5 decades: from clinical inferen evidence-based practice. Can J Psychiatry 2000;45:34–8. 3 Goldner EM, Abbass A, Leverette JS, et al. Evidence-based psychiatric practice: implications for education and continuing professional development Can J Psychiatry 2001;46:15 pages following page 424. on October 23, 2024 by guest. Protec http://mentalhealth.bmj.com/ er 2004. Downloaded from Even if EBM has these limitations, might it be relatively less inaccurate than pre-EBM medicine? I do not believe it is possible to draw such a conclusion at present given that few of the health interventions that have had the greatest impact on morbidity and mortality have been derived from EBM. Measures such as clean water, handwashing, vaccination, and prenatal care have dramatically improved the health of people. Most of these interventions have arisen through both historical accident and a variety of intellectual developments, including those most derided by EBM, such as pathophysio- logical reasoning and clinical observation. EBM’s ability to y y p g g p g 4 Sackett DL, Straus SE, Richardson WS, et al. Evidence-based medicine: how to practice and teach EBM, 2nd edition. Edinburgh: Churchill Livingstone, 2000. 6 Culpepper L, Gilbert TT. Evidence and ethics. The Lancet 1999;353:829–31. 7 Gilbody SM, Song F. Publication bias and integrity in psychiatric research. Psychol Med 2000;30:253–8. 6 Culpepper L, Gilbert TT. Evidence and ethics. The Lancet 1999;353:829–31. 7 Gilbody SM, Song F. Publication bias and integrity in psychiatric research. Psychol Med 2000;30:253–8. 8 Garland EJ. Facing the evidence: antidepressant treatment in children and adolescents. CMAJ 2004;170:489–91. 8 Garland EJ. Facing the evidence: antidepressant treatment in children and adolescents. CMAJ 2004;170:489–91. 9 Leeder SR, Rychetnik L. Ethics and evidence-based medicine. Med J Australia 2001;175:161–4. 9 Leeder SR, Rychetnik L. Ethics and evidence-based medicine. Med J Australia 2001;175:161–4. 10 Mickleburgh R. Antidepressants found ineffective on teenagers. Globe and Mail, 17 February 2004. 10 Mickleburgh R. Antidepressants found ineffective on teenagers. Globe and Mail, 17 February 2004. Evidence-based medicine: ethically obligatory or ethically suspect? Perhaps the best route to epistemological and ethical justification for EBM will begin with the recognition that all knowledge is tentative and subject to various distortions and error, including knowledge generated by EBM preferred methods. Eliminating distortion is probably impossible. This does not necessarily lead to a nihilistic conclusion that the pursuit of knowledge is pointless. Rather it suggests that what is needed in medical practice is an openness to a plurality of sources of knowledge. Employing the standards of transparency and explicitness championed by EBM, each piece of knowledge can be evaluated on its own terms rather than in accordance to a rigid set of rules which may not be applicable. In this way, we can acknowledge the legitimate challenge posed by EBM—that is, to critically examine the basis of our clinical decisions while at the same remaining open to the diverse means through which knowledge evolves. It is important to note that biases and interpretation are not unique to EBM. All knowledge is affected, to different degrees, by these types of factors. However, EBM ignores these issues. This means that the assumption upon which EBM rests—that it is more likely to yield accurate informa- tion than any other method—cannot be taken for granted. Indeed, we have good reasons to believe that EBM has significant potential to produce inaccurate information. Because the assumption of greater accuracy does not necessarily hold, then the ethical obligation to practice EBM is also thrown into question. MONA GUPTA, MD CM Department of Psychiatry and Behavioural Neurosciences, McMaster University, Ontario, Canada; mona.gupta@utoronto.ca The worm turns: publication bias and trial registers revisited R eaders of Evidence-Based Mental Health will be familiar with the fundamental rationale for systematic reviews: reviews should have adequate and well described methods and, in particular, avoid the biased selection of primary studies dependent on their results. A comprehensive and reproducible literature search is now recognised to be a key feature of systematic reviews. We know a lot about the advantages and limitations of electronic bibliographic data- bases, such as Medline.1 There is now a substantial body of empirical evidence about the various kinds of bias that can plague the identification and selection of studies for reviews, including publication bias (studies with significant results are more likely to get published than studies without significant results), English language bias (studies with significant results more likely to be published in English language journals), citation bias (studies with significant results more likely to be cited) and so on.2 As long as studies get published somewhere in journals that are indexed in one database or another, then they are potentially retrievable with an adequate search. Publication bias, however, remains the most devastating potential bias. The reasons for the non- publication of studies vary. The authors of a small study with unexciting results may have little interest or motivation in getting the study published. More pernicious is the situation where a substantial study is not published—and even purposefully suppressed—because the authors or funders do not like the look of the results. has provided a disturbing example of the potentially serious effects of the failure to publish trial data or to make them available. It has emerged that trials of paroxetine, a selective serotonin reuptake inhibitor, showing negative or neutral results in the treatment of depressive disorders in children and adolescents have been not been published by the trials’ sponsor, GlaxoSmithKline. The resulting distortions intro- duced into systematic reviews are illustrated in this issue of EBMH in the study by Whittington et al [see page 115]. This analysis shows clearly that the inclusion of unpublished data very much reduces the apparent efficacy and increases estimates of the harm of certain medications, in particular paroxetine. R The central problem of how to encourage pharmaceutical companies to become more open about their data has exercised the evidence-based community for a number of years. www.ebmentalhealth.com Response to Dr Gupta I t is not uncommon for proponents of evidence-based practice (EBP) to state that once a treatment has been found to be ineffective in a randomised control trial it is ‘‘unethical’’ to continue to practice it. Dr Gupta challenges this statement. She feels there is no justification for stating ‘‘we should practice evidence-based mental health because it is ethical’’. She buttresses her argument by stating that since there are so many concerns about the ‘‘truth’’ of the evidence provided in scientific studies that one should not base the practice of EBP on that foundation. empirical evidence becomes a closer and closer approximation to the truth. It is the ‘‘best available evidence’’, but it will never be the truth itself. Nobody who practices in this way should believe that the truth is a categorical phenomena; that is, true or untrue. Rather, it is ‘‘more’’ or ‘‘less’’ true. I Dr Gupta challenges us not to practice EB mental health because it is ethical. I would ask then, on what basis do I make a clinical decision? If I see a young child with autism, I have a choice: I can refer him to behaviour therapy or not. I would prefer to make my clinical decision on the basis of the best available evidence. What is the alternative to using evidence as a guide? I could do nothing, but that would be clinical paralysis and that surely is unethical. I could make a decision at random by simply flipping a coin, but that does not feel like an ethical thing to do if there is existing evidence (although randomisation in an N of 1 trial may be the best solution if there is inadequate evidence). I could make my decision on the basis on how I was trained. But as I was It is an interesting point but I think it may be dangerous to caricature EBM. I do not recollect any statement by an advocate of EBP saying that the evidence in a randomised controlled trial is the same as ‘‘truth’’. Indeed, I think that what we learn by practicing EBP is rather ‘‘the error of our ways’’. I would admit however, that many advocates of EBP would agree that as the evidence accumulates and insofar as it is consistent (as demonstrated in a meta-analysis), www.ebmentalhealth.com 98 trained many years ago, that information is now quite out of date. Response to Dr Gupta I could instead make my decision based on what I know about the pathophysiology of autism (just like many physicians make clinical decisions about prescribing medica- tion for depression on the basis of their knowledge of neurotransmitters in mood disorders). But as what we know about pathophysiology of autism is so limited (and, I would argue, is equally limited for all psychiatric disorders), I think it is very difficult, if not impossible, to make clinical decisions about treatment based on our very incomplete knowledge of aetiology. Finally, I could make my clinical decision based on my values. What values do I hold about the most appropriate, the most humane, the most empowering form of treatment? In fact, I personally do not find applied behavioural analysis (ABA) very humane. The use of massed discreet trials to teach simple tasks such as matching often looks intrusive and critical. I would much prefer a more developmental sociocognitive approach and indeed such treatments are available but they do not yet have the evidence to support them. But if I were to choose a treatment based on my values over the evidence, I would have to do so by the rules of informed consent. To be ethical, I would have to make that preference known to the parents of this child with autism and they would also have to choose (or not to choose) my values over the evidence. I wonder what a reasonable adult would do in such a circumstance? I bet they would choose treatment that is supported by the evidence over and above my own values and so would choose behaviour therapy. trained many years ago, that information is now quite out of date. I could instead make my decision based on what I know about the pathophysiology of autism (just like many physicians make clinical decisions about prescribing medica- tion for depression on the basis of their knowledge of neurotransmitters in mood disorders). But as what we know about pathophysiology of autism is so limited (and, I would argue, is equally limited for all psychiatric disorders), I think it is very difficult, if not impossible, to make clinical decisions about treatment based on our very incomplete knowledge of aetiology. Finally, I could make my clinical decision based on my values. What values do I hold about the most appropriate, the most humane, the most empowering form of treatment? Response to Dr Gupta In fact, I personally do not find applied behavioural analysis (ABA) very humane. The use of massed discreet trials to teach simple tasks such as matching often looks intrusive and critical. I would much prefer a more developmental sociocognitive approach and indeed such treatments are available but they do not yet have the evidence to support them. But if I were to choose a treatment based on my values over the evidence, I would have to do so by the rules of informed consent. To be ethical, I would have to make that preference known to the parents of this child with autism and they would also have to choose (or not to choose) my values over the evidence. I wonder what a reasonable adult Do we have an alternative? Instead of saying that it is ethical to practice EBM because the evidence leads one to the ‘‘truth’’, one could say ‘‘it is useful to practice EBM’’. More people tend to get better when a treatment has been shown to be effective by a randomised controlled trial than when an alternative is employed. In this circumstance, more people get better when they receive the ‘‘experimental’’ treatment than when they receive, say, a placebo or the standard treatment. It is not so much a matter of ‘‘truthfulness’’ as it is of ‘‘usefulness’’. Whether the results from a particular study are generalisable to my clinical case load is an open question. But choosing a treatment based on its ability to do good for the most people is a utilitarian approach to the truth, not an absolute categorical glimpse of the truth. It is moreover a value laden statement. EBP is ethical because it produces the most good for the largest number of people (whether or not it is also based on any knowledge of the truth). Surely that is a useful way to treat those with severe mental illness. PETER SZATMARI, MD Editor, EBMH Editorial The worm turns: publication bias and trial registers revisited The worm turns: publication bias and trial registers revisited Companies are clearly concerned about the effects of negative trials on sales of a drug in their drive to recoup the development costs and generate a profit for their share- holders. For this reason, voluntary initiatives to get negative results into the public domain have largely been unsuccess- ful. In 1998, a clinical trial amnesty was announced.3 Despite some initial support from companies (including the then GlaxoWellcome), the amnesty has largely failed—as compa- nies seem to have decided that openness about their products might damage their commercial prospects. It has long been acknowledged that the only satisfactory way of protecting against publication bias is by prospective registration of trials, but such registers remain unsatisfactory.4 Publication bias has been a perennial concern of reviewers and evidence-based practitioners, but a recent series of events 99 It is hardly surprising, therefore, that the current SSRI case has been seized upon by the general media, medical commentators, and regulatory bodies alike. Here is a great chance to put real pressure on the pharmaceutical companies to adopt a new policy of openness. On 2 June 2004, GlaxoSmithKline was sued by the Attorney General of New York for fraud over the suppression of trial data of importance in the estimation of the efficacy and the potential increase in suicidal risk of paroxetine in children and adolescents. The action was settled and, belatedly, the company has made full trial reports of the unpublished paroxetine trials available on its website (http://www. gsk.com/media/paroxetine.htm). The International Committee of Medical Journal Editors has recently announced that it will no longer accept trials that have not been entered onto a clinical trial database at inception.5 Sir Iain Chalmers, mean- while, has argued that only legislation can force drug companies to make all trial results available.6 transparent and open about their trial data. Failing to do so means, at best, that ineffective treatments are widely used in patients and, at worse, can lead to unnecessary illness and even death if the reported risks of harms are underestimated. We believe that the industry will want to avoid the possibility of increased legislation and cannot afford repeated public relations disasters of this kind. Lastly, while it is clearly important to make the most of the SSRI case to push for a general improvement in the accessibility of trial results, it is a tragedy that this has involved a treatment for a mental illness. The worm turns: publication bias and trial registers revisited We and our patients are continually fighting stigma, and public percep- tions of antidepressants are likely to have become even more negative as a result of this episode. JOHN GEDDES, MD, PETER SZATMARI, MD, DAVID STREINER, PHD Editors, EBMH JOHN GEDDES, MD, PETER SZATMARI, MD, DAVID STREINER, PHD Editors, EBMH JOHN GEDDES, MD, PETER SZATMARI, MD, DAVID STREINER, PHD Editors, EBMH From the point of view of the evidence-based practitioner, we are usually realistic enough in our expectations of our treatments to know that no drug provides benefits without costs. What we need, though, is reliable information about both benefits and costs to give our patients proper informa- tion about what they can expect from our recommended therapies. Evidence-Based Mental Health has abstracted two published trials that compared SSRIs with placebo in the treatment of childhood depression7 8 and a summary of the section on the treatment of childhood depression from Clinical Evidence.9 Although we believe that the coverage we have given the issue has been true to the status of the published evidence, we are of course unable to report evidence that is not published or is deliberately suppressed. In our view, publication bias subverts evidence-based practice and can harm patients. REFERENCES 1 Dickersin K, Scherer R, Lefebvre C. Identifying relevant studies for systematic reviews [see comments]. BMJ 1994;309:1286–91. 1 Dickersin K, Scherer R, Lefebvre C. Identifying relevant studies for systematic reviews [see comments]. BMJ 1994;309:1286–91. 2 Egger M, Smith GD. Bias in location and selection of studies. BMJ 1998;316:61–6. 2 Egger M, Smith GD. Bias in location and selection of studies. BMJ 1998;316:61–6. 3 Smith R, Roberts R. An amnesty for unpublished trials. BMJ 1997;315:622. 4 R i D T i l i i id i h f i d i i ibl 4 Rennie D. Trial registration: a great idea switches from ignored to irresistible. JAMA 2004. l ll l l l l 5 De Angelis C, Drazen JM, Frizelle FA, et al. Clinical trial registration: a statement from the International Committee of Medical Journal Editors. Lancet 2004;364:911–12. 6 Chalmers I. In the dark: drug companies should be forced to publish all the results of clinical trials. New Sci 2004;181:19. 7 Fleming J. Fluoxetine decreased depressive symptoms in children and adolescents with non-psychotic major depressive disorder. Evid Based Ment Health 1998;1:49. 7 Fleming J. Fluoxetine decreased depressive symptoms in children and adolescents with non-psychotic major depressive disorder. Evid Based Ment Health 1998;1:49. 8 McClure EB, Leibenluff E, Pine DS. Sertraline improves symptoms in children and adolescents with major depressive disorder. Evid Based Ment Health 2004;7:10. 8 McClure EB, Leibenluff E, Pine DS. Sertraline improves symptoms in children and adolescents with major depressive disorder. Evid Based Ment Health 2004;7:10. We at Evidence-Based Mental Health therefore join the calls on our partners in the pharmaceutical industry to be more 9 Hazell P. Depression in children and adolescents. Evid Based Ment Health 2003;6:103–4. www.ebmentalhealth.com
https://openalex.org/W3116233682	https://www.emerald.com/insight/content/doi/10.1108/PRR-01-2020-0001/full/pdf?title=corporate-characteristics-and-leverage-evidence-from-gulf-countries	English	null	Corporate characteristics and leverage: evidence from Gulf countries	PSU research review	2,020	cc-by	9,989	The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/2399-1747.htm The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/2399-1747.htm PRR 6,2 © Waleed M. Al-ahdal, Faozi A. Almaqtari, Dheya A. Zaid, Eissa A. Al-Homaidi and Najib H. Farhan. Published in PSU Research Review. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non- commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence maybe seen at http://creativecommons.org/licences/by/4.0/legalcode Corporate characteristics and leverage: evidence from Gulf countries Waleed M. Al-Ahdal Faculty of Commerce, Banaras Hindu University,Varanasi, India 120 Received 12 January 2020 Revised 26 August 2020 6 October 2020 Accepted 2 November 2020 Faozi A. Almaqtari Department of Accounting, Hodeidah University, Hodeidah, Yemen, and Dheya A. Zaid, Eissa A. Al-Homaidi and Najib H. Farhan Department of Commerce, Aligarh Muslim University, Aligarh, India Dheya A. Zaid, Eissa A. Al-Homaidi and Najib H. Farhan Department of Commerce, Aligarh Muslim University, Aligarh, India PSU Research Review Vol. 6 No. 2, 2022 pp. 120-140 EmeraldPublishingLimited 2399-1747 DOI 10.1108/PRR-01-2020-0001 1. Introduction Capital structure indicates to the mix of the various methods of funding sources that a ﬁrm maintains (Niu, 2008). Gomez et al. (2014) states that the way a ﬁrm is ﬁnanced is very relevant for investors, directors and all other stakeholders. Accordingly, the ﬁnancing decisions have a direct inﬂuence on the ﬁrm value. Prior evidences from ﬁnancial literature showed that a ﬁrm’s capital structure could be affected by different factors. Capital structure is one of the most broadly debatable areas of research in corporate ﬁnance. A central question was so long raised by Modigliani and Miller (1958), which evolve around how a mix of debt and equity in capital structure has an impact on ﬁrm value. Further, the determinants that can have an inﬂuence on a ﬁrm’s capital structure are arguable in prior ﬁnancial studies. 121 Bauer (2004) indicated that a ﬁrm’s leverage is not inﬂuenced by its size. Further, some studies believe that there is a negative correlation between a ﬁrm’s leverage and its proﬁtability (Pratheepkanth, 2011; Rouf, 2015; Zeitun and Tian, 2007). While Ahmed et al. (2010) found that there is a positive link between leverage and liquidity, Sharif et al. (2012) reported a negative relationship. Ahmed et al. (2010) argue that high liquidity provides ﬁrms with greater capability to pay off its debt accordingly, such ﬁrms should opt for debt ﬁnancing as a major contributor to their capital structure. In another quest, Miguel and Pindado (2001) advocated that capital structure determinants can be explained by business characteristics and the institutional settings of countries. Jong et al. (2008) investigated capital structure in different countries and found that enterprise-level determinants that affect corporate capital structure vary among countries. However, country-level determinants have an indirect inﬂuence on capital structure because they have an impact on enterprise-level factors. Li and Islam (2019) indicated that prior studies have made numerous attempts to investigate the corporate characteristics that may have an impact on the capital structure of ﬁrms operating within a speciﬁc country. However, modern studies have been conducted based on country comparison taking in consideration country-speciﬁc determinants to explain sample ﬁrms’ capital structure choice. Theoretically, Li and Islam, (2019) indicated that capital structure theories such as the pecking order theory, the market timing theory, the trade-off theory and the agency theory suggest different capital structure determinants. Jaisinghani et al. Abstract Purpose – This study aims to investigate the impact of corporate characteristics on leverage in the Gulf Cooperation Council (GCC) non-ﬁnancial listed ﬁrms. Design/methodology/approach – A sample comprising a balanced panel for eight years from 2009– 2016 for four Gulf countries is used. In total, 85 non-ﬁnancial listed companies have been selected using a non- probability sampling technique. Corporate characteristics are represented by return on assets (ROA), return on equity, return on capital employed, market value-added, Tobin-Q, liquidity and ﬁrm size. The study used ﬁxed and random effect models to estimate the results. Findings – The ﬁndings of the study revealed that both ROA and FSIZE have a signiﬁcant negative effect on leverage. However, market value-added, return on capital employed and Tobin-Q exhibited a statistically signiﬁcant positive effect on leverage. Further, the results indicated that Qatar is better than kingdom of Saudi Arabia (KSA), Oman and the UAE. In addition, evidence noted that KSA is better than both UAE and Oman in terms of the overall impact of corporate characteristics on the leverage. However, this effect is not statistically signiﬁcant. Practical implications – This study provides an open insight for managers, bankers, ﬁnancial analysts in the GCC countries and some other developing economies by highlighting the relationship between corporate characteristics and leverage in an emerging market. Originality/value – The current study provides an important insight into corporate characteristics and leverage. By so doing, it provides an attempt to identify the factors inﬂuencing corporate ﬁnancing behavior taking into consideration different issues such as different proxies of ﬁrms’ proﬁtability, market capitalization, market value added and liquidity, which provides original evidence from Gulf countries emerging markets. These countries are characterized by low tax rates and high liquidity. High liquidity may reduce the cost of borrowing and debt ﬁnancing may not be a huge burden on ﬁrms’ proﬁts. This makes the investigation of leverage and corporate characteristics, particularly, ﬁrms’ proﬁtability and liquidity, very important. Therefore, the study tries to bridge an existing gap in the body of literature of capital structure and debt ﬁnancing in Gulf countries emerging markets. PSU Research Review Vol. 6 No. 2, 2022 pp. 120-140 EmeraldPublishingLimited 2399-1747 DOI 10.1108/PRR-01-2020-0001 Keywords Leverage, GCC, Capital structure, Corporate characteristics Paper type Research paper Gulf countries 1. Introduction (2017) stated that the asymmetric information theory and the tradeoff theory are the prominent theories pertaining to optimal capital structure. However, Cappa et al. (2020) argued that there are a number of competing theories that pertain to capital structure formation, which are, for example, trade-off, asymmetric information, pecking order, market timing and agency costs theories. According to Jaisinghani et al. (2017), tradeoff theory postulates that a ﬁrm determines its capital structure depending on a trade-off between the costs and beneﬁts of debt. Miller (1977) indicated that there is a relationship between proﬁtability and debt ﬁnancing, based on the trade-off theory; ﬁrms that are more proﬁtable would prefer debt over equity ﬁnancing. In the same quest, pecking order theory suggests that a ﬁrm with higher proﬁtability would prefer to opt for a low debt level due to the information asymmetric element involved in the pecking order theory (Hamid et al., 2015). The asymmetric information theory proposed that a ﬁrm may use its capital structure as a signaling instrument to the market (Jaisinghani et al., 2017). Also, agency theory proposed that ﬁnancing decisions and capital structure mix depend on agency costs. The agency costs may arise from the conﬂict between shareholders and creditors accordingly, capital structure could be determined by minimizing such cost and balancing the interests of the parties involved. Further, the theory states that ﬁrms with high returns and great potential are more likely to prefer equity ﬁnancing over dept ﬁnancing (Li and Islam, 2019). parties involved. Further, the theory states that ﬁrms with high returns and great potential are more likely to prefer equity ﬁnancing over dept ﬁnancing (Li and Islam, 2019). y p q y g p g ( , ) Majority of prior research in this ﬁeld have concentrated on developed stock markets. Even though there are some studies that have been conducted in some emerging countries. For example, Bhaduri (2002) investigated the same issue in India, Chen and Strange (2005) conducted a study on some ﬁrms in China, Correa et al. (2007) provided an evidence from some Brazilian factories, Crnigoj and Mramor (2009) concentrated on ﬁrms in Slovenia, Kim et al. 1. Introduction (2006) examined this issue in the South Korean context, Fernandez (2005) introduced evidence from Chilean corporates, Mazur (2007) sampled Polish ﬁrms, Omet (2006) focused on Jordanian ﬁrms and Salawu and Agboola (2008), Shah and Khan (2007) and Vasiliou and Daskalakis (2009) provided evidence from Nigeria, Pakistan and Greek, respectively. However, the results of these studies are greatly varied and widespread. Hence, the present study aims to investigate the corporate characteristics and leverage in some Gulf Cooperation Council (GCC) countries. The current study contributes to ﬁnance literature in several ways. First, the study provides a comprehensive investigation of some selected large ﬁrms (85 ﬁrms), which are covering all sectors of the economy except ﬁnancial industry from 4 GCC countries over the years spanning from 2009 up to 2016 for 4 Gulf countries (Saudi Arabia, Oman, UAE and Qatar). Second, the study presents different statistical tools to understand the differences among the countries. Prior studies have used either the same or different statistical tools however, the differences among the GCC countries were not adequately provided. The present study used detailed descriptive statistics, and country wise effect comparison estimation. Third, the study contributes to the state of the art of capital structure, cost of ﬁnance, debt ﬁnancing and ﬁnance in general especially from the context of developing countries. The study links corporate characteristics to leverage, and thus, it provides an attempt to identify the factors inﬂuencing corporate ﬁnancing behavior taking into consideration, ﬁrm speciﬁc factors in the GCC. The study also provides evidence from different countries that are homogenous in their culture, economy and banking systems. Moreover, it attempts to provide evidence from oil-based and bank-based economies of the GCC countries. Several studies investigated different issues in debt ﬁnancing and capital structure in GCC. However, the present study provides an investigation into ﬁrm-speciﬁc factors on leverage of GCC ﬁrms taking into consideration different issues such as different proxies of ﬁrms’ proﬁtability, market capitalization, market value added (MVA) and liquidity, which makes the investigation of the present study unique and different from prior studies. 122 y q p The paper proceeds as follows. Section 2 explains the background of GCC. Section 3 discusses the literature review; this is followed by Section 4, which presents a collection of data, techniques and the study models. Section 5 provides empirical analysis and the related discussions. 3. Literature review Th l i hi b The relationship between capital structure and ﬁrm performance is widely discussed and tested in prior research. However, this ﬁeld of research still the most perplexing area in the strand literature of corporate ﬁnance (Brounen and Eichholtz, 2001). Different studies have been focused on the relationship between a ﬁrm’s performance and capital structure (Champion, 1999; Ghosh et al., 2000; Hadlock and James, 2002; Jensen and Meckling, 1976; Modigliani and Miller, 1963; Miller, 1977; Myers, 1977; Margaritis and Psillaki, 2010; Titman and Wessels, 1988). These studies contributed signiﬁcantly to the literature of corporate ﬁnance, which started early with the research of (Hirshleifer, 1958; Lintner, 1956; Modigliani and Miller, 1958). In the GCC context, different studies have highlighted the link between corporate characteristics and capital structure (El-Khatib, 2017; Sbeiti, 2010; Twairesh, 2014). Zeitun and Saleh, (2015) investigated the impact of ﬁnancial leverage on a ﬁrm’s performance in the GCC countries taking in consideration the effect of the last ﬁnancial crisis. The results revealed that ﬁrm’s performance is signiﬁcantly inﬂuenced by leverage and negatively affected by the ﬁnancial crisis. In the same context, El-Khatib (2017) studied conventional leverage determinants in some publicly listed ﬁrms in many GCC countries, namely; Qatar, Saudi Arabia and the United Arab Emirates over a period from 2005 up to 2014. The study found that conventional leverage is signiﬁcantly inﬂuenced by some determinants such as the tendency to pay dividends, ﬁrm’s size, tangibility, proﬁtability and age. Both ﬁrm’s size and Sharīʿah principles were found to have the most signiﬁcant effect but utilization of Sukuk as a ﬁnancing vehicle has no signiﬁcant effect. On the contrary, Sbeiti (2010) investigated the “determinants of capital structure in the context of three GCC countries and the impact of their stock markets’ development on the ﬁnancing choices of ﬁrms operating in these markets.” The results suggested that stock markets in the sampled countries are regarded as an important option for ﬁnancing decisions. Further, the results reveal that capital structure in the selected countries is not different from ﬁnance models of the developed countries. Although there are few studies that addressed the relationship between ﬁrms’ performance and ﬁnancial leverage in the GCC countries, there are numerous studies that investigated this issue in other countries (Muritala, 2012; Ojo, 2012; Rehman, 2013; Zeitun and Tian, 2007). Gomez et al. (2014) investigated capital structure determinants of non- ﬁnancial companies listed in the Stock Exchange of Lima. 1. Introduction Section 6 comprises the conclusion, recommendations and prospective avenues that future authors can pursue in line of this research. 2. Background to Gulf cooperation council The GCC region comprises fast-growing economies with government revenues fueled by sizeable oil rents, which are characterized by administrated consumer energy prices. Historically, GCC countries except Oman did not levy corporate taxes on owned domestic ﬁrms (Almutairi, 2014). The GCC ﬁnancial markets are small in size and less developed by international standards and its emerging peers, they account for about 0.8% of the global ﬁnancial markets (Zeitun and Saleh, 2015). In comparison of the GCC stock markets with the worldwide stock markets, they are considered young and small in size. They are lagging behind Asia and Latin America. They only account for 0.8% of the global ﬁnancial markets. Further, in 2010, they accounted for 1.3% of the global equity market capitalization. Furthermore, the GCC stock markets have some barriers such as low number of listed ﬁrms; 707 ﬁrms in 2013, restricted entry by foreigners and ownership concentration (Woertz, 2012). These characteristics have motivated the current study to investigate this issue to extend the evidence from prior studies to an emerging economy such as the GCC countries. The ﬁnancial crisis negatively affected the global liquidity, which, in turn, affected banks and other ﬁnancial instructions’ ability to offer credit facilities. Therefore, the cost of borrowing increased tremendously as the accessibility of liquidity decreased and banks created tighter credit policies, which affected ﬁrms’ performance negatively (Ellaboudy, 2010). While the majority of prior evidence on the relationship between corporate characteristics and leverage comes from capital structure determinants in both developed and developing countries, empirical research in this area did not speciﬁcally concentrate on the GCC countries (El-Khatib, 2017; Sbeiti, 2010; Twairesh, 2014). Further, the ambiguous results related to the relationship of corporate characteristics and leverage have motivated the present study to be conducted to investigate this relationship. 123 Gulf countries 3. Literature review Th l i hi b In the scope of trade-off theory and pecking order theory and with utilization of panel data with random effect model, the results reveal that the level of ﬁrms’ long-term debt is signiﬁcantly inﬂuenced by their size, proﬁtability, non-debt tax shields and collateral value of assets. Soumadi and Hayajneh (2011) investigated the “effect of capital structure on the performance of the public Jordanian ﬁrms listed in Amman stock market.” The results indicated that there is a signiﬁcant and negative relationship between both capital structure and ﬁrm performance and this relationship does not change in the case of ﬁrms with high or low ﬁnancial leverage. In the same line, (Pratheepkanth, 2011; Rouf, 2015; San and Heng, 2011; Zeitun and Tian, 2007) found that ﬁrms’ performance is negatively linked with capital structure. Salim and Yadav (2012) provided evidence on the link between 237 Malaysian ﬁrms’ performance and capital structure. They advocate a negative relationship between them. and pecking order theory and with utilization of panel data with random effect model, the results reveal that the level of ﬁrms’ long-term debt is signiﬁcantly inﬂuenced by their size, proﬁtability, non-debt tax shields and collateral value of assets. Soumadi and Hayajneh (2011) investigated the “effect of capital structure on the performance of the public Jordanian ﬁrms listed in Amman stock market.” The results indicated that there is a signiﬁcant and negative relationship between both capital structure and ﬁrm performance and this relationship does not change in the case of ﬁrms with high or low ﬁnancial leverage. In the same line, (Pratheepkanth, 2011; Rouf, 2015; San and Heng, 2011; Zeitun and Tian, 2007) found that ﬁrms’ performance is negatively linked with capital structure. Salim and Yadav (2012) provided evidence on the link between 237 Malaysian ﬁrms’ performance and capital structure. They advocate a negative relationship between them. 124 y g p Concerning liquidity, while Ahmed et al. (2010) indicated a positive link between both leverage and liquidity. Firms with high liquidity have a capital structure mix that is characterized by debt. This observation is attributed to the ability of such ﬁrms to repay the cost of borrowing with ﬁnancial ease. Sharif et al. (2012) advocated that the link between leverage and liquidity is negative. 3. Literature review Th l i hi b Despite the fact, the prior studies have investigated corporate characteristics and leverage in different countries, however, studies conducted in the GCC countries are unreliable as the stock markets in these countries are not well-developed, and cannot be benchmarked with international standards. Further, the tax system in these countries have a notable impact on capital structure. Therefore, the present study attempts to explore this impact. Referencing Table 1, prior studies have investigated different issues of capital structure and debt ﬁnancing; the present study is different from prior studies in several ways. For example, El-Khatib (2017) investigated tangibility, market to book ratio, proﬁtability, size, ﬁnancial deﬁcit and age of the company with relation to equity ﬁnancing. He further examined ﬁrm size, liquidity, proﬁtability, tangibility and growth opportunities and its relation with book leverage and market leverage. None of the studies conducted in GCC have investigated corporate characteristics such as liquidity, different proxies of proﬁtability, Tobin Q and MVA with its relation to leverage. Thus, the present study bridges an existing gap in the debt ﬁnancing and capital structure. Moreover, it provides a unique and comprehensive examination of the impact of corporate characteristics on leverage in emerging markets especially GCC countries. The study also extends the evidence on capital structure, leverage and debt ﬁnancing by re-visiting the impact of corporate characteristics on leverage in GCC countries. 4.1 Sample selection and data collection Thi d f i i h 4.1 Sample selection and data collection Thi d f i i h This study focuses on examining the associations of corporate characteristics and leverage in GCC non-ﬁnancial listed ﬁrms. We use a sample comprising balanced panel data for eight years from 2009 up to 2016 for four Gulf countries, which is covering all sectors of the economy except the ﬁnancial industry. In total, 85 non-ﬁnancial listed companies have been selected using a non-probability sampling technique. As a result, the ﬁnal sample consists of 23 companies from Saudi Arabia, 19 companies from Oman, 23 companies from the UAE and 20 companies from Qatar. The study uses a sample of large ﬁrms from Saudi Arabia, UAE, Qatar and Oman. According to KPMG (2015), these four countries have the highest total banks assets and net proﬁts. Further, according to The World Bank (2015), the gross domestic product annual growth in Saudi Arabia, UAE, Qatar and Oman is the highest among all GCC member states. Authors Country DV IV Sample Period Results Khan et al. (2020) Pakistan Leverage “Liquidity, proﬁtability, age, tangibility” 183 ﬁrms 2008– 2017 The research result found tangibility, proﬁtability and age to be positively related to leverage among listed ﬁrms in Pakistan. However, size and liquidity are negatively related to leverage Kyissima et al. (2019) China “Book leverage, market leverage and net leverage” “Size, proﬁtability and tangibility” 716 ﬁrms 1990– 2013 Capital structure is signiﬁcantly affected by proﬁtability, investment opportunities and ﬁrm’s size Yildirim et al. (2018) US, UK, Canada, Japan, Taiwan, South Korea and India “Book leverage and market leverage” “Proﬁtability, growth O, ﬁrm size, tangibility, business risk, GDP and growth” 756 2004– 2014 Most of the determinants do exhibit different effects among both ﬁrm types. Table 1. Review of related literature 4.1 Sample selection and data collection Thi d f i i h Depending on the leverage measure, the effect of different independent variables on ﬁrms’ capital structure varies Sofat and Singh (2017) India Debt equity ratio “Firm size, asset composition/tangibility, debt service, capacity, business risk and Proﬁtability” 100 ﬁrms 2003– 2012 Asset composition, business risk and return on assets are positively related to debt ratio whereas; ﬁrm size and debt service capacity are negatively related to debt ratio El-Khatib (2017) Saudi Arabia, United Arab Emirates and Qatar “Ratio of debt to market equity, debt to book equity, long term debt to market equity and long term debt to book equity” “Tangibility, market to book ratio, proﬁtability, size, ﬁnancial deﬁcit and age” 100 ﬁrms 2005– 2014 Conventional leverage is signiﬁcantly inﬂuenced by some determinants such as tendency to pay dividends, ﬁrm’s size, tangibility, proﬁtability and age (continued) 125 Authors Country DV IV Sample Period Results Güner (2016) Turkey Leverage “Size, growth opportunities, non-debt tax shields, proﬁtability and liquidity” 131 ﬁrms 2008– 2014 Companies that have a free ﬂoat rate between 50% and 75% have lower degrees of leverage Bandyopadhyay and Barua (2016) India “Total borrowings to total assets, short term bank, borrowings to total, borrowings, long term borrowings to total assets, bank borrowings to total assets, long term bank borrowings to total borrowings” “Firm age, ﬁrm size, tangibility, turnover, liquidity, price to book ratio, sales volatility, intercept” 1,594 ﬁrms 1998 to 2011 Financing decisions are widely inﬂuenced by macro-economic cycle Chadha and Sharma (2015) India “Total debt to total capital and total debt to total assets” “Size, age, growth, tang, proﬁtability, risk, dividend payout ratio, NDTS, liquidity, uniqueness, ICR, CFCR, ownership, inﬂation and GDP” 422 ﬁrms 2004– 2012 Leverage has a signiﬁcant relationship with “size, age, asset tangibility, growth, proﬁtability, non-debt tax shield, business risk, uniqueness and ownership structure Rouf (2015) Bangladesh “Total liabilities divided by total assets” “Total assets (TA), total sales (TSE), return on assets (ROA), return on sales (ROS), liquidity, age, debt-to-equity ratio, current debt ratio, proprietary of equity ratio” 106 ﬁrms 2011– 2015 There is a negative and signiﬁcant relationship between leverage and ROA, size and AGE Gomez et al. 4.1 Sample selection and data collection Thi d f i i h (2014) Peru “Long-term liabilities to total assets ratio” “Proﬁtability, size, business risk, collateral assets value, depreciation to total assets ratio, growth and liquidity” 64 ﬁrms 2004– 2008 The level of ﬁrms’ long-term debt is signiﬁcantly inﬂuenced by their size, proﬁtability, non- debt tax shields and collateral value of assets (continued) Table 1. PRR 6,2 126 126 Peru Authors Country DV IV Sample Period Results Benkraiem and Gurau (2013) French “Total, long-term and short-term debt” “Size, proﬁtability, growth and tangibility” 2003 and 2006 Capital structure is signiﬁcantly affected by size, proﬁtability, growth and tangibility of assets Matemilola et al. (2013) South Africa “Long term debt and total debt” “Fixed assets, net proﬁt, size, growth opportunity and non-debt tax shield” 600 2004– 2009 The empirical ﬁndings indicate that models that include unobservable ﬁrm- speciﬁc effects are correctly speciﬁed Sbeiti (2010) Kuwait, Saudi Arabia and Oman “Book leverage and market leverage” “Firm size, liquidity, proﬁtability, tangibility and growth opportunities” 142 ﬁrms 1998– 2005 Capital structure in the selected countries is not different from ﬁnance models of the developed countries Morri and Cristanziani, (2009) Europe “Total debt/total equity, total debt/total asset, total liabilities/total asset, total debt/capital, short-term debt/ total debt, long-term debt/total debt, short-term debt/total asset” “Size, proﬁtability, growth opportunities, cost of debt, ownership structure, risk and category” 97 companies Non-REIT companies are signiﬁcantly more leveraged than REITs. The negative relationship between operating risk and leverage demonstrates that the managers of riskier ﬁrms tend to reduce the overall company’s uncertainty by adopting a more careful capital structure Kim and Berger (2008) Korea and USA “Market value-based leverage ratio” “Proﬁt, company size, non-debt tax shields, growth and business-risk” 36 ﬁrms 1987– 1991 There is no signiﬁcant difference between Korean and Japanese ﬁrms Source: Prepared by the authors based on literature survey 127 4.3 Model speciﬁcation Consistent with previous literature (Al-ahdal et al., 2020; Almaqtari et al., 2019; Rouf, 2015), we developed the following model to investigate the impact of corporate characteristics on leverage in the GCC non-ﬁnancial listed ﬁrms: Y ¼ b 0 þ b fit þ eit (1) (1) Where Y is the dependent variable. b 0 is the constant, b is the coefﬁcient of the explanatory variable (corporate characteristics), ﬁt is the explanatory variable and eit is the error term. ( p ), p y By adopting the economic model as in equation (1) speciﬁcally in this study, we can estimate a ﬁxed effects model using equation (2) as follows: LEVit ¼ b 1 ROEit þ b 2 TQit þ b 3 LIQit þ b 4 ROCEit þ b 5 ROAit þ b 6 FSIZEit þ b 7 MVAit þ aiþuit (2) (2) Where all variables are as deﬁned in Table 2, ai (i = 1. . .. n) is the unknown intercept for each entity (n entity-speciﬁc intercepts) and uit is the error term. Where all variables are as deﬁned in Table 2, ai (i = 1. . .. n) is the unknown intercept for each entity (n entity-speciﬁc intercepts) and uit is the error term. Table 1. p Corporate characteristics represented by return on assets (ROA), return on equity (ROE), return on capital employed, MVA, Tobin-Q, liquidity and ﬁrm size are considered as the independent variables and leverage is the dependent variable. The relationship between corporate characteristics and leverage in the present study is viewed and motivated by different studies (Gomez et al., 2014; Kim and Berger, 2008; Rouf, 2015). 128 4.3 Model speciﬁcation Consistent with previous literature (Al-ahdal et al., 2020; Almaqtari et al., 2019; Rouf, 2015), we developed the following model to investigate the impact of corporate characteristics on leverage in the GCC non-ﬁnancial listed ﬁrms: 5.1 Descriptive statistics Table 3 provides a summary of panel descriptive statistics for the variables used by the current study using xtsum command in Stata. Xtsum generates additional descriptive statistics to the normal one. It provides an analysis for the variables into an overall, between and within descriptive statistics. The overall and within descriptive statistics are calculated over 680 (N = 680) ﬁrm-years observations. The between descriptive statistics is calculated over 85 ﬁrms (n = 85) and the average number of years a ﬁrm was observed in the variable data (T = 8). Overall, descriptive statistics reports common descriptive statistics in which the average value of a variable across all ﬁrms for the entire time period is calculated. Besides, minimum, maximum and the standard deviation values are given for the variables. While the minimum values of a variable denote the lowest value of a variable across the sample over the study time period, the maximum values are calculated as the highest values across the sampled ﬁrms during the time period. The average ROA of the sampled ﬁrms has a mean value of 8.57, which varies between a minimum of 65.13 and a maximum of 75.23 with a standard deviation (SD) of 9.83. This indicates that there is no high variation in ROA of the sampled companies. Further, the overall average of ROE for 680 ﬁrm years’ observations is 11.95 with an SD of 35.05 (min = 655.22 and max. = 106.54). The results also show that the mean value of return on capital employee (ROCE) across the 860 ﬁrm years observations is 9.72 with a minimum of 516.44, a maximum of 86.89 and SD of 27.61. MVA has an overall mean of 184,600 with a min. of 3,200,000 and a maximum of 12 million. Similarly, Tobin’s Q has Variable type Variable name Symbol Proxy measure Evidence from prior studies Dependent variable Leverage LEV “It is measured by dividing the total liabilities upon total assets” Güner (2016), Yildirim et al. (2018) Independent variables Return on equity ROE “ROE is calculated by proﬁt after tax divided total equity shares at the end of the year” Al-ahdal et al. (2020), Rouf (2015) Tobin-Q TQ “Tobin’s Q can calculate by the ratio of the market capitalization plus total debt divided by total asset of the company” Al-ahdal et al. (2020), Yameen et al. Table 2. Variables description Table 3. Descriptive statistics 5.1 Descriptive statistics (2019) Liquidity LIQ “Current assets divided by current liabilities” Güner (2016), Rouf (2015) Return on capital employed ROCE “It is calculated by comparing net proﬁt after tax with capital employed” Al-Matari et al. (2014), Yameen et al. (2019) Return on assets ROA “It is measured by the percentage of the net proﬁt of one year for the total assets of the same year” Almaqtari et al. (2019), Rouf (2015) Firm size FSIZE “The natural logarithm of total assets” Matemilola et al. (2013), Yildirim et al. (2018) Market value added MVA “It can be calculated by the difference between the market value and book value of equity” Al-Matari et al. (2014), Imberman and Lovenheim (2016) Gulf countries 129 PRR 6,2 Overall descriptive statistics Variable Panel Mean SD Min Max Variable Mean SD Min Max ROA overall 8.57 9.83 65.13 75.23 TQ 1.66 2.94 0.32 74.66 between 8.50 6.64 52.18 1.40 0.45 12.07 within 5.02 49.91 36.65 2.59 8.34 64.25 ROE overall 11.95 35.05 655.22 106.54 FSIZE 6.79 1.43 2.19 10.99 between 17.68 96.30 56.23 1.39 3.50 10.13 within 30.32 546.97 119.09 0.36 5.00 11.52 ROCE overall 9.72 27.61 516.44 86.89 LVR 47.81 110.51 2.51 2,869.03 between 15.43 66.94 75.25 44.47 8.32 411.46 within 22.94 439.78 84.33 101.27 343.56 2,505.37 MVA overall 184,600 1,203,803 3,200,000 12,000,000 LIQ 241.89 1,377.38 0.02 21,645.40 between 1,094,914 2,700,000 7,875,000 1,210.75 0.10 10,986.26 within 512,508 4,890,400 4,309,600 668.11 6,551.41 10,901.02 Notes: 1- Observations (Overall: N = 680, between: n = 85, within: T = 8) 2- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added 130 a minimum of 0.32, maximum of 74.66 with an average of 1.66. FSIZE shows a mean of 6.79 with a min. of 2.19 and a max. of 10.99. Importantly, LEV demonstrates a variation among the sampled companies. It has an average of 47.81 with SD of 110.51 (Min. = 2.51 and Max. = 2,869.03) (Figure 1). The value of SD is much higher than the mean value indicating that the mean does not represent the average values of the sampled ﬁrms. Finally, LIQ has an average value of 241.89 with a minimum of 0.02, a maximum of 2,1645.40 and SD of 1,377.38. 131 Table 4 reports country wise descriptive statistics. 5.1 Descriptive statistics As we have seen in Table 3, there is a variation between the standard deviation and the average values of some variables. This prompts us to conduct country-wise descriptive statistics to know the characteristics of the data of each country. Table 4 presents country-wise descriptive statistics in the form of mean, median, minimum, maximum and standard deviation for all the variables of the study. The results show the variation among the countries in LIQ, MVA and TOBINQ. All other variables have slight variation. Gulf countries 5.2 Unit root analysis Unit root analysis is applied to conﬁrm the stationarity of the variables as a pre- requisite requirement for analysis of the models of the study. Three tests were conducted to check the stationarity of the variables; Levin, Lin and Chu test, Im Pesaran and Shin test and Augmented Dickey-Fuller test. Each variable was tested individually at the ﬁrst level. As shown in Table 5, all variables used in the models are found to be stationary at level in all the tests applied. This leads to reject the null hypothesis of the unit root test at the level. 5.3 Correlation analysis and multicolinearity diagnostics 5.3 Correlation analysis and multicolinearity diagnostics Table 6 shows the results of Pearson correlation matrix for the variables of the present study. The results of Pearson correlation demonstrate that there is a positive and negative relationship between dependent and independent variables. While LEV has a signiﬁcant Figure 1. Average values of leverage across the countries Table 4. 5.2 Unit root analysis Country wise descriptive statistics Country wise descriptive statistics KSA Mean Median Maximum Minimum SD Mean Median Maximum Minimum SD KSA: observations = 184 Oman: observations = 182 FSIZE 6.67 6.84 10.00 3.98 1.33 6.88 6.76 10.36 2.19 1.79 LIQ 13.88 2.90 273.73 0.07 28.51 122.65 19.10 5,237.22 0.15 453.14 LEV 46.00 45.90 102.53 9.93 19.98 55.16 31.92 2,869.03 2.51 231.15 MVA (000) 95.74 0.004 3,000.00 1,500.00 485.99 320.28 0.011 6,900.00 1,100.00 1,064.69 ROA 8.97 6.96 75.23 28.86 11.70 8.46 6.88 37.30 65.13 10.55 ROCE 8.20 8.24 86.89 516.44 42.43 8.84 7.96 52.18 328.30 30.78 TQ 1.38 1.24 3.26 0.52 0.53 12.24 11.83 87.52 329.90 34.99 ROE 6.11 12.28 61.00 655.22 55.30 2.30 1.53 74.66 0.43 6.02 UAE: observations = 184 Qatar: observations = 160 FSIZE 6.88 6.92 10.05 4.69 1.31 6.72 6.96 10.99 3.76 1.28 LIQ 681.93 25.61 2,1645.40 0.15 2,557.08 111.32 2.06 1,185.24 0.02 255.42 LEV 42.46 37.80 95.34 7.30 23.06 49.06 50.64 84.61 16.27 15.65 MVA (000) 62.828 0.009 3,900.00 3,200.00 935.30 297.94 0.002 12,000.00 3,200.00 1,943.31 ROA 9.03 6.59 44.32 3.24 8.29 7.68 6.12 47.03 17.11 8.31 ROCE 11.64 7.90 44.85 4.27 10.40 13.20 10.18 64.97 65.77 16.74 ROE 16.46 14.85 106.54 17.71 13.78 10.11 7.34 51.41 27.24 12.30 TQ 1.59 1.46 5.64 0.32 0.85 1.45 1.10 4.13 0.40 0.88 Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added PRR 6,2 132 Table 5. Unit root test Variable Stationary tests AL level Im, Pesaran and Shin W-stat ADF – Fisher Chi-square PP – Fisher Chi-square Result FSIZE 0.0000 0.0000 0.0000 Reject null hypothesis ROA 0.0250 0.0016 0.0000 Reject null hypothesis LIQ 0.0000 0.0000 0.0002 Reject null hypothesis ROCE 0.0000 0.0000 0.0000 Reject null hypothesis LEV 0.4590 0.0393 0.0001 Reject null hypothesis ROE 0.0000 0.0000 0.0000 Reject null hypothesis MVA 0.0000 0.0000 0.0000 Reject null hypothesis TQ 0.0000 0.0000 0.0724 Reject null hypothesis Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added positive correlation at 1% level of signiﬁcance with TQ, it has a signiﬁcant negative correlation with all other independent variables. 5.2 Unit root analysis All independent variables have a maximum correlation coefﬁcient of 0.58 (0.58 < 0.70), which indicates that multicollinearity issues in this study does not exist. 5.4 Regression estimation 5.4 Regression estimation g Table 7 presents an estimation of the results of the models of the current study. An estimation of ﬁxed effect model is opted. At the initial step of the analysis, a redundant ﬁxed Table 6. Correlation analysis Variables FSIZE LIQ LVR TOBINQ MVA ROA ROCE ROE FSIZE 1 LIQ 0.01* 1 LVR 0.12 0.01 1 TQ 0.11* 0.05* 0.28* 1 MVA 0.04 0.11* 0.03 0.33* 1 ROA 0.11* 0.07* 0.08 0.51* 0.23* 1 ROCE 0.02 0.04 0.06 0.23* 0.10* 0.58* 1 ROE 0.03 0.03 0.04 0.26*** 0.07* 0.45* 0.54* 1 Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added; *, and *indicate at 1, 5 and 10% level of signiﬁcance respectively Table 7. Fixed effect model Variables Coef. Std. err. T-value P-value Cons 189.06 63.95 2.96 0.003 ROA 5.07 0.83 6.08 0.000 ROE 0.22 0.14 1.59 0.111 ROCE 0.32 0.18 1.75 0.080 MVA 0.00 0.00 5.21 0.000 FSIZE 28.13 9.25 3.04 0.002 LIQ 0.00 0.01 0.47 0.639 TQ 669.48 38.38 17.44 0.000 Husamn test 0.000 R-seq. within 0.3575 R-seq. between 0.1273 R-seq. overall 0.1416 F (7,588) 46.7300 Prob > F 0.0000 Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added Gulf countries 133 Table 7. Fixed effect model Variables Coef. Std. err. T-value P-value Cons 189.06 63.95 2.96 0.003 ROA 5.07 0.83 6.08 0.000 ROE 0.22 0.14 1.59 0.111 ROCE 0.32 0.18 1.75 0.080 MVA 0.00 0.00 5.21 0.000 FSIZE 28.13 9.25 3.04 0.002 LIQ 0.00 0.01 0.47 0.639 TQ 669.48 38.38 17.44 0.000 Husamn test 0.000 R-seq. within 0.3575 R-seq. between 0.1273 R-seq. overall 0.1416 F (7,588) 46.7300 Prob > F 0.0000 Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added Table 7. 5.2 Unit root analysis This result is in line with (Bandyopadhyay and Barua, 2016; Benkraiem and Gurau, 2013; Güner, 2016; Rouf, 2015), who found a negative and signiﬁcant impact of size on ﬁrms’ leverage. These results do not agree with the ﬁndings of (Alipour et al., 2015; Chadha and Sharma, 2015; El-Khatib, 2017; Gomez et al., 2014; Kyissima et al., 2019; Matemilola et al., 2013; Morri, and Cristanziani, 2009; Sbeiti, 2010), who advocated that size of a ﬁrm has a positive and signiﬁcant impact on ﬁrm’s leverage. PRR 6,2 134 134 MVA and TQ exhibit statistically signiﬁcant effect on LEV at the level of 1% (P-value = 0.000 < 0.01). This effect is positive indicated by (b = 0.01 and 699.48, respectively). Further, the results reveal that ROCE has a signiﬁcant positive impact (b = 0.32). However, this effect is statistically signiﬁcant at the level 10% (P-value = 0.08 < 0.10). y g ( ) Overall, the model is ﬁt with a signiﬁcance level of less than 1% (P-value = 0.000 < 0.01). The adjusted R2 is 13% indicating that the variables included in the model contribute about 13% of the variability of LEV. Table 8 illustrates a comparison on the overall impact of all explanatory variables included in the models on the dependent variable. A country wise effect comparison estimation is made based on kingdom of Saudi Arabia (KSA). KSA is considered as the basis of the comparison and other countries are compared with KSA. The results show that both Oman and the UAE have a negative difference from KSA (b = 18.24, 17.88, respectively) however, Qatar has a positive difference from KSA (b = 2.82). This means that Qatar is better than KSA, Oman and the UAE but KSA is better than both UAE and Oman in terms of the overall impact of all explanatory variables on the dependent variable. However, the P- value of the three countries (Oman, the UAE and Qatar) are insigniﬁcant (P-value > 0.10) indicating that there are no signiﬁcant differences among the countries in terms of the overall impact of all explanatory variables included in the models on the dependent variable. Table 8. Country wise effect comparison estimation Variables Coef. Std. err. 5.2 Unit root analysis Fixed effect model Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added effect estimation is made to choose between the panel set or pooled regression analysis. The results of the redundant ﬁxed effect test shows that the panel set is appropriate than the pooled set. Furthermore, the results of the redundant ﬁxed effect shows that the estimation should be a one-way cross-section ﬁxed effect model. In addition, an analysis is conducted to compare the relationship between ﬁxed or random effect models. Accordingly, Husman test is conducted for this purpose and the yielded result of Husman test was in favor of ﬁxed effect model (P-value < 0.00), which means a rejection of random effect model and the acceptance of ﬁxed effect model. effect estimation is made to choose between the panel set or pooled regression analysis. The results of the redundant ﬁxed effect test shows that the panel set is appropriate than the pooled set. Furthermore, the results of the redundant ﬁxed effect shows that the estimation should be a one-way cross-section ﬁxed effect model. In addition, an analysis is conducted to compare the relationship between ﬁxed or random effect models. Accordingly, Husman test is conducted for this purpose and the yielded result of Husman test was in favor of ﬁxed effect model (P-value < 0.00), which means a rejection of random effect model and the acceptance of ﬁxed effect model. The results of regression analysis of ﬁxed effect model shows that ROA has a signiﬁcant negative effect on leverage at the level of 1% level of signiﬁcance (b = 5.07, P-value = 0.000 < 0.01). This negative effect indicates that LEV is signiﬁcantly inﬂuenced by ROA, this result is consistent with (Bandyopadhyay and Barua, 2016; El-Khatib, 2017; Gomez et al., 2014; Güner, 2016; Kyissima et al., 2019; Matemilola et al., 2013; Rouf, 2015; Sbeiti, 2010), who found a negative and signiﬁcant impact of proﬁtability on leverage. The results contradict those of (Alipour et al., 2015; Chadha and Sharma, 2015), who documented a positive impact of proﬁtability on leverage. FSIZE has statistically signiﬁcant negative effect on LEV. This effect is statistically signiﬁcant at the level of 1% (b = 28.13, P-value = 0.000 < 0.01). 5.2 Unit root analysis Table 9 presents the results of robust regression. The results show consistent outputs to the results of the ﬁxed effect model except in the case of liquidity. Coefﬁcient estimates of both ﬁxed and robust regressions are not highly deviated. All variables exhibit same effect except in case of ROE, which was insigniﬁcant in case of the random effect model but it is signiﬁcant in the robust regression. This signiﬁes a proper estimation of the regression assumptions. Further, the outputs of robust regression signify that the data is not contaminated with outliers. 5.2 Unit root analysis T-value P-value Cons 103.91 25.59 4.06 0.000 ROA 4.017 0.63 6.37 0.000 ROE 0.01 0.14 0.10 0.922 ROCE 0.16 0.19 0.88 0.379 MVA 0.01 4.15e 3.60 0.000 FSIZE 8.20 3.41 2.40 0.016 LIQ 0.00 0.00 0.51 0.609 TQS 306.18 25.48 12.02 0.000 R.seq. within 0.3176 R.seq. between 0.1713 R.seq. overall 0.1705 Wald x 2(10) 161.0600 Prob > F 0.000 Country wise comparison effect (KSA is the base of comparison) Oman 18.24 14.36508 1.27 0.204 UAE 17.88 13.73376 1.30 0.193 Qatar 2.82 13.98649 0.20 0.84 Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added comparison estimation Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added Table 9. Robust regression Variables Coef. Std. err. T-value P-value C 51.341 5.322 9.646 0.000 ROA 3.069 0.087 35.436 0.000 ROE 0.000 0.000 1.107 0.269 ROCE 0.137 0.031 4.376 0.000 MVA 1.500 0.023 66.353 0.000 FSIZE 0.881 0.466 1.891 0.059 LIQ 0.200 0.054 3.729 0.000 TOBIN_Q 2.816 0.227 12.390 0.000 R2 0.192 Adjusted R2 0.184 Rw-squared 0.472 Adjust Rw-squared 0.472 Akaike info criterion 1,001.933 Prob(Rn-squared stat.) 0.000 Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added Gulf countries 135 Table 9. Robust regression Notes: 1- LEV is leverage, ROE is return on equity, TQ is Tobin-Q, LIQ is liquidity, ROCE is return on capital employed, ROA is return on assets, FSIZE is ﬁrm size and MVA is market value added Table 9 presents the results of robust regression. The results show consistent outputs to the results of the ﬁxed effect model except in the case of liquidity. Coefﬁcient estimates of both ﬁxed and robust regressions are not highly deviated. All variables exhibit same effect except in case of ROE, which was insigniﬁcant in case of the random effect model but it is signiﬁcant in the robust regression. This signiﬁes a proper estimation of the regression assumptions. Further, the outputs of robust regression signify that the data is not contaminated with outliers. 6. Conclusion and policy implications The main objective of the present research was to investigate the impact of corporate characteristics on leverage. These characteristics include ROE, Tobin-Q (TQ), Liquidity (LIQ), ROCE, ROA, ﬁrm size (FSIZE) and MVA. The ﬁnding of the study revealed that both ROA and FSIZE have a signiﬁcant negative effect on leverage. However, MVA, ROCE and TQ exhibited statistically signiﬁcant positive effect on leverage. Further, the results indicated that Qatar is better than KSA, Oman and the UAE but KSA is better than both UAE and Oman in terms of the overall impact of corporate characteristics on the leverage. However, this effect in not statistically signiﬁcant. , y g Despite the effort made by prior studies to investigate the issue of corporate characteristics and its relationship with leverage, this study focused on the GCC context, which open an insight of different research implications. The study realizes that the GCC countries have some characteristics such as immature stock markets, tax systems and Sharīʿah ﬁnancing, which may have a signiﬁcant impact on ﬁnancing decisions of ﬁrms. This study calls upon managers, bankers, inventors, ﬁnancial analysts and regulators in the GCC stock markets to reconsider capital structure components and try to rebalance between debts and equities depending on corporate characteristics. In addition, future research in the GCC countries should investigate some other determinants such as country-level factors, stock market characteristics, Sharīʿah ﬁnancing and some areas of capital structure mix. This study contributes to knowledge by providing new insights into the corporate characteristics and leverage. By so doing, it provides an attempt to identify the factors inﬂuencing corporate ﬁnancing behavior taking into consideration different issues such as different proxies of ﬁrms’ proﬁtability, market capitalization, MVA and liquidity, which provides original evidence from Gulf countries emerging markets. These countries are characterized by low tax rates and high liquidity. High liquidity may reduce the cost of borrowing and debt ﬁnancing may not be a huge burden on ﬁrms’ proﬁts. This makes the investigation of leverage and corporate characteristics, particularly, ﬁrms’ proﬁtability and liquidity, very important. Therefore, the study tries to bridge an existing gap in the body of literature of capital structure and debt ﬁnancing in Gulf countries emerging markets. The ﬁndings of this study will enable regulatory agencies to aim toward greater compliance with the local and international standards and will also enable them to enforce penalties for non-compliance. 6. Conclusion and policy implications The limitation of the study is that only non-ﬁnancial companies have been used as a sample. Hence, the results may not extend across all listed companies in Gulf countries. 136 References 3-14, doi: 10.1108/JAMR- 08-2014-0051. Gulf countries Champion, D. (1999), “Finance: the joy of leverage”, Harvard Business Review, Vol. 77 No. 4, pp. 19-22 Chen, J. and Strange, R. (2005), “The determinants of capital structure: evidence from Chinese listed companies”, Economic Change and Restructuring, Vol. 38 No. 1, pp. 11-35, doi: 10.1007/s10644- 005-4521-7. 137 Correa, C.A. Basso, L.F.C. and Nakamura, W.T. (2007), “What determines the capital structure of the largest Brazilian ﬁrms? An empirical analysis using panel data”, available at: https://ssrn.com/ abstract=989047 Crnigoj, M. and Mramor, D. (2009), “Determinants of capital structure in emerging European economies: evidence from Slovenian ﬁrms”, Emerging Markets Finance And Trade, Vol. 45 No. 1, pp. 72-89, doi: 10.2753/REE1540-496X450105. El-Khatib, R. (2017), “Determinants of corporate leverage in publicly listed GCC companies – conventional versus Sukuk”, Global Corporate Governance (Advances in Financial Economics), pp. 77-102, doi: 10.1108/S1569-373220160000019004. Ellaboudy, S. (2010), “The global ﬁnancial crisis: economic impact on GCC countries and policy implications”, International Research Journal of Finance and Economics, Vol. 41, pp. 180-193. Fernandez, V. (2005), “Determinants of ﬁrm leverage in Chile: evidence from panel data”, Estudios de Administracion, Vol. 12 No. 1, pp. 41-85, doi: 10.5354/0719-0816.2020.56455. Ghosh, C., Nag, R. and Sirmans, C.F. (2000), “The pricing of seasoned equity offerings: evidence from REITs”, Real Estate Economics, Vol. 28 No. 3, pp. 363-384, doi: 10.1111/1540-6229.00805. Gomez, G., Rivas, A.M. and Bolaños, E.R.L. (2014), “The determinants of capital structure in Peru”, Academia Revista Latinoamericana de Administracion, Vol. 27 No. 3, pp. 341-354, doi: 10.1108/ ARLA-01-2014-0007. Güner, A. (2016), “The determinants of capital structure decisions”, Procedia Economics and Finance, Vol. 38, pp. 84-89, doi: 10.1016/S2212-5671(16)30180-0. Hadlock, C. and James, C. (2002), “Do banks provide ﬁnancial slack?”, The Journal of Finance, Vol. 57 No. 3, pp. 1383-1420, doi: 10.1111/1540-6261.00464. Hamid, M.A., Abdullah, A. and Kamaruzzaman, N.A. (2015), “Capital structure and proﬁtability in family and non-family ﬁrms: Malaysian evidence”, Procedia Economics and Finance, Vol. 31, pp. 44-55. Hirshleifer, J. (1958), “On the theory of optimal investment decision”, Journal of Political Economy Vol. 66 No. 4, pp. 329-352, doi: 10.1086/258057. Imberman, S.A. and Lovenheim, M.F. (2016), “Does the market value value-added? Evidence from housing prices after a public release of school and teacher value-added”, Journal of Urban Economics, Vol. 91, pp. 104-121, doi: 10.1016/j.jue.2015.06.001. Jaisinghani, D., Batra, D.K. and Tandon, D. References Ahmed, N., Ahmed, Z. and Ahmed, I. (2010), “Determinants of Capital structure: a case of life insurance sector of Pakistan. European journal of economics”, Finance and Administrative Sciences, Vol. 24 No. 24, pp. 7-12. Al-Ahdal, W.M., Alsamhi, M.H., Tabash, M.I. and Farhan, N.H.S. (2020), “The impact of corporate governance on ﬁnancial performance of Indian and GCC listed ﬁrms: an empirical investigation”, Research in International Business and Finance, Vol. 51, pp. 1-13, doi: 10.1016/j. ribaf.2019.101083. Alipour, M., Mohammadi, M.F.S. and Derakhshan, H. (2015), “Determinants of capital structure: an empirical study of ﬁrms in Iran”, International Journal of Law and Management, Vol. 57 No. 1, pp. 53-83, doi: 10.1108/IJLMA-01-2013-0004. Almaqtari, F.A., Al-Homaidi, E.A., Tabash, M.I. and Farhan, N.H. (2019), “The determinants of proﬁtability of Indian commercial banks: a panel data approach”, International Journal of Finance and Economics, Vol. 24 No. 1, pp. 168-185, doi: 10.1002/ijfe.1655. Al-Matari, E.M., Al-Swidi, A.K. and Fadzil, F.H.B. (2014), “The measurements of ﬁrm performance’s dimensions”, Asian Journal of Finance and Accounting, Vol. 6 No. 1, pp. 24-49, doi: 10.5296/ajfa. v6i1.4761. Almutairi, H. (2014), “Competitive advantage through taxation in GCC countries”, International Business and Economics Research Journal, Vol. 13 No. 4, pp. 769-778. Bandyopadhyay, A. and Barua, N.M. (2016), “Factors determining capital structure and corporate performance in India: studying the business cycle effects”, The Quarterly Review of Economics and Finance, Vol. 61, pp. 160-172, doi: 10.1016/j.qref.2016.01.004. Bauer, P. (2004), “Determinants of capital structure: empirical evidence from the Czech Republic”, Cz Journal of Economics and Finance, Vol. 54 Nos 1/2, pp. 2-21. Benkraiem, R. and Gurau, C. (2013), “How do corporate characteristics affect capital structure decisions of French SMEs?”, International Journal of Entrepreneurial Behavior and Research, Vol. 19 No. 2, pp. 149-164, doi: 10.1108/13552551311310356. Bhaduri, S.N. (2002), “Determinants of capital structure choice: a study of the Indian corporate sector”, Applied Financial Economics, Vol. 12 No. 9, pp. 655-665, doi: 10.1080/09603100010017705. Brounen, D. and Eichholtz, P.M. (2001), “Capital structure theory: evidence from European property companies’ capital offerings”, Real Estate Economics, Vol. 29 No. 4, pp. 615-632, doi: 10.1111/ 1080-8620.00025. Cappa, F., Cetrini, G. and Oriani, R. (2020), “The impact of corporate strategy on capital structure: evidence from Italian listed ﬁrms”, The Quarterly Review of Economics and Finance, Vol. 76, pp. 379-385, doi: 10.1016/j.qref.2019.09.005. Chadha, S. and Sharma, A.K. (2015), “Determinants of capital structure: an empirical evaluation from India”, Journal of Advances in Management Research, Vol. 12 No. 1, pp. References (2017), “Capital expenditure and persistence of ﬁrm performance: an empirical study for the Indian automobiles industry”, International Journal of Indian Culture and Business Management, Vol. 16 No. 1, pp. 39-54, doi: 10.1504/ ijicbm.2018.10009212. Jensen, M.C. and Meckling, W.H. (1976), “Theory of the ﬁrm: managerial behavior, agency costs and ownership structure”, Journal of Financial Economics, Vol. 3 No. 4, pp. 305-360, doi: 10.1016/ 0304-405X(76)90026-X. Jong, A., Kabir, M.R. and Nguyen, T.T. (2008), “Capital structure around the world: the roles of ﬁrm- and country-speciﬁc determinants”, Journal of Banking & Finance, Vol. 32 No. 9, pp. 1954-1969, available at: https://doi. org/10.1016/j.jbankﬁn.2007.12.034 Khan, K., Qu, J., Shah, M.H., Bah, K. and Khan, I.U. (2020), “Do ﬁrm characteristics determine capital structure of Pakistan listed ﬁrms? A quantile regression approach”, The Journal of Asian Finance, Economics and Business, Vol. 7 No. 5, pp. 61-72, doi: 10.13106/jafeb.2020.vol7. no5.061. PRR 6,2 Kim, H. and Berger, P.D. (2008), “A comparison of capital structure determinants: the United States and the Republic Of Korea”, Multinational Business Review, Vol. 16 No. 1, pp. 79-100, doi: 10.1108/ 1525383X200800004. 138 Kim, H., Heshmati, A. and Aoun, D. (2006), “Dynamics of capital structure: the case of Korean listed manufacturing companies”, Asian Economic Journal, Vol. 20 No. 3, pp. 275-302, doi: 10.1111/ j.1467-8381.2006.00236.x. KPMG (2016), “GCC listed Banks results”, available at: https://assets.kpmg/content/dam/kpmg/qa/pdf/ qa-gcc-listed-banks-results.pdf (accessed 17 November 2019). Kyissima, K.H., Xue, G.Z., Yapatake Kossele, T.P. and Abeid, A.R. (2019), “Analysis of capital structure stability of listed ﬁrms in China”, China Finance Review International, Vol. 10 No. 2, pp. 213-228, doi: 10.1108/CFRI-05-2018-0044. Li, L. and Islam, S.Z. (2019), “Firm and industry speciﬁc determinants of capital structure: evidence from the Australian market”, International Review of Economics and Finance, Vol. 59, pp. 425-437, doi: 10.1016/j.iref.2018.10.007. Lintner, J. (1956), “Distribution of incomes of corporations among dividends, retained earnings, and taxes”, The American Economic Review, Vol. 46 No. 2, pp. 97-113. Margaritis, D. and Psillaki, M. (2010), “Capital structure, equity ownership and ﬁrm performance”, Journal of Banking and Finance, Vol. 34 No. 3, pp. 621-632, doi: 10.1016/j.jbankﬁn.2009.08.023. Matemilola, B.T., Bany-Arifﬁn, A.N. and B. McGowan, C. (2013), “Unobservable effects and ﬁrm’s capital structure determinants”, Managerial Finance, Vol. 39 No. 12, pp. 1124-1137, doi: 10.1108/ MF-08-2012-0187. Mazur, K. (2007), “The determinants of capital structure choice: evidence from polish companies”, International Advances in Economic Research, Vol. 13 No. 4, pp. 495-514, doi: 10.1007/s11294- 007-9114-y. Miguel, A. and Pindado, J. References (2001), “Determinants of capital structure: new evidence from Spanish panel data”, Journal of Corporate Finance, Vol. 7 No. 1, pp. 77-99, doi: 10.1016/S0929-1199(00) 00020-1. Miller, M.H. (1977), “Debt and taxes”, The Journal of Finance, Vol. 32, pp. 261-276. Modigliani, F. and Miller, M. (1958), “The cost of capital, corporation ﬁnance and the theory of investment”, The American Economic Review, Vol. 48 No. 3, pp. 261-297, available at: www.jstor. org/stable/1809766 (accessed 25 August 2020). Modigliani, F. and Miller, M. (1963), “Corporate income taxes and the cost of capital: a correction”, The American Economic Review, Vol. 53 No. 3, pp. 433-443, available at: www. jstor.org/stable/ 1809167 (accessed 25 August 2020) Morri, G. and Cristanziani, F. (2009), “What determines the capital structure of real estate companies?”, Journal of Property Investment and Finance, Vol. 27 No. 4, pp. 318-372, doi: 10.1108/ 14635780910972288. Muritala, T. (2012), “An empirical analysis of capital structure on ﬁrms’ performance in Nigeria”, International Journal of Advances in Management and Economics, Vol. 1 No. 5, pp. 116-124. Myers, S.C. (1977), “Determinants of corporate borrowing”, Journal of Financial Economics, Vol. 5 No. 2, pp. 147-175, doi: 10.1016/0304-405X(77)90015-0. Niu, X. (2008), “Theoretical and practical review of capital structure and its determinants”, International Journal of Business and Management, Vol. 3 No. 3, pp. 133-139, doi: 10.5539/ijbm. v3n3p133. Ojo, A.S. (2012), “The effect of ﬁnancial leverage on corporate performance of some selected companies in Nigeria”, Canadian Social Science, Vol. 8 No. 1, pp. 85-91, doi: 10.3968/j. css.1923669720120801.700. Gulf countries Omet, G. (2006), “Ownership structure and capital structure: evidence from the Jordanian capital market (1995-2003)”, Corporate Ownership and Control, Vol. 3 No. 4, pp. 99-107, doi: 10.22495/ cocv3i4p8. Pratheepkanth, P. (2011), “Capital structure and ﬁnancial performance: evidence from selected business companies in Colombo stock exchange Sri Lanka”, Journal of Arts, Science and Commerce, Vol. 2 No. 2, pp. 1-13. 139 Rehman, S.S.F.U. (2013), “Relationship between ﬁnancial leverage and ﬁnancial performance: empirical evidence of listed sugar companies of Pakistan”, Global Journal of Management and Business Research, Vol. 13 No. 8, pp. 33-40. Rouf, D. (2015), “Capital structure and ﬁrm performance of listed non-ﬁnancial companies in Bangladesh”, The International Journal of Applied Economics and Finance, Vol. 9 No. 1, pp. 25-32. Salawu, R.O. and Agboola, A.A. (2008), “The determinants of capital structure of large non-ﬁnancial listed ﬁrms in Nigeria”, The International Journal of Business and Finance Research, Vol. 2 No. 2, pp. 75-84. Salim, M. and Yadav, R. References (2012), “Capital structure and ﬁrm performance: evidence from Malaysian listed companies”, Procedia – Social and Behavioral Sciences, Vol. 65, pp. 156-166, doi: 10.1016/j. sbspro.2012.11.105. San, O.T. and Heng, T.B. (2011), “Capital structure and corporate performance of Malaysian construction sector”, International Journal of Humanities and Social Science, Vol. 1 No. 2, pp. 28-36. Sbeiti, W. (2010), “The determinants of capital structure: evidence from the GCC countries”, International Research Journal of Finance and Economics, Vol. 47 No. 2, pp. 56-82, doi: 10.2478/ remav-2019-0019. Shah, A. and Khan, S. (2007), “Determinants of capital structure: evidence from Pakistani panel data” International Review of Business Research Papers, Vol. 3 No. 4, pp. 265-282. Sharif, B., Naeem, M.A. and Khan, A.J. (2012), “Firms characteristics and capital structure: a panel data analysis of Pakistans insurance sector”, African Journal of Business Management, Vol. 6 No. 14, pp. 4939-4947. Sofat, R. and Singh, S. (2017), “Determinants of capital structure: an empirical study of manufacturing ﬁrms in India”, International Journal of Law and Management, Vol. 59 No. 6, pp. 1029-1045, doi: 10.1108/IJLMA-05-2016-0051. Soumadi, M. and Hayajneh, O. (2011), “Capital structure and corporate performance: empirical study on the public Jordanian shareholdings ﬁrms listed in the Amman stock market”, European Scientiﬁc Journal, Vol. 8 No. 22, pp. 173-189. The World Bank (2015), GDP Growth (Annual %), available at: https://data.worldbank.org/indicator/ NY.GDP.MKTP.KD.ZG?page=3 (accessed 17 November 2019). Titman, S. and Wessels, R. (1988), “The determinant of capital structure choice”, The Journal of Finance, Vol. 43 No. 1, pp. 1-19, doi: 10.1111/j.1540-6261.1988.tb02585.x. Twairesh, A.E.M. (2014), “The impact of capital structure on ﬁrm’s performance evidence from Saudi Arabia”, Journal of Applied Finance and Banking, Vol. 4 No. 2, pp. 183-193. Vasiliou, D. and Daskalakis, N. (2009), “Institutional characteristics and capital structure: a cross-national comparison”, Global Finance Journal, Vol. 19 No. 3, pp. 286-306, doi: 10.1016/j.gfj.2008.09.002. Woertz, E. (2012), “GCC ﬁnancial markets: the world’s new money centers”, Gerlach Press, available at: www.jstor.org/stable/j.ctt1s474r3 (accessed 24 August 2020). Yameen, M., Farhan, N.H. and Tabash, M.I. (2019), “The impact of corporate governance practices on ﬁrm’s performance: an empirical evidence from Indian tourism sector”, Journal of International Studies, Vol. 12 No. 1, pp. 208-228, doi: 10.14254/2071-8330.2019/12-1/14. PRR 6,2 Yildirim, R., Masih, M. and Bacha, O.I. (2018), “Determinants of capital structure: evidence from Shari’ah compliant and non-compliant ﬁrms”, Paciﬁc-Basin Finance Journal, Vol. 51, pp. 198-219, doi: 10.1016/j.pacﬁn.2018.06.008. Zeitun, R. and Saleh, A.S. References (2015), “Dynamic performance, ﬁnancial leverage and ﬁnancial crisis: evidence from GCC countries”, EuroMed Journal of Business, Vol. 10 No. 2, pp. 147-162, doi: 10.1108/EMJB-08-2014-0022. 140 140 Zeitun, R. and Tian, G. (2007), “Capital structure and corporate performance: evidence from Jordan”, Australasian Accounting, Business and Finance Journal, Vol. 1 No. 4, pp. 40-61, doi: 10.14453/ aabfj.v1i4.3. Further reading Akbiyikli, R., Eaton, D. and Turner, A. (2006), “Project ﬁnance and the private ﬁnance initiative (PFI)”, The Journal of Structured Finance, Vol. 12 No. 2, pp. 67-75. 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 Corresponding author i A Al i b Corresponding author Faozi A. Almaqtari can be contacted at: fouzi_gazim2005@yahoo.com p g Faozi A. Almaqtari can be contacted at: fouzi_gazim2005@yahoo.com 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
https://openalex.org/W2778960380	https://bioone.org/journals/mountain-research-and-development/volume-37/issue-4/MRD-JOURNAL-D-17-00030.1/Social-and-Labor-Integration-of-Asylum-Seekers-in-Rural-Mountain/10.1659/MRD-JOURNAL-D-17-00030.1.pdf	English	null	Social and Labor Integration of Asylum Seekers in Rural Mountain Areas—A Qualitative Study	Mountain research and development	2,017	cc-by	6,534	Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Social and Labor Integration of Asylum Seekers in Rural Mountain Areas—A Qualitative Study Authors: Marcher, Anja, Kofler, Ingrid, and Streifeneder, Thomas Philipp Source: Mountain Research and Development, 37(4) : 388-395 Published By: International Mountain Society URL: https://doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. 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MountainDevelopment Transformation knowledge MountainDevelopment Transformation knowledge Mountain Research and Development (MRD) Mountain Research and Development (MRD) An international, peer-reviewed open access journal published by the International Mountain Society (IMS) www.mrd-journal.org Social and Labor Integration of Asylum Seekers in Rural Mountain Areas—A Qualitative Study Anja Marcher, Ingrid Kofler, and Thomas Philipp Streifeneder Corresponding author: anja.marcher@eurac.edu Eurac Research, Viale Druso 1, 39100 Bolzano, Italy 2017 Marcher et al. This open access article is licensed under a Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/). Please credit the authors and the full source. Anja Marcher, Ingrid Kofler, and Thomas Philipp Streifeneder Corresponding author: anja.marcher@eurac.edu Eurac Research, Viale Druso 1, 39100 Bolzano, Italy Eurac Research, Viale Druso 1, 39100 Bolzano, Italy 2017 Marcher et al. This open access article is licensed under a Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/). Please credit the authors and the full source. 2017 Marcher et al. This open access article is licensed under a Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/). Please credit the authors and the full source. Tyrol) in which reception facilities for asylum seekers have been established. A qualitative research approach offered empirical insights into the functioning of the reception system and governance in these communities. Our social network analysis of the research data, focusing on the labor integration of asylum seekers, indicated that stronger relational linkages among actors in rural mountain communities may facilitate access to the labor market for asylum applicants. Migration into Europe affects the Alps in various ways. The recent influx of refugees and a higher number of asylum requests has presented governance challenges for mountain communities. In Italy, the responsibility of regions to host asylum seekers increased when a national system was implemented to distribute asylum seekers throughout the country. This study explored the impact of current distributions through the analysis of 2 rural mountain municipalities in the northeast Italian Alps in the Autonomous Province of Bolzano (also known as South Keywords: Asylum seeker; labor integration; refugee; mountain area; social network analysis; South Tyrol; Italy. Peer-reviewed: June 2017 Accepted: October 2017 structure. Under the new system, rural regions that had been of little interest to migrants and had barely been affected by the migrant crisis—including remote areas in the Italian Alps that have experienced depopulation (Membretti and Dematteis 2016)—have begun to host migrants and asylum seekers and thus confront issues relating to migration, integration, and inclusion. Introduction Ongoing refugee and migration movements are among the governance-related challenges of our time. According to the Ofﬁce of the United Nations High Commissioner for Refugees (UNHCR 2016: 2), in 2015, about 65.3 million people ﬂed conﬂicts, war, prosecution, and/or human rights violations. Only a comparably small share of these forcibly displaced people came to Europe—about 4.4 million refugees in 2015. In that year, ‘‘Europe witnessed a dramatic increase in the number of refugees and migrants arriving by the sea’’ (UNHCR 2016: 7). More than 1 million people entered the European Union from the Mediterranean Sea. The central Mediterranean route via Italy is one of the main routes to Europe, especially for African migrants. In 2015, about 153,800 people came across that route. One year later, the number had risen to 181,400 (UNHCR 2016, 2017c). Italy and Greece have Europe’s highest inﬂow of irregular migrants, some of whom are in transit to central or northern European countries while others are willing to stay in the country of arrival. g g g This study explored the experiences of 2 rural communities in the Italian Alps (in the Autonomous Province of Bolzano, also known as South Tyrol) to which asylum seekers have recently been assigned. It focused on labor integration, which is an essential factor in personal autonomy and independence (Konle-Seidl and Bolits 2016). Because most refugees are likely to stay in the host country for a long time (Worbs and Bund 2016), early access to the labor market, including during the process of application for asylum, can be crucial. We sought to understand how these communities are dealing with the new situation and how they organize the labor integration process for the newcomers. 388 Mountain Research and Development Vol 37 No 4 Nov 2017: 388–395 Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Theoretical background Because of these growing numbers, Italy decided to ease the burden on migrant destinations in the southern part of the country and to pursue a more balanced distribution of asylum seekers within its territory (Italian Ministry of the Interior 2017). By means of a national distribution formula, based on each province’s portion of the country’s total population, the applicants were distributed nationally through a decentralized reception This investigation focused on international migrants with a low degree of voluntariness to migrate who are seeking asylum. We use the United Nations Educational, Scientiﬁc and Cultural Organization’s deﬁnition of asylum seeker as a person who ‘‘move[s] across borders in search of protection’’ nd ‘‘h s pplied for protection s ref gee protection’’ and ‘‘has applied for protection as a refugee http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 MountainDevelopment MountainDevelopment and is awaiting the determination of his or her status’’ (UNESCO 2017). Asylum seekers can therefore become refugees with a protected status. This deﬁnition is in turn based on the 1951 Geneva Convention on Refugees, which deﬁnes the criteria to qualify for protection, the rights of the displaced, and the legal obligations of the receiving countries (Hillmann 2016). The deﬁnition focuses on ‘‘the protection of persons from political or other forms of persecution’’ (UNHCR 2017a). Italy ratiﬁed the 1951 Convention, but only after many years. Through the so- called Martelli Act in 1990 (Gazzetta ufﬁciale 2017), the ﬁrst speciﬁc regulations regarding asylum procedures and the reception of third-country nationals and stateless people emerged (UNHCR 2017b). According to Petrovic (2013), the Italian state has issued many decrees in recent years, but mostly as a reaction to emergencies or to increasing European pressure. Italy still has no comprehensive asylum policy. to other foreign migrants who can choose where to settle, rarely know anyone in their new community (Cheung and Phillimore 2013: 10) and must rely on its receptiveness. Research has shown that social capital and social networks can have a positive effect on the integration of migrants and on labor integration in particular (Portes 1995; Chou and Chow 2009: 341; Cheung and Phillimore 2013; Morosanu 2016). By assigning asylum seekers to smaller communities and rural mountain areas with denser social networks, it may be possible to manage integration more easily and in a more targeted way. However, this could involve governance challenges because of the different institutional levels involved (Benz 2007; Wald and Jansen 2007). Choice of study area We selected 2 communities in the northeast Italian Alps in the Autonomous Province of Bolzano, also known as South Tyrol, for our case studies. We selected South Tyrol because of its autonomous status as an Italian province and its opportunities to shape the process of integration through different strategies. South Tyrol is an example of local autonomy in the European context. Its autonomy is anchored in an international treaty, the Degasperi-Gruber Pact of 1946, and in the Italian Constitution; this status was revised in 1972. This province qualiﬁes as a predominantly rural area under both the Organisation for Economic Co-operation and Development (OECD) rural–urban typology (Eurostat 2017) and Italian Law 991/1952, which classiﬁes all South Tyrolian municipalities as mountain areas (ISTAT 2015). The province’s autonomous status gives it legislative competence to offer a range of integration strategies. The region established an integration act in 2011 and new guidelines for integration policies in 2016, which among other things, deﬁne the role of municipalities (Mitterhofer et al 2016). National and regional regulations also provide a scope for municipalities to contribute to the integration of new arrivals. Sørensen (2016: 392) argued that social capital is different in rural and urban contexts, with bonding social capital higher in rural areas, because of ‘‘localized trust, [the] rate of passive and active participation in local civic associations, and. . .local reciprocity.’’ Social capital can be seen as the actual or potential resources arising from a network of relationships at either the personal level (Bourdieu 2012) or the collective level (Putnam et al 1994). It can promote bonding within a group, as well as bridging from one group to another (Putnam 2001). By considering integration a long-term process, smaller villages may therefore offer more possibilities for new population groups, where networks and cooperation among actors (associations and organizations inside and outside the village) are crucial for the integration of asylum seekers. Social interrelations and networks in a small, deﬁned space could be easier to manage and could consequently encourage contact between the newcomers and the receiving society. Refugees and asylum seekers, in contrast South Tyrol is required to accommodate at least 0.9% of the people who are searching for protection in Italy. Applicants are ﬁrst registered in the provincial capital and then accommodated in a reception facility within the province; these are considered their ﬁrst and second receptions (APB-ST and Eurac Research 2017). 389 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Theoretical background We investigated, in the 2 study areas, the inﬂuence of social networks and of individual and organizational actors on the labor integration of the asylum seekers living in the established reception centers. Our primary research question was: How do social relations and the governance of labor integration inﬂuence asylum seekers’ access to the (local) labor market? p y p y Italy’s distribution of asylum seekers was intended to prevent urban concentrations. In contrast to urban areas, mountain and rural areas are often interpreted as places where time and development seem to be slower. Members of communities in such areas often know one another, share experiences, and live together in a limited territorial space, which promotes a certain type of solidarity and identity (Sørensen 2016). Because of their poor accessibility, mountain areas are also characterized by small villages with a speciﬁc social structure, which makes integration and migration processes different from those in urban areas (L€ofﬂer et al 2016; Membretti and Dematteis 2016). Haller (2016) assumes that the social structure and mentality of the Alpine region differ from those of other regions for 3 reasons: the importance of agriculture and strength of people’s attachment to the soil and to tradition, the economic structure (less accessibility and the predominance of small and medium-size enterprises in the artisanal, tourism, and service sectors), and the establishment of political autonomy. http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 Choice of study area The numbers of asylum seekers in South Tyrol increased from 137 in 2012 to about 1700 in 2017 (APB-ST 2012; APB-ST http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 MountainDevelopment MountainDevelopment TABLE 1 General characteristics of the case-study villages. (Source: ISTAT 2017) TABLE 1 General characteristics of the case-study villages. (Source: ISTAT 2017) Demographics CS1 CS2 Population in 2015 .5000 ,5000 Non-Italian citizens 6.1% 8.1% Non-EU citizens 2.6% 5.7% Natural population growth Positive Negative Net immigration Balanced Positive Dominant economic sectors in 2011a) Manufacturing 8.1% 29.2% Construction 13.5% 6.3% Wholesale and retail trade and repair of motor vehicles and motorcycles 18.0% 19.0% Accommodation and food service 24.3% 29.2% No. of farmers in 2010b) .350 ,60 a) Italian Census of Employment 2011. b) Agricultural Census 2010. reception centers, as well as secondary data from the regional statistical institute (ASTAT 2017). Central actors or groups of actors and their relationships were analyzed using interpretive and exploratory techniques. The qualitative data (interviews, protocols, ﬁeld notes, and verbal remarks) allowed conclusions to be drawn regarding the context of action, linkages of meaning, and shared references (Hollstein 2010; Fuhse 2016). Subsequently, a network matrix was created to conduct a social network analysis of the local governance structure (Benz et al 2007). The special feature of network analysis is that it is based on relational data (ie on different relationships [ties] among actors or groups of actors [nodes] and to a lesser extent on the actors’ characteristics). The network is measured through the relationships and dynamics between 2 actors (dyads), 3 actors (triads), cliques, or groups (Wasserman and Faust 1994; Jansen 2006). These analyses gave us additional insight into the relations among key actors and groups of actors, because it is based on the principle of interdependency (Rank 2015), meaning that there exists a kind of reciprocity and exchange, as well as indirect connections, among the related actors. Using this technique, we focused on relations among actors at the different governance levels of each village. 2017). This increase led to the creation of new reception centers in rural mountain areas. g g We used UCINET software (Borgatti et al 2013) for analysis and NetDraw for visualization (Borgatti 2002). One way to assess networks is based on their density, which is measured as the existing number of ties divided by the total number of all possible network ties and ranges between 0 (no relationships) and 1 (maximum possible relationships). Data gathering and analysis The data were anonymized, and the communities (Table 1) are referred to here as case study (CS) 1 and 2. We used a mix of methods to capture the complexity of the topic (Bohnsack et al 2006; Flick 2008), using a participatory approach (Gerring 2007; Bergold and Thomas 2012) for data collection within the scope of a project, with both municipalities aiming to identify the communities’ speciﬁc needs for increasing support for helping asylum seekers to enter the labor market. The mix of methods made it possible to consider different aspects of the research target during an observation period of about 1 year and provided additional insights. We conducted 5 semistructured key-informant interviews regarding the reception of asylum seekers at the local level, documented face-to-face interactions and project meetings between researchers and municipal actors, and collected demographic data on the asylum seekers from the 390 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Choice of study area Another important parameter is centrality, which can be measured in different ways. We focused on 2 measures: (1) the average degree, which is the average number of relations that each node (actor) has with other actors of the network, and (2) the betweenness centrality, which is based on the number of shortest paths that one actor has to get to another actor (direct and indirect relations); betweenness centrality measures broker positions within the network (Jansen 2006; Fuhse 2016). The 2 case-study communities were selected based on (1) their status as rural mountain communities under Italian and OECD standards, (2) a high share of people working in tourism and agriculture (ISTAT 2017), (3) their level of experience with reception facilities and asylum seekers, and (4) a higher (or lower) number of asylum applicants hosted in comparison to the number within South Tyrolian rural reception facilities. Both communities are more than 30 km from the closest urban center and are situated about 1000 m above sea level. http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 Two South Tyrolian mountain villages in comparison In 2015 and 2016, both municipalities opened reception facilities for asylum seekers. Since autumn 2015, CS1’s reception center has been hosting 50 people, and since the beginning of 2016, 25 asylum applicants have been living in CS2. Facility records indicate that, as of April 2017, of these 75 asylum seekers, only a few had received a ﬁnal decision on their applications for protection (19 had been granted a protected status, 14 were still awaiting a decision, and 42 were appealing a decision); nobody had left the facilities. According to facility records, residents were mostly (82%) male; almost 60% were younger than 26 (5% were unaccompanied minors), and 97% were from http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 TABLE 2 Work experience gained by asylum seekers during their application process.a) TABLE 2 Work experience gained by asylum seekers during their application process.a) CS1 CS2 Statistics In April 2017, of 50 asylum seekers, 43 had gained work experience during their stay and 19 were working. In March 2017, of 25 asylum seekers, 24 had gained work experience during their stay (no information provided for current working status). Main work areas Agriculture (seasonal harvester, gardener) Crafts (carpenter, welder) Industry (factory worker) Tourism (kitchen assistant, cleaner, barkeeper) Voluntary work for the municipality Agriculture (seasonal harvester, gardener) Social services (babysitter, companionship for disabled people) Tourism (kitchen assistant, cleaner, dishwasher, receptionist) Voluntary work for the municipality and a retirement home a) Data sources are the respective reception centers. a) Data sources are the respective reception centers. end, they were able to gain their initial work experience in the host country and demonstrate that ‘‘they are hardworking. . .and. . .able to work’’ (social adviser, CS1). It was important for the 2 highly interconnected communities to get to know the new residents personally. sub-Saharan Africa, with English or French as their main language. Residents had a limited education (average number of school years was estimated at 6 years for CS1 and 2 years for CS2), and their prior work experience was mainly in agriculture and crafts. y g The 2 study communities had various differences and similarities. First, having or not having prior experiences with past reception facilities and asylum seekers leads to the assumption that they may be enriching for the local community. Two South Tyrolian mountain villages in comparison CS1 has had such experiences since the 1990s, when it hosted hundreds of asylum seekers from the former Yugoslavia, whereas this was CS2’s ﬁrst such experience. The 2 host communities had different reactions to the experience. For CS1, it was a chance to develop further knowledge and experiences. The community followed an open communication strategy, backed by a high level of residents’ trust in the experienced people in charge. A social adviser said, ‘‘The experience in the 1990s showed that this challenge can be mastered. . .. The community trusts us.’’ A reception center coordinator said, ‘‘There was a huge receptiveness among the population. The whole region is open-minded and receptive.’’ In contrast, the establishment of the reception center in CS2 led to severe polarization, with mistrust exacerbated because of provincial authorities’ lack of consultation with the local community. An integration adviser said, ‘‘There were big differences within the population, positive and negative reactions; especially some small right-wing extremist groups were against it.’’ But, the mayor said, ‘‘overall the reception was a positive experience, the community is more open-minded than before.’’ g p y The 2 study villages faced similar problems with employment—for example, seasonal job offers in agriculture and tourism were predominant, mostly because for ‘‘short-term work there are fewer barriers for the employers to hire asylum seekers’’ (center coordinator, CS2). Another barrier to hiring asylum seekers were the legally required courses for work safety, which are obligatory in Italy and are normally paid by the employer. To lower employers’ hiring costs and thus increase asylum seekers’ chances of getting a job, the NGO offered these courses to the asylum seekers. Most job opportunities for asylum seekers were identiﬁed by the volunteers or through direct connections among other key local actors, such as the social advisers’ ties to local enterprises: ‘‘They found jobs most of all through the volunteers. Without them we wouldn’t be in such good position as we are now’’ (social adviser, CS1). This shows the importance of personal ties within the rural communities. Table 2 summarizes asylum applicants’ work experiences, both paid and volunteer. p p Focusing on the labor integration of the present asylum seekers, we identiﬁed 17 actors or groups of actors in CS1 and 18 in CS2, working at 3 administrative levels: municipal, intermunicipal, and provincial (Table 3). 391 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use Two South Tyrolian mountain villages in comparison Actor Institutional level Comments Autonomous Province of Bolzano Provincial Responsible for assigning asylum seekers to reception facilities Police Provincial NGO Provincial Responsible for the first and second receptions at the provincial level; commissioned by the provincial government Social services office Intermunicipal Employment office Intermunicipal Local offices commissioned by the provincial department of labor Schools Intermunicipal District government Intermunicipal Provides centralized services for a group of municipalities Trade associations Intermunicipal Enterprises Municipal, intermunicipal Potential and actual employers of asylum seekers Social adviser Municipal Responsible for health, housing, the elderly, education and integration; sits on the municipal council; collaborates with the reception center; communicates with the local population Integration adviser Municipal Responsible for integration; sits on the municipal council; collaborates with the reception center; communicates with the local population (CS2 only) Mayor Municipal Communicates with the local population Other municipal departments Municipal Center coordinator Municipal Responsible for the reception facility; employee of the commissioned NGO; collaborates with key municipal actors Center staff Municipal Employees of the commissioned NGO Volunteers Municipal Help search for jobs for the asylum seekers; offer language courses Local population Municipal Local associations Municipal Support the job search integration adviser, and the mayor—which means a more balanced distribution of responsibilities. In contrast, the social adviser in CS1 has both a high number of ties and an overload of responsibilities. Furthermore, these key actors emphasized a lack of transparency and information sharing among the different institutional levels, which demonstrates the difﬁculties of multilevel governance. Both study communities had a density value of 1. The denser a network, the higher the level of social control within it, which also means a high presence of trust-based relationships and risk minimization (Wasserman and Faust 1994; Jansen 2006, 2007; Rank 2015). The average degree of centrality of the nodes was 3.88 for the CS1 network and 4.06 for CS2. Both study villages had a dense network characterized mostly by strong ties, which is characteristic of small mountain villages in general. This could be an advantage for integration in general, as well as in the job market. As the mayor of village CS2 said, ‘‘Within this rural context it is easier to integrate the people. It is an advantage, because it’s easy to get in touch with the local community.’’ The CS2 center coordinator said, ‘‘Small structures are ideal. Here you have a direct contact with the population and volunteers. Two South Tyrolian mountain villages in comparison Each actor can be considered a node in a network of social relations relevant to employment. In both networks (Figures 1 and 2), the collaboration between the municipality and the reception center was crucial. The key actors work at the municipal level; they include the reception center coordinator and the people responsible for social affairs (mayor and social adviser and/or integration adviser). Center coordinators oversee the administrative affairs of each center resident. Within the network, they play a central role and are connected to almost all other actors. The second most important person is the social adviser of each municipality. In CS1, these 2 ﬁgures connect all other actors. In CS2, there are more municipal actors involved—a social adviser, an In both communities, long-term volunteers (37 in CS1 and 20 in CS2) assisted the local authorities and the assigned nongovernmental organization (NGO) in welcoming the asylum seekers and providing basic services. In addition to cultural and informational events, they offered free literacy and language courses in German and Italian as a supplement to the courses offered by the Autonomous Province of Bolzano. They were also among the ﬁrst local contacts for the asylum seekers and thus served as a bridge to the community. The asylum seekers also did volunteer work in both communities. At ﬁrst, it was difﬁcult for them to forgo remuneration, but in the http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 TABLE 3 Key actors in the case-study networks. 392 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 Two South Tyrolian mountain villages in comparison You have more possibilities to ﬁnd work.’’ Nevertheless, there is also high social control, and newcomers have to deal with high expectations from the receiving society that they will maintain cultural and social norms: ‘‘They greet, are kind, The CS1 network had 66 ties, and the CS2 network had 73 ties. Ties between individuals can be of different intensity and have different objectives. Granovetter (1973) differentiated between weak ties and strong ties that are central to the concept of social capital and embeddedness in a community. Individuals act based not only on their preferences, motivations, and rational choices but also on their embeddedness in a social system, which has institutional rules, norms, and relations and is inﬂuenced by dominance, both at the individual level (relational embeddedness) and at the structural level (structural embeddedness). http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 MountainDevelopment FIGURE 1 Network analysis of the first case-study village. Node size represents betweenness centrality. and shake hands’’ (integration adviser, CS2); ‘‘by doing voluntary work in the municipality, people see that they are able to work’’ (social adviser, CS1). Moreover, the asylum seekers in the reception centers are highly dependent on the local community and the engagement of individuals. In both cases, community institutions play an important role for the asylum seekers as bridges between them and the local community. As the CS1 center coordinator said, ‘‘In our small community it is [also easier] for the locals. They don’t see a critical and undeﬁned mass of refugees; they see human beings.’’ The contact with local residents was additionally supported by volunteers. Besides providing free services for asylum seekers that supplemented state services, volunteers serve as mediators between the newly arrived and the local people and enterprises, including potential employers. This requires good collaboration among the different actors serving the migrants. new community. The results also show that prior experiences and familiarity with the migration phenomenon, as well as acceptance of the local community, may facilitate the integration process for the new inhabitants. These ﬁndings depend strongly on the trust a local community has in the person or people responsible—in CS1, this is the social adviser, who has had this position since the 1990s. The social capital of the central actors in the networks (an individual good) was an added value for the people within the centers. Two South Tyrolian mountain villages in comparison Besides the negative element of higher social control because of the spatial (and social) proximity and the lack of anonymity, we found that strong ties could support integration and facilitate access to the labor market. They seem indispensable for getting a job, which is illustrated by the statement of the integration adviser in CS2: ‘‘You give your personal warranty.’’ Stronger relationships and trust within rural communities may facilitate integration during asylum seekers’ application process and enhance local social embeddedness, with volunteers making a signiﬁcant difference in this regard. 393 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 The importance of social capital for asylum seekers Social capital can play a positive role in the acquisition of other forms of capital (Palloni et al 2001) and for the integration process in general. The 2 case studies presented here demonstrate that integration is multidimensional, connects different governance levels, and is a long-term project. Nonetheless, full access to the labor market during the application process helped some Social capital can play a positive role in the acquisition of other forms of capital (Palloni et al 2001) and for the integration process in general. The 2 case studies presented here demonstrate that integration is The establishment of reception facilities in the 2 study villages can be considered an example of integration of asylum seekers in small mountain villages. This had a positive impact on their labor integration and the integration process as a whole. The people who got asylum status already had a job or work experience and could take advantage of the strong relationships within their multidimensional, connects different governance levels, and is a long-term project. Nonetheless, full access to the labor market during the application process helped some http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 393 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use MountainDevelopment FIGURE 2 Network analysis of the second case-study village. Node size represents betweenness centrality. applicants who were later formally recognized as refugees to overcome barriers. The competences newly acquired through language and training courses, working experiences, and embeddedness in the community during that time was crucial. Without a strong local network bridging local institutions and the community, the integration of the new arrivals would have been more difﬁcult. First results led to the assumption that relations with volunteers and the local population (social networks) in particular helped the asylum seekers to enter the labor market. This aspect needs further investigation. In this case, the bonding social capital of the rural community had a positive inﬂuence on the occupational status of the asylum seekers. In addition, the low numbers of asylum seekers made it easier for local residents to experience the integration process in a positive way and thus increased their acceptance of the newcomers. area, especially if they had a job, although new problems may arise in the long term, for example, with ﬁnding housing. Short-term and emergency strategies play a predominant role in Italy’s migrant policy. The importance of social capital for asylum seekers The early investments in a sustainable and positive integration process may help to reduce problems that might arise after an asylum application is accepted. However, this would require a paradigm shift in integration policy and a different attitude regarding immigration and diversity, namely, one of openness and intercultural competence that ‘‘facilitates learning and change processes from and among different people, ways of life and forms of organization, thus removing barriers to access and delimitation mechanisms in the organizations’’ (Schr€oer 2015: 10). Multicultural relations are probably more effective if integration is given top priority in public administration and leading actors (Aum€uller and Gesemann 2014: 171), which also requires recognition of the central role of local actors. According to the center coordinators, asylum seekers whose applications were approved wanted to stay in the http://www.provinz.bz.it/news/de/news.asp?news_action¼4&news_ article_id¼396834; accessed on 21 February 2017. APB-ST [Autonomous Province of Bolzano—South Tyrol]. 2017. 25 Asylbewerber €ubersiedeln von Bozen nach Welschnofen. S€udtiroler Landesverwaltung/Amministrazione Provincia Bolzano. http://www.provinz.bz. http://www.provinz.bz.it/news/de/news.asp?news_action¼4&news_ article_id¼396834; accessed on 21 February 2017. APB-ST [Autonomous Province of Bolzano—South Tyrol]. 2017. 25 Asylbewerber €ubersiedeln von Bozen nach Welschnofen. S€udtiroler Landesverwaltung/Amministrazione Provincia Bolzano. http://www.provinz.bz. 394 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use R E F E R E N C E S Bohnsack R, Marotzki W, Meuser M, editors. 2006. Hauptbegriffe Qualitative Sozialforschung: Ein W€orterbuch. Leverkusen, Germany: Verlag Barbara Budrich, Opladen & Farmington Hills. g ; Morosanu L. 2016. Professional bridges: Migrants’ ties with natives and occupational advancement. Sociology 50:349–365. Palloni A, Massey DS, Ceballos M, Espinosa K, Spittel M. 2001. Social capital and international migration: A test using information on family networks. American Journal of Sociology 106:1262–1298. Borgatti SP. 2002. NetDraw Software for Network Visualization. Lexington, KY: Analytic Technologies. https://sites.google.com/site/netdrawsoftware/ home; accessed on 1 March 2017. gy Petrovic N. 2013. Rifugiati, profughi, sfollati. Breve storia del diritto d’asilo in Italia. Milano, Italy: Franco Angeli. Borgatti SP, Everett MG, Johnson JC. 2013. Analyzing Social Networks. Los Angeles, CA: Sage. Portes A, editor. 1995. The Economic Sociology of Immigration. Essays on Networks, Ethnicity, and Entrepreneurship. New York, NY: Russell Sage Foundation. Bourdieu P. 2012. ¨Okonomisches Kapital, kulturelles Kapital, soziales Kapital. In: Bauer U, Bittlingmayer UH, Scherr A, editors. Handbuch Bildungs- und Erziehungssoziologie. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften pp 229 242 Putnam RD. 2001. Bowling Alone: The Collapse and Revival of American Community. New York, NY: Touchstone, Simon & Schuster. Sozialwissenschaften, pp 229–242. Cheung SY, Phillimore J. 2013. Social Networks, Social Capital and Refugee Integration. Research Report. Birmingham, United Kingdom: University of Birmingham. http://www.nufﬁeldfoundation.org/role-social-capital-refugee- integration; accessed on 20 February 2017. Putnam RD, Leonardi R, Nanetti RY. 1994. Making Democracy Work: Civic Traditions in Modern Italy. Princeton, NJ: Princeton University Press. Putnam RD, Leonardi R, Nanetti RY. 1994. Making Democracy Work: Civic Traditions in Modern Italy. Princeton, NJ: Princeton University Press. Rank O. 2015. Unternehmensnetzwerke: Erfassung, Analyse und erfolgreiche Nutzung. Wiesbaden, Germany: Springer Fachmedien. ¨ Rank O. 2015. Unternehmensnetzwerke: Erfassung, Analyse und erfolgreiche Nutzung. Wiesbaden, Germany: Springer Fachmedien. Chou K-L, Chow NWS. 2009. The roles of human capital and social capital in the economic integration of new arrivals from Mainland China to Hong Kong. Habitat International 33:340–346. Schr€oer H. 2015. Interkulturelle ¨Offnung und Diversity Management. In: Genkova P, Ringeisen T, editors. Handbuch Diversity Kompetenz: Gegenstandsbereiche. Wiesbaden, Germany: Springer Fachmedien, pp 1–12. http://link.springer.com/referenceworkentry/10.1007/978-3-658-08932- 0_1-1; accessed on 27 March 2017. Eurostat. 2017. Urban–rural typology. Eurostat—Statistics Explained. http:// ec.europa.eu/eurostat/statistics-explained/index.php/Urban-rural_typology; accessed on 17 February 2017. Sørensen JFL. 2016. Rural–urban differences in bonding and bridging social capital. Regional Studies 50:391–410. y Flick U. 2008. Triangulation. Eine Einf€uhrung. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften. UNESCO [United Nations Educational, Scientiﬁc and Cultural Organization]. 2017. Asylum seeker. Glossary of Migration-Related Terms. http://www. R E F E R E N C E S APB-ST [Autonomous Province of Bolzano—South Tyrol]. 2012. Weltﬂ€uchtlingstag: 137 Fl€uchtlinge in S€udtirol—LR Theiner: ‘‘Pﬂicht zu helfen.’’ S€udtiroler Landesverwaltung/Amministrazione Provincia Bolzano. http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 394 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use MountainDevelopment MountainDevelopment it/news/de/news.asp?news_action¼4&news_article_id¼596519; accessed on 14 October 2017. it/news/de/news.asp?news_action¼4&news_article_id¼596519; accessed on 14 October 2017. Jansen D. 2006. Einf€uhrung in die Netzwerkanalyse: Grundlagen, Methoden, Forschungsbeispiele. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften. APB-ST [Autonomous Province of Bolzano—South Tyrol], Eurac Research. 2017. Asyl und Fl€uchtlinge in S€udtirol. Information brochure. S€udtiroler Landesverwaltung/Amministrazione Provincia Bolzano. http://www.provinz. bz.it/familie-soziales-gemeinschaft/soziale-notlagen/asylantragsteller- ﬂuechtlinge/allgemeine-informationen.asp; accessed on 23 April 2017. Jansen D. 2007. Theoriekonzepte in der Analyse sozialer Netzwerke. Entstehung und Wirkungen, Funktionen und Gestaltung sozialer Einbettung. https://dopus. uni-speyer.de/frontdoor/index/index/docId/519; accessed on 6 February 2017. g g y g ﬂuechtlinge/allgemeine-informationen.asp; accessed on 23 April 2017. Konle-Seidl R, Bolits G. 2016. Labour Market Integration of Refugees: Strategies and Good Practices. Policy Department—Economic and scientiﬁc policy. Brussels, Belgium: European Parliament. http://www.europarl.europa.eu/ thinktank/it/document.html?reference¼IPOL_STU(2016)578956; accessed on 14 October 2017. ASTAT [Landesinstitut f€ur Statistik—Istituto provinciale di statistica]. 2017. Statistisches Jahrbuch 2016. http://astat.provinz.bz.it/de/statistisches- jahrbuch.asp; accessed on 14 October 2017. Aum€uller J, Gesemann F. 2014. Integrationspotenziale l€andlicher Regionen im Strukturwandel. Final Project Report. Darmstadt, Germany: Schader-Stiftung. L€ofﬂer R, Walder J, Beismann M, Warmuth W, Steinicke E. 2016. Amenity migration in the Alps: Applying models of motivations and effects to 2 case studies in Italy. Mountain Research and Development 36:484–493. Benz A. 2007. Multilevel governance. In: Benz A, L€utz S, SchimankU, Simonis G, editors. Handbuch Governance: Theoretische Grundlagen und empirische Anwendungsfelder. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften, pp 229–242. Benz A. 2007. Multilevel governance. In: Benz A, L€utz S, SchimankU, Simonis G, editors. Handbuch Governance: Theoretische Grundlagen und empirische Anwendungsfelder. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften, pp 229–242. Membretti A, Dematteis M. 2016. Montanari per forza. L’immigrazione straniera nelle montagne italiane, tra emergenza e inclusione sociale. http://issuu.com/ dislivelli/docs/64_webmagazine_febbraio16/1; accessed 9 March 2017. pp Benz A, L€utz S, Schimank U, Simonis G, editors. 2007. Handbuch Governance. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften. Mitterhofer J, Wisthaler V, Stawinoga AE. 2016. Zusammenleben in S€udtirol: Vielfalt in den Gemeinden. Ein €Uberblick €uber Integrations- und Inklusionspolitiken auf Gemeindeebene. Bolzano, Italy: Eurac Research. http://docplayer.org/36520547-Zusammenleben-in-suedtirol-vielfalt-in-den- gemeinden.html; accessed on 14 October 2017. ergold J, Thomas S. 2012. Participatory research methods: A Bergold J, Thomas S. 2012. Participatory research methods: A methodological approach in motion. Forum: Qualitative Social Research/ Sozialforschung 13(1). http://www.qualitative-research.net/index.php/fqs/ article/view/1801; accessed on 21 February 2017. 395 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use R E F E R E N C E S unesco.org/new/en/social-and-human-sciences/themes/international- migration/glossary/asylum-seeker/; accessed on 1 March 2017. Fuhse JA. 2016. Soziale Netzwerke: Konzepte und Forschungsmethoden. Konstanz, Germany: Uni-Taschenb€ucher. Gazzetta ufﬁciale [Gazzetta ufﬁciale della Repubblica italiana]. 2017. LEGGE 28 febbraio 1990, n.39. Roma, Italy. http://www.gazzettaufﬁciale.it/eli/id/ 1990/02/28/090G0075/sg; accessed on 14 October 2017. UNHCR [Ofﬁce of the United Nations High Commissioner for Refugees]. 2016. Global Trends. Forced displacement in 2015. http://www.unhcr.org/ statistics/unhcrstats/576408cd7/unhcr-global-trends-2015.html; accessed on 29 April 2017. Gerring J. 2007. Case Study Research: Principles and Practices. New York, NY: Cambridge University Press. Granovetter M. 1973. The strength of weak ties. American Journal of Sociology 78:1360–1380. http://dx.doi.org/10.1086/225469; accessed on 25 August 2016. € UNHCR [Ofﬁce of the United Nations High Commissioner for Refugees]. 2017a. The 1951 Refugee Convention. http://www.unhcr.org/1951-refugee- convention.html; accessed on 20 February 2017. Haller M. 2016. Forschungsstand, Uberlegungen und Hypothesen f€ur S€udtirol. In: Atz H, Haller M, Pallaver G, editors. Ethnische Differenzierung und soziale Schichtung in der S€udtiroler Gesellschaft. Baden Baden, Germany: Nomos, pp 41–60. UNHCR [Ofﬁce of the United Nations High Commissioner for Refugees]. 2017b. Legislazione nazionale. http://www.unhcr.it/cosa-facciamo/ protezione/il-diritto-dasilo/asilo-in-italia/legislazione-nazionale; accessed on 1 March 2017. Hillmann F. 2016. Migration. Eine Einf€uhrung aus sozialgeographischer Perspektive Stuttgart Germany: Franz Steiner Verlag Hillmann F. 2016. Migration. Eine Einf€uhrung aus sozialgeographischer UNHCR [Ofﬁce of the United Nations High Commissioner for Refugees]. 2017c. Mediterranean situation. Operational Portal: Refugee Situations. http://data2.unhcr.org/en/situations/mediterranean; accessed on 17 February 2017. Perspektive. Stuttgart, Germany: Franz Steiner Verlag. Perspektive. Stuttgart, Germany: Franz Steiner Verlag. Hollstein B. 2010. Qualitative Methoden und Mixed-Method-Designs. In: Stegbauer C, H€außling R, editors. Handbuch Netzwerkforschung. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften, pp 459–470. Hollstein B. 2010. Qualitative Methoden und Mixed-Method-Designs. In: Stegbauer C, H€außling R, editors. Handbuch Netzwerkforschung. Wiesbaden, Hollstein B. 2010. Qualitative Methoden und Mixed-Method-Designs. In: Stegbauer C, H€außling R, editors. Handbuch Netzwerkforschung. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften pp 459–470 g , g , g , Germany: VS Verlag f€ur Sozialwissenschaften, pp 459–470. Wald A, Jansen D. 2007. Netzwerke. In: Handbuch Governance: Theoretische Grundlagen und empirische Anwendungsfelder. Wiesbaden, Germany: VS Verlag f€ur Sozialwissenschaften, pp 93–106. ISTAT [Istituto nazionale di statistica]. 2015. Precisazione sulla classiﬁcazione dei comuni montani. http://www.istat.it/it/archivio/147760; accessed on 12 December 2016. Wasserman S, Faust K. 1994. Social Network Analysis: Methods and Applications. Cambridge, United Kingdom: Cambridge University Press. ISTAT [Istituto nazionale di statistica]. 2017. Statistiche Istat. Online Database. http://dati.istat.it/; accessed on 2 March 2017. ISTAT [Istituto nazionale di statistica]. 2017. Statistiche Istat. Online Database. http://dati.istat.it/; accessed on 2 March 2017. Italian Ministry of the Interior. 2017. R E F E R E N C E S Immigrazione e asilo. Rome, Italy: Ministry of the Interior. http://www.interno.gov.it/it/temi/immigrazione-e- asilo; accessed on 14 October 2017. Worbs S, Bund E. 2016. Asylberechtigte und anerkannte Fl€uchtlinge in Deutschland. Qualiﬁkationsstruktur, Arbeitsmarktbeteiligung und Zukunftsorientierungen. Report No. 1/2016. Germany: BAMF [Bundesamt f€ur Migration und Fl€uchtlinge]. Italian Ministry of the Interior. 2017. Immigrazione e asilo. Rome, Italy: Ministry of the Interior. http://www.interno.gov.it/it/temi/immigrazione-e- asilo; accessed on 14 October 2017. http://dx.doi.org/10.1659/MRD-JOURNAL-D-17-00030.1 395 Mountain Research and Development Downloaded From: https://bioone.org/journals/Mountain-Research-and-Development on 23 Oct 2024 Terms of Use: https://bioone.org/terms-of-use
https://openalex.org/W2991630182	https://www.mdpi.com/2073-4395/9/12/825/pdf?version=1575103178	English	null	Field Evaluation of RD6 Introgression Lines for Yield Performance, Blast, Bacterial Blight Resistance, and Cooking and Eating Qualities	Agronomy	2,019	cc-by	10,175	Received: 24 September 2019; Accepted: 25 November 2019; Published: 30 November 2019 Abstract: Glutinous rice cultivar “RD6” is well known for its fragrance and high cooking and eating qualities, and is the most popular glutinous cultivar in the north and northeastern regions of Thailand. However, it’s susceptible to blast and bacterial blight (BB) diseases. Previously, four blast resistance QTLs on chromosomes 1, 2, 11, and 12, and a single BB resistance gene xa5 pyramided to the background of the RD6 cultivar were tested for a broad spectrum of disease resistance under greenhouse conditions. In the present study, a ﬁeld experiment was conducted during the rainy seasons of 2015, 2016, 2017, and 2018, across three locations, for performance evaluations of promising lines in terms of disease reaction, agronomical characteristics, grain yield, and quality attributes. The results revealed that the ILs (BC2F5 2-7-5-36, BC2F5 2-7-5-43, BC2F5 2-8-2-25, and BC2F5 6-1/15-2-11) exhibited higher level resistance to leaf blast and neck blast disease. The BC2F5 2-8-2-52 showed resistance to both blast and BB diseases and, like all ILs, exhibited superior yield compared to the original RD6. Furthermore, the agronomic traits and grain qualities were similarly displaced, and were therefore recommended as near-isogenic lines to the RD6. This clearly demonstrated that farm phenotypic selection plays an important role in achieving not only NIL resistance to diseases, but also high yield potential, as well as representing an eﬀective way in which to enhance BB, leaf blast, and neck blast resistance in rice planting in the north and northeastern regions of Thailand. Keywords: marker-assisted selection; resistance gene; quantitative trait locus; pseudo-backcrossing Myo San Aung Nan, Jirayoo Janto, Arthit Sribunrueang, Tidarat Monkham, Jirawat Sanitchon and Sompong Chankaew * Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand; myo081159@gmail.com (M.S.A.N.); kamong285@gmail.com (J.J.); arthitsribunrueang@gmail.com (A.S.); tidamo@kku.ac.th (T.M.); jirawat@kku.ac.th (J.S.) * Correspondence: somchan@kku.ac.th; Tel.: +66-8512-40427 Agronomy 2019, 9, 825; doi:10.3390/agronomy9120825 agronomy agronomy agronomy agronomy Article Field Evaluation of RD6 Introgression Lines for Yield Performance, Blast, Bacterial Blight Resistance, and Cooking and Eating Qualities Myo San Aung Nan, Jirayoo Janto, Arthit Sribunrueang, Tidarat Monkham, Jirawat Sanitchon and Sompong Chankaew * 1. Introduction Rice (Oryza sativa L.) is a crucial food crop for global food security. Approximately 90% of the world’s rice is produced and consumed in Asia [1]. Globally, Thailand ranks as the second largest rice exporter, grown on some 11M ha of land; more than half of the rice grown in Thailand is grown in the north and northeastern regions [2]. Additionally, Thailand has huge varietal diversity and varied rice growing ecologies. Among them, the RD6 improved glutinous rice variety, released in 1977, is one of the most popular lowland varieties for domestic consumption, especially in Thailand’s north and northeastern regions [3]. This variety, representing 83% of the northeastern region’s total glutinous rice in 1995 [4], is preferred among Thais due to its cooking and eating characteristics, e.g., slender kernel grain, aroma, stickiness, and taste. Currently, it is being cultivated in large areas of lowland, rain-fed terrain in north and northeastern Thailand [3]. However, RD6 rice contains several production constraints, including biotic stresses, among which rice blast, caused by the fungus Magnaporthe oryzae [5], and bacterial blight disease, caused by Agronomy 2019, 9, 825; doi:10.3390/agronomy9120825 www.mdpi.com/journal/agronomy www.mdpi.com/journal/agronomy 2 of 15 Agronomy 2019, 9, 825 Agronomy 2019, 9, 825 Xanthomonas oryzae pv. oryzae [6], are the most severe, causing high losses in both yield and quality. Rice blast disease can infect all parts of the rice plant including the roots [7]; however, the leaves and neck panicles are the most seriously aﬀected. Leaf blast lesions can reduce the net photosynthetic rate per leaf. Neck blast, considered to be the most destructive phase of the disease, can occur without being preceded by severe leaf blast, resulting in major losses of yield and grain quality [8]. The speciﬁc genetic mechanisms of resistance to leaf blast and panicle neck blast may be varied, as well as independently controlled by diﬀerent R genes [9]. Therefore, evaluations of seedling blasts (ignoring panicle blast) have proven inconclusive. Likewise, BB is also a serious disease which aﬀects the seedling to the tillering stage [10]. Previous studies have reported that both diseases occur in more than 80 rice growing countries, resulting in yield losses estimated at more than 50% of production [10,11]. Both blast and BB diseases can be controlled with chemicals, but the spraying of chemicals is hazardous to the environment, causes land degradation, and in turn, increases production costs. 1. Introduction While cultural practices, such as the reduction of nitrogen fertilizers and water management, can minimize damage, these practices cannot control the infection completely. Therefore, the utilization of rice varieties carrying resistance genes has proven to be one of the most economical, eﬀective, and environment-friendly strategies for the management of rice diseases [12]. Gene backcross pyramiding combines one or more desirable genes from multiple parents into a single genotype, without compromising on agronomic performance and grain qualities [13]. In earlier studies, Suwannual et al. [14] and Pinta et al. [15] successfully introgressed one BB resistant gene (xa5) and four blast resistant QTLs in chromosomes 1, 2, 11, and 12 through marker-assisted selection. Broad spectrum resistance was then tested using artiﬁcial inoculum with eight blast isolates and ten BB isolates under greenhouse conditions [15]. However, IL information on the agromorphology traits, disease resistance levels, grain yield, as well as cooking and eating quality, has not yet been reported. Therefore, the performance of a farm-level, ﬁeld-based trial emphasizing agromorphological traits, disease resistance levels, and grain and cooking quality is essential. The objective of this study was therefore to investigate the performance of introgression lines in paddy ﬁelds, and their grain quality attributes following the concept of backcross selection, in order to select superior, improved RD6 NIL lines which are resistant to blast and BB diseases, and which oﬀer high yield potentials and attribute qualities. 2.1. Plant Materials and Experimental Design The ten selected RD6 introgression lines (ILs) [14,15], ILs without QTLs, four check lines including three donor parents (Jao Hom Nin (JHN), P0489, and IR62266), and a recurrent parent (RP) RD6, were evaluated for blast, BB diseases reactions, yield, yield components, and grain quality attributes (Table 1). Field experiments were conducted in 2015, 2016, 2017, and 2018 across three locations (Figure 1). All entries were assigned in a randomized complete block design (RCBD) with three replications during the rainy season of each year and location. Natural infection was applied by sowing the KDML105 as a susceptible variety in the experiment surroundings one month prior to transplantation. Each plot contained ﬁve rows, 1.5 m in length, with 25 cm × 25 cm between and within each row. To ensure a uniform spread of disease, the initial and the ﬁve interval plots in each replication were inserted with the susceptible RD6. Fertilizer was applied at a rate of 23 kg/ha (N: P: K) at 30 and 60 days after planting. Plants were protected through hand weeding and the use of common pesticides. Field water was maintained during the tillering stage at around 10 cm until ten days before harvesting. Daily minimum and maximum temperature, rainfall, and relative humidity during the experiment period, 2015–2018, were recorded. 2.2. Evaluation for Leaf Blast, Neck Blast, and Bacterial Blight Diseases Resistance 2.2. Evaluation for Leaf Blast, Neck Blast, and Bacterial Blight Diseases Resistance Leaf blast, neck blast, and BB disease reactions were observed individually from the inner ten plants, and scored through the Standard Evaluation System (IRRI, 1996) [16] on a scale of 0–9. Leaf blast 3 of 15 Agronomy 2019, 9, 825 and bacterial blight were evaluated at the tillering stage, and neck blast disease evaluations were done at the ripening stage. Individual scores were averaged. Lines with a score of 0–3 were considered resistant, 4–5 moderately resistant, 6 moderately susceptible, and 7–9 susceptible. Table 1. List of pyramided lines, and parent and check varieties used in this study. Line/Cultivar Name Types of Lines QTL on Chromosome a BC F 2 7 5 36 B di li Bl t (1 2 11 12) d 5 Table 1. List of pyramided lines, and parent and check varieties used in this study. Line/Cultivar Name Types of Lines QTL on Chromosome a BC2F3 2-7-5-36 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2-8-2-36 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2-7-5-43 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2-8-2-19 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2-8-2-25 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 9-1/15-1-28 b Breeding line Blast (1, 2,11,12) BC2F3 2-8-2-27 c Breeding line - BC2F3 2-8-2-52 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2-8-2-24 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 6-1/15-2-11 b Breeding line Blast (1, 2, 12) RD6 Recurrent parent - Jao Hom Nin Donor parent Blast (1, 11) P0489 Donor parent Blast (2, 12) IR62266 Donor parent xa5 KDML105 Susceptible check - PLT Susceptible check - IR64 Resistance check - PLD Resistance check - Note: a The number in round brackets indicates the location of the resistant QTL. b Suwannual et al. [14]; c Pinta et al. [15]. 2.2. Evaluation for Leaf Blast, Neck Blast, and Bacterial Blight Diseases Resistance g ( , , , ) BC2F3 2‐8‐2‐36 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2‐7‐5‐43 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2‐8‐2‐19 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2‐8‐2‐25 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 9‐1/15‐1‐28 b Breeding line Blast (1, 2,11,12) BC2F3 2‐8‐2‐27 c Breeding line ‐ BC2F3 2‐8‐2‐52 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 2‐8‐2‐24 c Breeding line Blast (1, 2, 11, 12) and xa5 BC2F3 6‐1/15‐2‐11 b Breeding line Blast (1, 2, 12) RD6 Recurrent parent ‐ Jao Hom Nin Donor parent Blast (1, 11) P0489 Donor parent Blast (2, 12) IR62266 Donor parent xa5 KDML105 Susceptible check ‐ PLT Susceptible check ‐ IR64 Resistance check ‐ PLD Resistance check ‐ Note: a The number in round brackets indicates the location of the resistant QTL. b Suwannual et al [14]; c Pinta et al. [15]. Table 1. List of pyramided lines, and parent and check varieties used in this study. g ( ) F3 2‐8‐2‐36 c Breeding line Blast (1, 2, 11, 12) a Note: a The number in round brackets indicates the location of the resistant QTL. b Suwannual et al. [14]; c Pinta et al. [15]. [14]; c Pinta et al. [15]. Fi 1 S h i di f h f h i b d l i i l il i Figure 1. Schematic diagram of the farm phenotypic-based selection in multilocation target environments (2015–2018). AP, Ampawan farm; KKU, Khon Kaen University; KRC, Khon Kaen Rice Research center. Figure 1. Schematic diagram of the farm phenotypic-based selection in multilocation target environments (2015–2018). AP, Ampawan farm; KKU, Khon Kaen University; KRC, Khon Kaen Rice Research center. Figure 1. Schematic diagram of the farm phenotypic-based selection in multilocation target environments (2015–2018). AP, Ampawan farm; KKU, Khon Kaen University; KRC, Khon Kaen Rice Research center. Figure 1. Schematic diagram of the farm phenotypic-based selection in multilocation target environments (2015–2018). AP, Ampawan farm; KKU, Khon Kaen University; KRC, Khon Kaen Rice Research center. Figure 1. Schematic diagram of the farm phenotyp environments (2015–2018). AP, Ampawan farm; KKU, K 2.3. Evaluation for Agronomic Performance and Grain Yield 2.4. Evaluation for Grain and Cooking Quality Characterization of grain and cooking quality traits was performed on the grains harvested from the BC2F5 generation, Khon Kaen University site, planted in 2018. For grain characteristics, such as length, breadth, and length breadth ratio (L/B), data were obtained from ten fully-developed paddies and milled rice grain, and the means were again calculated. For cooking quality, the gelatinization temperature (GT) was measured indirectly by alkali spreading value (ASV) using six milled grains, as suggested by Little et al. [17]. Fragrance was determined following the sensory test protocol of Yi et al. [18], and the percentage of amylose content (AC %) was estimated following the method of Juliano [19]. The percentages of milled rice recovery (MRR %) and head rice recovery (HRR %) were calculated using 100g of paddy grains in each line, milled in a mini-milling machine, as per the formula given by SES, IRRI [16]. Figure 1. Schematic diagram of the farm phenotyp environments (2015–2018). AP, Ampawan farm; KKU, K 2.3. Evaluation for Agronomic Performance and Grain Yield Agronomy 2019, 9, x; doi: FOR PEER REVIEW www.mdpi.com/journal/agronomy Research center. The agronomical characteristics were observed from the Khon Kaen University site, planted in 2018. Agronomical traits were measured according to the same evaluation system, in which four plants between the second and sixth row in each plot were selected for the following agronomic traits: days 4 of 15 Agronomy 2019, 9, 825 to (50%) ﬂowering (DTF), plant height (PH), number of tillers (NT), number of panicles (NP), panicle length (PL), panicle neck length (PNL), branching per panicle (BPP), ﬁlled grains per panicle (FGPP), unﬁlled grains percentage per panicle (UFG%), 1000-grain weight (GW), harvest index (HI), and grain yield (GY). All data were compiled through the use of raw measurements for data analysis except UFG% (UFG% = (number of unﬁlled grains per panicle/number of all grains per panicle) × 100) and HI (HI = total grain weight/total plant dry weight), which were calculate prior to data analysis. 2.5. Sensory Analysis Milled rice samples weighing 300g were washed three times and soaked for six hours. All samples were cooked in Thai Bamboo sticky rice steamers for 40 minutes, as is the local method. Textures were evaluated twice, i.e., in the morning (9:00 am) and in the afternoon (1:00 pm). Twelve local consumers, ﬁve males and seven females, were asked to measure the texture of the cooked rice, utilizing a three points hedonic scale: 1 = low, 2 = middle, and 3 = high. Their overall preferences were based upon their like or dislike of the rice, as well as their reasons. The mean scores from each individual consumer were compared. 2.6. Data Analysis The traits examined in each experiment were subjected to statistical analysis via the Statistics 10© (1985–2013) program (Analytical Software, Tallahassee, FL, USA), which utilized replication as a random eﬀect and genotypes as a ﬁxed eﬀect within the statistic model. Multiple comparisons were analyzed by Duncan’s Multiple Range Test (DMRT) using the Statistical Tool for Agricultural Research (STAR) software (http://bbi.irri.org/products), developed by the IRRI’s Biometrics and Breeding Informatics team. 3.1. Evaluation of Leaf Blast, Neck Blast, and Bacterial Blight Resistance Note: The number above the traits is the number of the environment, Signiﬁcant at 0.01 probability levels, respectively. 1 Not including the AP18 location, which ﬂooded before harvesting. We also found a serious BB infection at AP in 2017, in which the IL BC2F3 2-8-2-52 produced the lowest BB infection score (3.33) compared to the other ILs and the susceptible lines, including RP RD6 (Table 4). Surprisingly, the DP IR62266 used for introgression xa5 displayed a susceptible rating (6.00), similar to that of the RP RD6 (6.67) and susceptible check KDML105 (6.00). Furthermore, the JHN, used in introgression blast resistance genes, proved resistant to BB (2.00), with less infection than the multidisease resistant IR64 (3.33). Table 3. Disease reaction scores for leaf blast in diﬀerent years, under diﬀerent environmental conditions, and at diﬀerent locations. Table 3. Disease reaction scores for leaf blast in diﬀerent years, under diﬀerent environmental conditions, and at diﬀerent locations. 3.1. Evaluation of Leaf Blast, Neck Blast, and Bacterial Blight Resistance 3.1. Evaluation of Leaf Blast, Neck Blast, and Bacterial Blight Resistance 3.1. Evaluation of Leaf Blast, Neck Blast, and Bacterial Blight Resistance Based on the combined ANOVA results, both leaf blast and neck blast diseases presented highly signiﬁcant diﬀerences in infection levels among the diﬀerent environmental conditions; however, the G × E interactions proved to be highly signiﬁcant only for leaf blast disease (Table 2). Evaluations of the ILs, along with the recurrent parent (RP) RD6, donor parent (DP), and check lines for leaf blast, neck blast, and bacterial blight disease reaction during the 2015–2018 seasons, are shown in Tables 3 and 4. In 2015, both leaf blast and neck blast were infected at Ampawan (AP). The resulting blast scores showed that the selected ILs with resistant QTLs/genes presented complete resistance reactions for leaf blast (0.33–1.70) and neck blast (1.10–1.97), similar to those of both donor parents JHN (0.60) and P0489 (1.05), as well as the IR-64 (resistant check) (0.80), whereas the RP RD6 was displaced as susceptible (6.57), i.e., similar to that of the KDML105 (susceptible check), with a score of 6.43. However, in the 5 of 15 Agronomy 2019, 9, 825 nondisease year of 2016 at AP, leaf blast infected all of the study genotypes, including RD6, in which the susceptible check lines produced a score of two or less. nondisease year of 2016 at AP, leaf blast infected all of the study genotypes, including RD6, in which the susceptible check lines produced a score of two or less. Table 2. Summary of ANOVA results mean square values and percentage of the sum of squares (in parenthesis) for leaf blast, neck blast, and the grain yield variable for combined environment/location. SOV df Leaf Blast Neck Blast Grain Yield (kg ha−1) Environment (E) 1 4 27.5728 (11.21) 31.4693 (4.56) 95094161.66 (57.56) Replication/Envi. 10 0.6354 (0.69) 1.2839 (0.74) 2261223.22 (3.42) Genotype (G) 10 23.4592 (54.07) 25.7281 (63.41) 662851.63 (1.00) G × E 39 4.1482 (27.56) 3.5731 (7.77) 2093750.29 (12.36) Error 97 0.3647 (6.48) 2.5744 (23.51) 1748007.61 (25.66) Note: The number above the traits is the number of the environment, Signiﬁcant at 0.01 probability levels, respectively. 1 Not including the AP18 location, which ﬂooded before harvesting. Table 2. Summary of ANOVA results mean square values and percentage of the sum of squares (in parenthesis) for leaf blast, neck blast, and the grain yield variable for combined environment/location. 3.1. Evaluation of Leaf Blast, Neck Blast, and Bacterial Blight Resistance Genotype t QTL on Chromosome Leaf Blast Mean AP15 AP16 AP18 KKU18 BC2F3 2-7-5-36 Blast (1, 2, 11, 12) and xa5 0.87cd 0.03c 0.00c 0.07c 0.24b BC2F3 2-8-2-36 Blast (1,2, 11, 12) and xa5 0.33d 0.03c 0.00c 0.00c 0.09b BC2F3 2-7-5-43 Blast (1,2, 11, 12) and xa5 0.40cd 0.03c 0.00c 0.03c 0.12b BC2F3 2-8-2-19 Blast (1,2, 11, 12) and xa5 0.83cd 0.07c 0.00c 0.00c 0.23b BC2F3 2-8-2-25 Blast (1,2, 11, 12) and xa5 0.77cd 0.03c 0.17c 0.07c 0.26b BC2F3 9-1/15-1-28 Blast (1, 2, 11, 12) 1.70c 0.03c 0.00c 0.00c 0.43b BC2F3 2-8-2-52 Blast (1,2, 11, 12) and xa5 1.03cd 0.03c 0.00c 0.00c 0.27b BC2F3 2-8-2-24 Blast (1,2, 11, 12) and xa5 0.87cd 0.13c 0.00c 0.07c 0.27b BC2F3 6-1/15-2-11 Blast (1, 2, 12) 0.47cd 0.10c 0.00c 0.00c 0.14b BC2F3 2-8-2-27 No QTL 5.10b 1.03b 2.33b 4.07b 3.13a RD6 (RP) - 6.57a 1.77a 4.03a 4.73b 4.28a Jao Hom Nin (DP) Blast (1, 11) 0.60cd 0.03c 0.00c 0.00c 0.16b P0489 (DP) Blast (2, 12) 1.05cd 0.10c 0.00c 0.00c 0.29b IR62266 (DP) xa5 0.47cd 0.10c 0.00c 0.00c 0.14b PLD (LRC) - ND 0.10c 0.00c 0.77c 0.29b IR64 (IRC) - 0.80cd 0.07c 0.00c 0.00c 0.22b KDML105 (SC) - 6.43a 1.10b 0.53c 7.53a 3.89a PLT (SC) - ND 0.27c 0.10c 6.33a 2.23a Mean 1.77 0.28 0.4 1.32 0.959 F-Test CV (%) 38.0 104.5 147.5 57.9 119.6 Note: RP: recurrent parent; DP: donor parent; LRC: local resistance check; IRC: international resistance check; SC: susceptible check; ND: no data; KKU18: Khon Kaen University during 2018; AP15, 16, 18, Ampawan during 2015, 2016, and 2018; CV (%): coeﬃcient of variation percentage; t: the generation of introgression lines were BC2F3, BC2F4, and BC2F4 in 2015, 2016, and 2018, respectively. Signiﬁcant at p ≤0.05. The letter after each value indicates the signiﬁcance within each column by DMRT. Disease reaction scores were evaluated following the Standard Evaluation System [16]. Note: RP: recurrent parent; DP: donor parent; LRC: local resistance check; IRC: international resistance check; SC: susceptible check; ND: no data; KKU18: Khon Kaen University during 2018; AP15, 16, 18, Ampawan during 2015, 2016, and 2018; CV (%): coeﬃcient of variation percentage; t: the generation of introgression lines were BC2F3, BC2F4, and BC2F4 in 2015, 2016, and 2018, respectively. Signiﬁcant at p ≤0.05. The letter after each value indicates the signiﬁcance within each column by DMRT. 3.1. Evaluation of Leaf Blast, Neck Blast, and Bacterial Blight Resistance Note: RP: recurrent parent; DP: donor parent; LRC: local resistance check; IRC: international resistance check; SC: susceptible check; ND: no data; ns: not signiﬁcant; BB: Bacterial Blight; KKU18: Khon Kaen University during 2018; AP15, 17, Ampawan in 2015 and 2017; CV (%): coeﬃcient of variation percentage; 1: The generation of introgression lines were BC2F3, BC2F4, and BC2F4 in 2015, 2017, and 2018, respectively. Signiﬁcant at p ≤0.05. The letter after each value indicates the signiﬁcance within each column by DMRT. Disease reaction scores were evaluated following the Standard Evaluation System [15]. In 2018, leaf blast and neck blast infected the site at KKU, whereas leaf blast alone occurred at AP (Tables 3 and 4). The infections which occurred at KKU were stronger than those at AP. At KKU, the ILs with resistant QTLs/genes were found to be completely resistant to both leaf blast (0.00–0.07) and neck blast (0.00–0.33), whereas the RP RD6 produced disease reaction scores of 4.73 for leaf blast, and 5.37 for neck blast. The RD6 produced a higher infection rate (4.03) than that of the KDML105 susceptible check line (0.53) and PLT (0.10) at AP; however, the ILs with resistant QTLs/genes were found once again to have complete resistance (0.00–0.17), similar to the donor and resistant check IR64. In general, the selected ILs with resistant QTLs/genes were found to be completely resistant for the leaf blast and neck blast, whereas only the IL BC2F3 2-8-2-52 demonstrated resistance to both blast and BB diseases. 3.1. Evaluation of Leaf Blast, Neck Blast, and Bacterial Blight Resistance Disease reaction scores were evaluated following the Standard Evaluation System [16]. 6 of 15 Agronomy 2019, 9, 825 Table 4. Disease reaction scores for neck blast and bacterial blight in diﬀerent years, under diﬀerent environmental conditions, and at diﬀerent locations. Genotype 1 QTL on Chromosome Neck Blast Mean BB AP15 KKU18 AP17 BC2F3 2-7-5-36 Blast (1, 2, 11, 12) and xa5 1.33c 0.00c 0.67d 5.00abc BC2F3 2-8-2-36 Blast (1, 2, 11, 12) and xa5 1.97c 0.00c 0.99d 6.00ab BC2F3 2-7-5-43 Blast (1, 2, 11, 12) and xa5 1.80c 0.00c 0.90d 6.00ab BC2F3 2-8-2-19 Blast (1, 2, 11, 12) and xa5 1.70c 0.00c 0.85d 5.33abc BC2F3 2-8-2-25 Blast (1, 2, 11, 12) and xa5 1.10c 0.00c 0.55d 6.00ab BC2F3 9-1/15-1-28 Blast (1, 2,11, 12) 1.00c 0.00c 0.50d 4.33a-d BC2F3 2-8-2-52 Blast (1, 2, 11, 12) and xa5 1.47c 0.33c 0.90d 3.33cd BC2F3 2-8-2-24 Blast (1, 2, 11, 12) and xa5 1.70c 0.00c 0.85d 6.00ab BC2F3 6-1/15-2-11 Blast (1, 2, 12) 1.30c 0.00c 0.65d 5.33abc BC2F3 2-8-2-27 No QTL 5.43b 5.07b 5.25bc 4.67abc RD6 (RP) - 6.53b 5.37b 5.95b 6.67a Jao Hom Nin (DP) Blast (1, 11) 1.30c 0.00c 0.65d 2.00d P0489 (DP) Blast (2, 12) 1.96c 0.00c 0.98d 4.33cd IR62266 (DP) xa5 1.10c 0.00c 0.55d 6.00ab PLD (LRC) - ND 0.00c 0.00d 4.00bcd IR64 (IRC) - 1.30c 0.00c 0.65d 3.33a-d KDML105 (SC) - 8.23a 8.27a 8.25a 6.00ab PLT (SC) - ND 5.77b 5.77b 3.67bcd Mean 2.45 1.38 2.01 4.89 F-Test CV (%) 28.5 151.57 18.9 25.54 Note: RP: recurrent parent; DP: donor parent; LRC: local resistance check; IRC: international resistance check; SC: susceptible check; ND: no data; ns: not signiﬁcant; BB: Bacterial Blight; KKU18: Khon Kaen University during 2018; AP15, 17, Ampawan in 2015 and 2017; CV (%): coeﬃcient of variation percentage; 1: The generation of introgression lines were BC2F3, BC2F4, and BC2F4 in 2015, 2017, and 2018, respectively. Signiﬁcant at p ≤0.05. The letter after each value indicates the signiﬁcance within each column by DMRT. Disease reaction scores were evaluated following the Standard Evaluation System [15]. Table 4. Disease reaction scores for neck blast and bacterial blight in diﬀerent years, under diﬀerent environmental conditions, and at diﬀerent locations. 3.3. Grain Quality, Cooking and Eating Quality of the Introgression Lines with RD6 The superior grain quality of the ILs (Table 7) demonstrated grain characteristics similar to those of the RP RD6. The milled grain of the ILs with QTLs ranged 6.5–6.8 cm in length, and 2.1–2.2 cm in breath, with an L/B (3.00–3.1) similar to that of the RP RD6. Furthermore, the ILs with resistant QTLs/genes produced an MRR % (66.7–69.1) and HRR% (58.8–64.9) greater than that of the RD6 (67.1 and 58.9), respectively (Figure 2). The cooking quality results of the ILs with RP RD6 are presented in Table 7. The mean sensory fragrance of the ILs (1.5–2.3) was very near that of the RD6 (2.1). The ILs produced an ASV (4.9–5.7) or GT [(55–69) −(70–74)], similar to the RD6 ASV (5.3) or GT (70–74), as well as a low AC % (3.5–6.9), i.e., like that of the RP RD6 (5.2). This indicates that undesirable quality trait alleles were not located in QTLs 1, 2, 11, or 12, or in the xa5, which were introduced into the RD6 background. The eating quality sensory performances of the ILs with RD6 are compared in Table 8. Similarly, the morning and afternoon tests showed that IL BC2F5 2-7-5-36 was preferred for its swelling capacity, sweetness, aroma, grain shape and size, and stickiness, being like that of the original RD6. The IL BC2F5 2-8-2-52 scored second, followed by the other ILs (Table 8). Table 5. Agronomic performance of introgression lines possessing blast- and bacterial blight-resistant genes/QTLs and the recurrent parent RD6. 3.2. Agronomic and Yield Performance of the Introgression Lines with RD6 The agronomic performance of the ILs with RP RD6 is presented in Table 5. There were no signiﬁcant diﬀerences (p ≤0.05) between the selected ILs and RP RD6 for agronomic traits, TN, PN, PL, FGPP, TSW, and HI. The IL mean values ranged between 6.7–9.0 (TN), 5.7–7.3 (PN), 27.4–30.5cm (PL), 194.3–243.7 (FGPP), 26.6–28.7g (TSW), and 28.0–42.7% (HI). However, the ILs BC2F3 2-8-2-19 and BC2F3 2-7-5-36 showed early signiﬁcance for (DTF) 103.0 and 116.0, respectively compared with the RD6 (136.7). Other ILs displaced ranges (131.0–135.0) similar to that of the RD6. Additionally, the PH of the ILs exhibited ranges (177.0–189.5) shorter than those of the RD6 (204.9). Moreover, all of the ILs were low for PNL (3.8–5.3) versus the RD6 (6.7). Unexpectedly, the UFG % of the ILs were higher (7.4–18.1) than the RD6 (6.6). In general, the selected ILs with resistant QTLs/genes, with the exception of the DTF, PH, PNL, and UFG % were similar to the original RD6. Agronomy 2019, 9, 825 7 of 15 Agronomy 2019, 9, 825 The grain yield performances of the ILs with RP RD6 within the six environments are presented in Table 6. In 2016, the lower yield at AP may have been due to the high amount of rainfall during the tilling stage aﬀecting the water level in the ﬁeld as evidenced by the low tiller number. Additionally, the average GY at KKU in 2018 may have been lower due to the sandy soil conditions. Due to the late planting season, the rice yield was low again at the Rice Research Center (KRC) in 2018. The pooled mean of GY in the ILs with resistant QTLs/genes ranged from BC2F3 2-8-2-19 (4566.66 kg/ha) to BC2F3 2-8-2-25(5592.39 kg/ha), as opposed to the RD6 (4998.02 kg/ha). Some of the ILs exhibited production rates higher than the RD6, particularly the BC2F3 2-8-2-25 and BC2F3 2-7-5-36, which produced 5592.39 and 5400.37 kg/ha, respectively (Table 6). In general, most of the ILs have plant types similar to the original RD6, and are well adapted to the rain-fed lowland environment of northeast Thailand. 3.3. Grain Quality, Cooking and Eating Quality of the Introgression Lines with RD6 Genotype Tiller PN PH (cm) DTF TSW (g) PL (cm) PNL (cm) BPP FGPP UFG (%) HI BC2F4 2-7-5-36 8.4 6.4 187.9abc 116.0bc 26.6 29.8 5.0b 12.6 210.9 7.4d 0.28 BC2F4 2-8-2-36 7.3 6.0 177.8bc 131.0ab 27.3 27.6 4.1b 13.4 201.6 12.8a-d 0.30 BC2F4 2-7-5-43 7.0 5.7 177.0bc 132.7ab 28.1 29.2 3.8b 13.7 204.9 18.1a 0.21 BC2F4 2-8-2-19 6.6 5.8 167.7c 103.0c 28.7 27.4 4.4b 14.7 194.2 16.3ab 0.43 BC2F4 2-8-2-25 7.0 6.6 189.5ab 131.0ab 27.3 30.5 4.6b 14.6 243.4 11.3a-d 0.29 BC2F4 9-1/15-1-28 8.0 6.1 187.4abc 133.0a 27.7 30.2 5.2b 13.7 204.5 15.7abc 0.36 BC2F4 2-8-2-27 7.6 6.0 204.6a 135.0a 27.4 28.8 5.3b 13.7 198.2 9.6bcd 0.29 BC2F4 2-8-2-52 9.0 7.1 184.1bc 132.0ab 27.7 29.5 4.0b 13.9 197.6 8.1cd 0.35 BC2F4 2-8-2-24 7.2 6.1 179.8bc 130.7ab 28.3 29.2 4.1b 13.3 220.8 10.4a-d 0.30 BC2F4 6-1/15-2-11 7.5 6.4 180.9bc 132.7ab 28.7 28.0 4.5b 13.6 174.9 11.2a-d 0.33 RD6 (RP) 9.0 6.3 204.9a 136.7a 27.4 29.0 6.7a 12.5 201.3 6.6d 0.25 Mean 7.7 6.2 185.6 128.5 27.7 29.0 4.7 13.6 204.8 11.59 0.31 F-Test ns ns * ns ns ns ns * ns CV (%) 12.34 8.46 5.78 6.71 3.14 4.62 16.06 6.16 12.7 34.49 28.7 Note: RP: recurrent parent; PN: panicle number per hill; PH (cm): plant height in centimeter; DTF: day to ﬂowering; TSW (g): thousand seed weight in gram; PL (cm): panicle length in centimeter; PNL (cm): panicle neck length in centimeter; FGPP: number of ﬁlled grain per panicle; UFG (%): unﬁlled grain percentage per panicle; HI: harvest index; CV (%): coeﬃcient of variation percentage; ns: * and ** are not signiﬁcant and signiﬁcantly diﬀerent at p ≤ 0.05 and p ≤0.01, respectively. The letter after each value indicates the signiﬁcance within each column by DMRT. Table 5. Agronomic performance of introgression lines possessing blast- and bacterial blight-resistant genes/QTLs and the recurrent parent RD6. Note: RP: recurrent parent; PN: panicle number per hill; PH (cm): plant height in centimeter; DTF: day to ﬂowering; TSW (g): thousand seed weight in gram; PL (cm): panicle length in centimeter; PNL (cm): panicle neck length in centimeter; FGPP: number of ﬁlled grain per panicle; UFG (%): unﬁlled grain percentage per panicle; HI: harvest index; CV (%): coeﬃcient of variation percentage; ns: * and ** are not signiﬁcant and signiﬁcantly diﬀerent at p ≤ 0.05 and p ≤0.01, respectively. 3.3. Grain Quality, Cooking and Eating Quality of the Introgression Lines with RD6 The letter after each value indicates the signiﬁcance within each column by DMRT. 8 of 15 Agronomy 2019, 9, 825 Table 6. Grain Yield (kg ha−1) of introgression lines and the recurrent parent RD6 over diﬀerent years and locations. Genotype a Ampawan 2018 Pool Mean 2015 2016 2018 KKU KRC BC2F3 2-7-5-36 7573.47 3950.37 7730.00 2792.67 4955.33 5400.37 BC2F3 2-8-2-36 4787.87 3942.23 6370.00 3793.20 6070.00 4992.66 BC2F3 2-7-5-43 7953.33 2541.97 7003.33 3327.07 5599.47 5285.03 BC2F3 2-8-2-19 5510.40 3764.10 ND 3672.93 5319.20 4566.66 BC2F3 2-8-2-25 7718.53 2714.90 8440.00 3235.60 5852.93 5592.39 BC2F3 9-1/15-1-28 5444.93 3218.87 6770.00 4232.13 5525.07 5038.20 BC2F3 2-8-2-27 5574.53 4587.73 7473.33 3388.80 5389.87 5282.85 BC2F3 2-8-2-52 5252.00 2760.87 8903.33 4127.20 4606.00 5129.88 BC2F3 2-8-2-24 6746.13 3911.17 6653.33 3088.27 3862.53 4852.29 BC2F3 6-1/15-2-11 7019.87 3195.97 6386.67 3907.33 5305.60 5163.09 RD6 (RP) 6480.27 3799.03 7140.00 2953.87 4616.93 4998.02 Mean 6369.21 3489.75 7286.99 3501.73 5191.18 5118.31 F-Test ns ns ns ns ns ns CV (%) 32.71 32.07 18.33 21.73 16.45 16.17 Note: a The generation of introgression lines were BC2F3, BC2F4, and BC2F4 in 2015, 2016, and 2018, respectively. RP, recurrent parent; KKU, Khon Kaen University; KRC, Khon Kaen Rice Research Center; ND, no data; ns, not signiﬁcant; CV (%), coeﬃcient of variation percentage. Table 6. Grain Yield (kg ha−1) of introgression lines and the recurrent parent RD6 over diﬀerent years and locations. Note: a The generation of introgression lines were BC2F3, BC2F4, and BC2F4 in 2015, 2016, and 2018, respectively. RP, recurrent parent; KKU, Khon Kaen University; KRC, Khon Kaen Rice Research Center; ND, no data; ns, not signiﬁcant; CV (%), coeﬃcient of variation percentage. Table 7. Cooking quality of introgression lines and the recurrent parent RD6. 3.3. Grain Quality, Cooking and Eating Quality of the Introgression Lines with RD6 Genotype QTL on Chromosome Aroma ASV GT AC BC2F5 2-7-5-36 Blast (1, 2, 11, 12) and xa5 2.3 5.7 55–69 3.5 BC2F5 2-8-2-36 Blast (1, 2, 11, 12) and xa5 1.8 5.7 55–69 5.4 BC2F5 2-7-5-43 Blast (1, 2, 11, 12) and xa5 1.8 4.9 70–74 6.9 BC2F5 2-8-2-19 Blast (1, 2, 11, 12) and xa5 1.7 5.3 70–74 4.7 BC2F5 2-8-2-25 Blast (1, 2, 11, 12) and xa5 2.0 5.3 70–74 5.1 BC2F5 9-1/15-1-28 Blast (1, 2, 11, 12) 1.9 5.3 70–74 4.7 BC2F5 2-8-2-27 No QTL 2.0 5.7 55–69 6.5 BC2F5 2-8-2-52 Blast (1, 2, 11, 12) and xa5 1.9 5.7 55–69 5.1 BC2F5 2-8-2-24 Blast (1, 2, 11, 12) and xa5 2.0 5.3 70–74 5.2 BC2F5 6-1/15-2-11 Blast (1, 2, 12) 1.5 5.3 70–74 5.3 RD6 (RP) - 2.1 5.3 70–74 5.2 Mean 1.9 5.4 - 5.2 F-test ns ns - ns CV (%) 18.14 10.03 - 22.71 Note: RP: recurrent parent; ASV: alkali spreading value; GT: gelatinization temperature; AC: amylose content; ns: nonsigniﬁcant; CV (%): coeﬃcient of variation percentage. Table 7. Cooking quality of introgression lines and the recurrent parent RD6. Note: RP: recurrent parent; ASV: alkali spreading value; GT: gelatinization temperature; AC: amylose content; ns: nonsigniﬁcant; CV (%): coeﬃcient of variation percentage. Note: RP: recurrent parent; ASV: alkali spreading value; GT: gelatinization temperature; AC: amylose content; ns: nonsigniﬁcant; CV (%): coeﬃcient of variation percentage. Agronomy 2019, 9, 825 was preferred fo like that of the or 9 of 15 ess, being (Table 8) (a) Seed Length (mm) 0 2 4 6 8 10 12 Genotypes BC2F5 2-7-5-36 BC2F5 2-8-2-36 BC2F5 2-7-5-43 BC2F5 2-8-2-19 BC2F5 2-8-2-25 BC2F5 9-1/15-1-28 BC2F5 2-8-2-27 BC2F5 2-8-2-52 BC2F5 2-8-2-24 BC2F5 6-1/15-2-11 RD6 (RP) (b) Seed Breadth (mm) 0 2 4 6 8 10 12 (c) Length/Breadth ratio 0 1 2 3 4 5 Genotypes BC2F5 2-7-5-36 BC2F5 2-8-2-36 BC2F5 2-7-5-43 BC2F5 2-8-2-19 BC2F5 2-8-2-25 BC2F5 9-1/15-1-28 BC2F5 2-8-2-27 BC2F5 2-8-2-52 BC2F5 2-8-2-24 BC2F5 6-1/15-2-11 RD6 (RP) (d) HRR and MRR (%) 0 20 40 60 80 100 HRR MRR Milled seed Whole seed ab b ab ab b b a a b ab ab c abc ab c ab ab bc bc a ab bc ab a-d d ab cd cd ab abc bcd cd a Figure 2. 3.3. Grain Quality, Cooking and Eating Quality of the Introgression Lines with RD6 Grain quality attributes of introgression lines in comparison to the recurrent parent RD6; (a) seed length of milled and whole seed, (b) seed breadth of milled and whole seed, (c) seed length/breadth ratio of milled and whole seed, and (d) milled rice recovery percent (MRR) and head rice recovery percent (HRR); RP: recurrent parent; the different letter at each graph show significant different among genotypes in each trait at p < 0.05. Figure 2. Grain quality attributes of introgression lines in comparison to the recurrent parent RD6; (a) seed length of milled and whole seed, (b) seed breadth of milled and whole seed, (c) seed length/breadth ratio of milled and whole seed, and (d) milled rice recovery percent (MRR) and head rice recovery percent (HRR); RP: recurrent parent; the diﬀerent letter at each graph show signiﬁcant diﬀerent among genotypes in each trait at p < 0.05. (a) Seed Length (mm) 0 2 4 6 8 10 12 Genotypes BC2F5 2-7-5-36 BC2F5 2-8-2-36 BC2F5 2-7-5-43 BC2F5 2-8-2-19 BC2F5 2-8-2-25 BC2F5 9-1/15-1-28 BC2F5 2-8-2-27 BC2F5 2-8-2-52 BC2F5 2-8-2-24 BC2F5 6-1/15-2-11 RD6 (RP) (b) Seed Breadth (mm) 0 2 4 6 8 10 12 Milled seed Whole seed ab b ab ab b b a a b ab ab c abc ab c ab ab bc bc a ab bc (c) Length/Breadth ratio 0 1 2 3 4 5 Genotypes BC2F5 2-7-5-36 BC2F5 2-8-2-36 BC2F5 2-7-5-43 BC2F5 2-8-2-19 BC2F5 2-8-2-25 BC2F5 9-1/15-1-28 BC2F5 2-8-2-27 BC2F5 2-8-2-52 BC2F5 2-8-2-24 BC2F5 6-1/15-2-11 RD6 (RP) (d) HRR and MRR (%) 0 20 40 60 80 100 HRR MRR ab a-d d ab cd cd ab abc bcd cd a Figure 2. Grain quality attributes of introgression lines in comparison to the recurrent parent RD6; (a seed length of milled and whole seed, (b) seed breadth of milled and whole seed, (c) see length/breadth ratio of milled and whole seed, and (d) milled rice recovery percent (MRR) and hea rice recovery percent (HRR); RP: recurrent parent; the different letter at each graph show significan different among genotypes in each trait at p < 0.05. Figure 2. Note: RP: recurrent parent; PR: Preference rank, with 1 being the most appealing and 7 the least. 4. Discussion Conventional breeding for disease resistance has proven to be time consuming and highly dependent on environmental conditions, in comparison to molecular breeding. The MAS, in particular, is simpler, more eﬃcient, and accurate. For crop improvement in both durability and multiple disease resistance, gene pyramiding is emphasized to integrate many complex biochemical pathways within a plant [13]. However, the introgression of resistant genes without maintaining or improving yield levels and the grain quality would oﬀer no reward; as such, varieties would not be adopted by farmers. Diﬀerent MAS approaches are now, in practice, using background and foreground selection [20,21], as well as combined approaches utilizing foreground selection with stringent phenotypic selection [22], and foreground selection coupled with stringent phenotypic selection and background analysis [21]. Such practices have aided in accurate and eﬃcient gene transfer, including gene pyramiding, into desired varietal backgrounds. In the present study, phenotypic selection was employed on the selected ILs for a higher degree of background recovery of the donor parent. g g g y p Blast disease caused by M. oryzae mainly occurs in two forms: seedling blast and panicle blast. In this study, the ILs with resistant QTLs genes showed outstanding resistance to leaf blast, as well as eﬀective resistance against panicle neck blast (Tables 3 and 4). On the other hand, the ILs without resistant QTLs genes, the RP RD6, and the susceptible check lines, proved susceptible to both leaf blast and neck blast under favorable environmental conditions. The results obtained through multitrials indicated that lines lacking resistant QTLs genes were susceptible to both blast and BB diseases, as evidenced through blast conidia, brown spots, and yellow blight when grown in the open ﬁeld under favorable weather conditions. Furthermore, the variations in disease incidence from one year to another indicated that weather conditions played an important role in both blast and BB incidence and development. Rainfall at the early ﬂowering stages signiﬁcantly aﬀected blast disease, especially neck blast, which occurred only in 2015 and 2018. Although high amounts of rainfall occurred in the ﬂowering stages in 2016, low plant density (low tiller number due to high water levers in the ﬁeld) eradicated neck blast disease (Figure 3b). Rainfall and relative humidity are the most important factors aﬀecting the sporulation, release, and germination of blast conidia as the microclimate within the plant canopy [10,23]. 3.3. Grain Quality, Cooking and Eating Quality of the Introgression Lines with RD6 Grain quality attributes of introgression lines in comparison to the recurrent parent RD6; (a) seed length of milled and whole seed, (b) seed breadth of milled and whole seed, (c) seed length/breadth ratio of milled and whole seed, and (d) milled rice recovery percent (MRR) and head rice recovery percent (HRR); RP: recurrent parent; the diﬀerent letter at each graph show signiﬁcant diﬀerent among genotypes in each trait at p < 0.05. 10 of 15 Agronomy 2019, 9, 825 Table 8. Mean Sensory scores for four attribute eating qualities of the introgression lines with RD6. Genotype Morning PR Afternoon PR Aroma Stiﬀness Softness Stickiness Aroma Stiﬀness Softness Stickiness BC2F5 2-7-5-36 1.9 1.8 1.9 2.1 1 1.7 1.0 1.6 1.8 1 BC2F5 2-8-2-36 2.0 1.5 1.8 2.2 3 1.6 1.7 1.2 1.5 5 BC2F5 2-7-5-43 1.7 1.5 1.4 1.3 4 1.8 1.6 1.5 1.5 3 BC2F5 2-8-2-19 1.8 2.3 1.9 1.8 4 1.6 2.2 1.6 1.6 6 BC2F5 2-8-2-25 1.6 1.7 1.3 2.0 5 1.6 1.8 1.0 1.0 7 BC2F5 9-1/15-1-28 1.6 1.5 1.8 1.7 4 1.7 1.7 1.5 1.0 5 BC2F5 2-8-2-27 2.1 1.0 2.5 2.3 2 1.8 1.0 2.0 1.6 2 BC2F5 2-8-2-52 1.5 1.0 2.4 1.9 2 1.7 1.0 1.9 1.7 2 BC2F5 2-8-2-24 1.5 2.0 1.8 1.9 3 1.6 1.5 1.6 1.8 5 BC2F5 6-1/15-2-11 1.6 1.4 1.6 1.8 4 1.5 1.4 1.5 1.8 4 RD6 (RP) 1.8 1.5 2.2 2.3 1 1.8 1.8 1.4 1.6 3 Note: RP: recurrent parent; PR: Preference rank, with 1 being the most appealing and 7 the least. Table 8. Mean Sensory scores for four attribute eating qualities of the introgression lines with RD6. Agronomy 2019, 9, 825 11 of 15 11 of 15 4. Discussion (a) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 June July August September Octuber November R l ti h idit (%) 0 20 40 60 80 100 Flowering period Sowing Transplanting Relative humidity (%) (b) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 Rainfall June July August September Octuber November Relative humidity (%) 0 20 40 60 80 100 Relative humidity (%) Flowering period Sowing Transplanting (d) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 Rainfall June July August September Octuber November Relative humidity (%) 0 20 40 60 80 100 Relative humidity (%) Flowering period S1 T1 S2 T2 S1=Sowing date1 S2=Sowing date2 T1=Transplanting date1 T2=Transplanting date2 (c) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 Rainfall June July August September Octuber November Relative humidity (%) 0 20 40 60 80 100 Sowing Transplanting Flooded Figure 3. Daily minimum and maximum temperature, rainfall, and relative humidity during the experimental years of 2015(a), 2016(b), 2017(c), and 2018(d). Figure 3. Daily minimum and maximum temperature, rainfall, and relative humidity during the experimental years of 2015(a), 2016(b), 2017(c), and 2018(d). As expected, most of the agronomical and quality traits (TN, PN, TSW, FGPP, PL, BPP, HI, GL, GW, L/B, MRR %, and HRR %) of the ILs with resistant QTLs genes remained similar to those of the RD6 (Table 5 and Figure 2). However, some of the ILs showed slight differences to the RD6 for some traits, such as PH, DTF, PNL, and UFG% (Table 5). Panicle neck length, known as ‘panicle exertion’ (PE), is considered to be essential for enhancing total sink capacity and grain filling by improving the transport efficiency of assimilates [29]. Incomplete PE (also referred to as ‘sheathed panicle’) is an adverse symptom observed in rice plants which is associated with almost all cytoplasmic male sterile lines and which damages grain yield and raises disease incidence [30]. In our study, all of the ILs exhibited complete panicle neck lengths lower than those of the RD6 (Table 5) within similar parameters of ideal rice panicle types [31]. 4. Discussion Interestingly, Jao Hom Nin, the donor parent for the blast resistance QTL on chromosome 1 and 11, also proved to be resistant to bacterial blight (Table 4). This may be because the gene contains a form of resistance to some BB pathogens strains, further indicating that this variety may be useful as a multidisease resistance source. Incredibly, DP IR62266, used for introgression xa5 gene, as well as most of the ILs studied, were susceptible to BB diseases, perhaps because a single resistance gene was introgressed for resistance against BB, and easily overcome by the pathogen, which can be explained by the gene-for-gene theory [24]. Furthermore, Kosawang et al. [25] reported that the pathogen is highly diverse in nature, particularly in Thailand, and several studies have indicated that cultivars with single BB-resistant genes do not provide broad-spectrum resistance [26–28]. Because several races of a pathogen within a rice-growing region can breakdown resistance, we have attempted to identify a new source of BB resistant genes (or combinations of xa5 with other BB resistant genes) necessary for the further improvement of rice varieties in Thailand. In the present study, we deployed a phenotypic selection for agromorphological traits in the later generations to identify backcross-resistant plants which are not only the closest to RD6, but also superior to the elite mega-varieties. Data from multilocations and numerous trial years demonstrated that the ILs had a potential yield similar or higher than the original RD6 (Table 6), as well as being well adapted to the rain-fed lowland environment in Thailand. We found that the ILs BC2F3 2-8-2-25, BC2F3 2-7-5-36, and BC2F3 2-7-5-43 produced higher grain yields (5592.39, 5400.37, and 5285.03 kg/ha, respectively) than those of the RP RD6 and other ILs through each season. Also, the percentage of milling and head rice recovery were more displaced than the RP RD6 (Figure 2). Furthermore, despite an incidence of blast infection at both AP (2015) and KKU (2018), rice yields were still higher than those of the RP RD6 and the nonresistant ILs (Table 6). This may be due to the similar genome content of the 12 of 15 e to the Agronomy 2019, 9, 825 were still higher th line with the recipient parent and the lower severity index of the blast and bacterial blight diseases of the genotype, as compared to the RP RD6. 4. Discussion This also implies that the MAS is an eﬀective RP RD6 approach to improve resistance to biotic stress in Jasmine rice [14,15]). These high levels of resistance to blast and BB, as well as the variations in yield potential, are a successful example of the integrated approach of selection at both the molecular and phenotypic level. g p p y and bacterial blight diseases of the genotype, as compared to the RP RD6. This also implies that the MAS is an effective RP RD6 approach to improve resistance to biotic stress in Jasmine rice [14,15]). These high levels of resistance to blast and BB, as well as the variations in yield potential, are a successful example of the integrated approach of selection at both the molecular and phenotypic level. (a) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 Rainfall June July August September Octuber November Relative humidity (%) 0 20 40 60 80 100 Relative humidity (%) Flowering period Sowing Transplanting (b) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 Rainfall June July August September Octuber November Relative humidity (%) 0 20 40 60 80 100 Relative humidity (%) Flowering period Sowing Transplanting (c) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 Rainfall June July August September Octuber November Relative humidity (%) 0 20 40 60 80 100 Relative humidity (%) Sowing Transplanting Flooded (d) Month 1/6/2015 1/7/2015 1/8/2015 1/9/2015 1/10/2015 1/11/2015 Amount of rainfall (mm.) 0 10 20 30 40 50 60 70 80 90 Rainfall June July August September Octuber November Relative humidity (%) 0 20 40 60 80 100 Relative humidity (%) Flowering period S1 T1 S2 T2 S1=Sowing date1 S2=Sowing date2 T1=Transplanting date1 T2=Transplanting date2 Figure 3. Daily minimum and maximum temperature, rainfall, and relative humidity during the experimental years of 2015(a), 2016(b), 2017(c), and 2018(d). Figure 3. Daily minimum and maximum temperature, rainfall, and relative humidity during the experimental years of 2015(a), 2016(b), 2017(c), and 2018(d). 4. Discussion These agronomical trait variations might be due to a linkage drag of the donor segments with the target trait [32]. This is because some genes associated with undesirable traits are tightly linked with R genes on the rice genome [33]. As expected, most of the agronomical and quality traits (TN, PN, TSW, FGPP, PL, BPP, HI, GL, GW, L/B, MRR %, and HRR %) of the ILs with resistant QTLs genes remained similar to those of the RD6 (Table 5 and Figure 2). However, some of the ILs showed slight diﬀerences to the RD6 for some traits, such as PH, DTF, PNL, and UFG% (Table 5). Panicle neck length, known as ‘panicle exertion’ (PE), is considered to be essential for enhancing total sink capacity and grain ﬁlling by improving the transport eﬃciency of assimilates [29]. Incomplete PE (also referred to as ‘sheathed panicle’) is an adverse symptom observed in rice plants which is associated with almost all cytoplasmic male sterile lines and which damages grain yield and raises disease incidence [30]. In our study, all of the ILs exhibited complete panicle neck lengths lower than those of the RD6 (Table 5) within similar parameters of ideal rice panicle types [31]. These agronomical trait variations might be due to a linkage drag of the donor segments with the target trait [32]. This is because some genes associated with undesirable traits are tightly linked with R genes on the rice genome [33]. Marker‐assisted selection (MAS) is a highly‐efficient breeding strategy for the introduction of disease R genes from both cultivated and exotic germplasms into adaptive genetic backgrounds Marker-assisted selection (MAS) is a highly-eﬃcient breeding strategy for the introduction of disease R genes from both cultivated and exotic germplasms into adaptive genetic backgrounds across generations. However, if the donor parent used is a native landrace, the linkage drag problem may be 13 of 15 Agronomy 2019, 9, 825 compounded [34]. In our study, three of the DPs are nonglutinous rice, possessing nonaromatic grains. However, we found that after the backcross of two terms, the most important factor determinants in grain and cooking quality, such as grain size and shape, aroma, GT, and AC (%), were comparable to those of the RP RD6 (Figure 2 and Table 7). 4. Discussion They therefore achieved consumer preference due to their excellent swelling capacity, sweetness, aroma, grain shape and size, and stickiness, i.e., like that of the original RD6 (Table 8). Our research clearly proved that a cross between glutinous and nonglutinous varieties [14,15] was possible through the conversion of glutinous RD6 NIL, resulting in eﬀective resistance to blast and BB, as well as possessing excellent qualities. In general, it requires six to eight backcross cycles to gain the background of the recurrent parent. However, in later generations (i.e., after BC2), it may be impossible to discriminate between backcross progeny and the RP based on individual plants [35]. In the present study, ILs BC2F3 2-8-2-25, BC2F3 2-7-5-43, BC2F3 9-1/15-1-28, BC2F3 2-7-5-36, and BC2F3 2-8-2-52 expressed eﬀective resistance to both leaf blast and neck blast. Some ILs showed resistant and moderately resistant reactions to BB disease, as well as having higher grain yields than those of the RP RD6. Furthermore, the agronomic characteristics and grain and cooking quality attributes were similar to those of the original RD6. Hence, we expect that it could be easily promoted among farmers who have shown a preference for the original RD6, thus negating the need to further backcross with RD6 again. Similar to the ﬁndings of Suwannual et al. [14] and Pinta et al. [15], we observed that the use of a pseudo-backcrossing design and MAB for two generations without genetic background selection was suﬃcient to recover the majority of the desired characteristics of the original RD6 within the BC2F5 generation. The development of broad-spectrum and multiple disease resistance against blast and BB in Thailand is a major challenge, as rice cultivation areas have the presence of a number of genetically-distinct virulent pathogen strains in diﬀerent geographical areas of the country. The present study demonstrated that the deployment of a four-QTL combination provided broad-spectrum resistance for blast, whereas a single xa5 was unable to achieve durable and broad-spectrum resistance in many BB-prone rice growing areas in Thailand. Author Contributions: Data curation: M.S.A.N., J.J., A.S. and T.M.; Formal analysis: M.S.A.N. and S.C.; Methodology: M.S.A.N., T.M., S.C. and J.S.; Resources: S.C., T.M., and J.S.; Software: M.S.A.N. and S.C.; Supervision: S.C., T.M. and J.S.; Validation: M.S.A.N., S.C., T.M. and J.S.; Preparation of original draft: M.S.A.N. and S.C.; Writing, review, and editing: T.M., S.C. and J.S. 5. Conclusions The ILs with resistant QTLs genes (BC2F3 2-7-5-36, BC2F3 2-7-5-43, BC2F3 2-8-2-25, BC2F3 2-8-2-52, and BC2F3 6-1/15-2-11) are shown to be promising NILs, due to their superior characteristics in terms of yield, morphology, physico-chemical properties, and resistance. Our results indicate that BC2F3 2-8-2-25 generated greater yield output compared to other ILs. It also produced long and slender grains, satisfactory milled and head rice recovery, aroma, GT, and an AC % similar to those of the original RD6. These lines also demonstrated better resistance against leaf blast and neck blast. However, only the IL BC2F3 2-8-2-52 showed resistant against bacterial blight disease. Thus, these new rice lines deliver the prospect of successful combination with xa5, as well as other BB resistance genes, in order to achieve durable, broad-spectrum resistance in many BB-prone rice growing areas in Thailand. Author Contributions: Data curation: M.S.A.N., J.J., A.S. and T.M.; Formal analysis: M.S.A.N. and S.C.; Methodology: M.S.A.N., T.M., S.C. and J.S.; Resources: S.C., T.M., and J.S.; Software: M.S.A.N. and S.C.; Supervision: S.C., T.M. and J.S.; Validation: M.S.A.N., S.C., T.M. and J.S.; Preparation of original draft: M.S.A.N. and S.C.; Writing, review, and editing: T.M., S.C. and J.S. Funding: This research received no external funding. Acknowledgments: This research was supported by The Plant Breeding Research Center for Sustainable Agriculture and The Salt-Tolerance Rice Research Group, Khon Kaen University, Khon Kaen, Thailand. Our gratitude is also extended to the Kachin State Agriculture Institute (Laiza) for providing ﬁnancial support on behalf of Mr. Myo San Aung Nan. Conﬂicts of Interest: The authors declare no conﬂict of interest. s of Interest: The authors declare no conﬂict of interes 14 of 15 Agronomy 2019, 9, 825 References 1. Khush, G.S. What it will take to Feed 5.0 Billion Rice consumers in 2030. Plant Mol. Biol. 2005, 59, 1–6. [CrossRef] 2. Oﬃce of Agricultural Economics. Report on Survey of Rice Growing Areas in Thailand Crop Year 2012/2013; Center for Agricultural Information, Oﬃce of Agricultural Economics, Ministry of Agri-culture and Cooperation: Bangkok, Thailand, 2013. 3. Kharkwal, M.C.; Shu, Q.Y. The role of induced mutations in world food security. In Induced Plant Mutations in the Genomics Era; Shu, Q.Y., Ed.; Food and Agriculture Organization of the United Nations: Rome, Italy, 2009; pp. 33–38. pp 4. Mapnet. Sub-District Level Agricultural Survey of North-East Thailand, 1997; North-eastern Regional Agricultural Extension Oﬃce: Nakhon Ratchasima, Thailand, 1999. 5. Wongsaproma, C.; Sirithunyab, P.; Vanavichitc, A.; Pantuwand, G.; Jongdeee, B.; Sidhiwongf, N.; Lanceras-Siangliwa, J.; Toojindaa, T. Two introgressed quantitative trait loci confer a broad-spectrum resistance to blast disease in the genetic background of the cultivar RD6 a Thai glutinous jasmine rice. Field Crops Res. 2010, 119, 245–251. [CrossRef] 6. June. International Rice Research Notes; International Rice Research Institute: Los Banos, Philippines, 1994; Volume 19, pp. 1–42. 7. Sesma, A.; Osbourn, A.E. The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi. Nature 2004, 431, 582–586. [CrossRef] 8. Titone, P.; Mongiano, G.; Tamborini, L. Resistance to neck blast caused by Pyricularia oryzae in Italian rice cultivars. Eur. J. Plant Pathol. 2015, 142, 49–59. [CrossRef] 9. Sirithunya, P.; Tragoonrung, S.; Vanavichit, A.; Pa-In, N.; Vongsaprom, C.; Toojinda, T. Quantitative trait loci associated with leaf and neck blast resistance in recombinant inbred line population of rice (Oryza sativa). DNA Res. 2002, 9, 79–88. [CrossRef] 10. Ou, S.H. Rice Diseases, 2nd ed.; Common Wealth Mycological Institute: Surrey, UK, 1985; pp. 61–96 11. Mew, T.W. Current status and future prospects of research on bacterial blight of rice. Annu. Rev. Phytopathol. 1987, 25, 359–382. [CrossRef] 12. Zheng, C.K.; Wang, C.L.; Yu, Y.J.; Liang, Y.T.; Zhao, K.J. Identiﬁcation and Molecular Mapping of Xa32(t), a Novel Resistance Gene for Bacterial Blight (Xanthomonas oryzae pv. oryzae) in Rice. Acta Agron. Sin. 2009, 35, 1173–1180. [CrossRef] 13. Kumar, J.R.; Nayak, S. Gene pyramiding-A broad spectrum technique for developing durable stress resistance in crops. Biotechnol. Mol. Biol. Rev. 2010, 5, 51–60. 14. Suwannual, T.; Chankaew, S.; Monkham, T.; Saksirirat, W.; Sanitchon, J. Pyramiding of four blast resistance QTLs into Thai rice cultivar RD6 through marker-assisted selection. Czech J. Genet. Plant Breed. 2009, 53, 1–8. References [CrossRef] 15. Pinta, W.; Toojinda, T.; Thummabenjapone, P.; Sanitchon, J. Pyramiding of blast and bacterial leaf blight resistance genes into rice cultivar RD6 using marker assisted selection. Afr. J. Biotechnol. 2013, 12, 4432–4438. 15. Pinta, W.; Toojinda, T.; Thummabenjapone, P.; Sanitchon, J. Pyramiding of blast and bacterial leaf blight resistance genes into rice cultivar RD6 using marker assisted selection. Afr. J. Biotechnol. 2013, 12, 4432–4438. 16. International Rice Research Institute. Standard Evaluation System for Rice; International Rice Research Institute: Los Banos, Philippines, 1996; Volume 19, pp. 1–42. 16. International Rice Research Institute. Standard Evaluation System for Rice; International Rice Research Institute: Los Banos, Philippines, 1996; Volume 19, pp. 1–42. 17. Little, R.R.; Hilder, G.B.; Dawson, E.H. Diﬀerential eﬀect of dilute alkali on 25 varieties of milled white rice. Cereal Chem. 1958, 35, 111–126. 18. Yi, M.; Nwe, K.T.; Vanavichit, A.; Chai-arree, W.; Toojinda, T. Marker assisted backcross breeding to improve cooking quality traits in Myanmar rice cultivar Manawthukha. Field Crops Res. 2009, 113, 178–186. [CrossRef] 19. Juliano, B.O. A simpliﬁed assay for milled-rice amylose. Cereal Foods World 1971, 16, 334–340. 19. Juliano, B.O. A simpliﬁed assay for milled-rice amylose. Cereal Foods World 1971, 16, 334–340. 20. Basavaraj, S.H.; Singh, V.K.; Singh, A.; Singh, A.; Singh, A.; Anand, D.; Yadav, S.; Ellur, R.K.; Singh, D.; Gopalakrishnan, S.; et al. Marker-assisted improvement of bacterial blight resistance in parental lines of Pusa RH10, a superﬁne grain aromatic rice hybrid. Mol. Breed. 2010, 26, 293–305. [CrossRef] 21. Singh, V.K.; Singh, A.; Singh, S.P.; Ellur, R.K.; Singh, D.; Gopala Krishnan, S.; Bhowmick, P.K.; Nagarajan, M.; Vinod, K.K.; Singh, U.D.; et al. Marker-assisted simultaneous but stepwise backcross breeding for pyramiding blast resistance genes Piz5 and Pi54 into an elite Basmati rice restorer line “PRR78”. Plant Breed. 2013, 132, 486–495. 15 of 15 15 of 15 Agronomy 2019, 9, 825 22. Gopalakrishnan, S.; Sharma, R.K.; Anand, R.K.; Joseph, M.; Singh, V.P.; Singh, A.K.; Bhat, K.V.; Singh, N.K.; Mohapatra, T. Integrating marker assisted background analysis with foreground selection for identiﬁcation of superior bacterial blight resistant recombinants in Basmati rice. Plant Breed. 2008, 127, 131–139. [CrossRef] 23. Ghini, R.; Hamada, E.; Bettiol, W. Climate change and plant diseases. Sci. Agric. 2008, 65, 98–107. [C 4. Flor, H.H. Current status of the gene-for-gene. Annu. Rev. Phytopathol. 1971, 9, 275–296. [CrossRef] 25. Kosawang, C.; Smitamana, P.; Toojinda, T.; Nilpanit, N.; Sirithunya, P. © 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/). References Ampliﬁed fragment length polymorphism ﬁngerprinting diﬀerentiates genetic diversity of Xanthomonas oryzae pv. oryzae from northern Thailand. J. Phytopathol. 2006, 154, 550–555. [CrossRef] 26. Mew, T.W.; Cruz, V.C.M.; Medalla, E.S. Changes in race frequency of Xanthomonas oryzae pv. oryzae in response to rice cultivars planted in the Philippines. Plant Dis. 1992, 76, 1029–1032. [CrossRef] 27. Nelson, R.J.; Baraoidan, M.R.; Vera Cruz, C.M.; Yap, I.V.; Leach, J.E.; Mew, T.W.; Leung, H. Relationship between phylogeny and pathotype for the bacterial blight pathogen of rice. Appl. Environ. Microbiol. 1994, 60, 3275–3283. [PubMed] 28. George, M.L.C.; Bustamam, M.; Cruz, W.T.; Leach, J.E.; Nelson, R.J. Movement of Xanthomonas oryzae pv. oryzae in Southeast Asia Detected Using PCR-Based DNA Fingerprinting. Phytopathology 1997, 87, 302–309. [CrossRef] [PubMed] Cruz, R.P.D.; Milach, S.C.K.; Federizzi, L.C. Inheritance of pinnacle exertion in rice. Sci. Agric. 2008, 65 502–507. [CrossRef] 30. Shen, Z.T.; Yang, C.D.; He, Z.H. Studies on eliminating panicle enclosure in WA type MS line of rice Oryza sativa subsp. indica. Chin. J. Rice Sci. 1987, 1, 95–99. 31. Xu, Z.; Chen, W.; Zhang, L.; Yang, S. Design principles and parameters of rice ideal panicle type. Chin. Sci. Bull. 2005, 50, 2253–2256. [CrossRef] 32. Neeraja, C.N.; Maghirang-Rodriguez, R.; Pamplona, A.; Heuer, S.; Collard, B.C.Y.; Septiningsih, E.M.; Vergara, G.; Sanchez, D.; Xu, K.; Ismail, A.M.; et al. A marker-assisted backcross approach for developing submergence-tolerant rice cultivars. Theor. Appl. Genet. 2007, 115, 767–776. [CrossRef] 33. Fukuoka, S.; Norikuni, S.; Hironori, K.; Kazuko, O.; Takehiko, S.; Kaworu, E.; Nagao, H.; Takahashi, A.; Hirochika, H.; Okuno, K.; et al. Loss of function of a proline-containing protein confers durable disease resistance in Rice. Science 2009, 325, 998–1001. [CrossRef] 34. Stam, P.; Zeven, A.C. The theoretical proportion of the donor genome in near-isogenic lines of self-fertilizers bred by backcrossing. Euphytica 1981, 30, 227–238. [CrossRef] 35. Hasan, M.M.; Raﬁi, M.Y.; Ismail, M.R.; Mahmood, M.; Rahim, H.A.; Alam, M.A.; Ashkani, S.; Malek, M.A.; Latif, M.A. Marker-assisted backcrossing: A useful method for rice improvement. Biotechnol. Biotechnol. Equip. 2015, 29, 237–254. [CrossRef] © 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/).
W1984319693.txt	https://digibuo.uniovi.es/dspace/bitstream/10651/23938/1/Adult%20Patients.pdf	en		Adult Patients with Eosinophilic Esophagitis Do Not Show an Increased Frequency of the HLA-DQ2/DQ8 Genotypes Predisposing to Celiac Disease	Digestive diseases and sciences	2,010	cc-by	3,193	Dig Dis Sci (2011) 56:1107–1111 DOI 10.1007/s10620-010-1383-2 ORIGINAL ARTICLE Adult Patients with Eosinophilic Esophagitis Do Not Show an Increased Frequency of the HLA-DQ2/DQ8 Genotypes Predisposing to Celiac Disease Alfredo J. Lucendo • Ángel Arias • Isabel Pérez-Martı́nez • Antonio López-Vázquez • Jesús Ontañón-Rodrı́guez • Sonia González-Castillo • Livia C. De Rezende • Luis Rodrigo Received: 1 June 2010 / Accepted: 29 July 2010 / Published online: 20 August 2010 Ó Springer Science+Business Media, LLC 2010 Abstract Background Recent articles have described patients that share eosinophilic esophagitis (EoE) and celiac disease (CD) suggesting a true relationship between both diseases. Aims The purpose of this study was to investigate whether HLA DQ2 and DQ8 predisposing to CD are increased in adult patients with EoE. Methods HLA alleles conferring risk for CD was assessed in 75 adult EoE patients attended at two hospitals located in different Spanish regions over the past 2 years. We compared the frequencies to the registered data of 421 healthy kidney and bone marrow donors from our hospitals for the following alleles: (a) DR3-DQ2 haplotype; (b) the combination of DR3-DQ2 and DR4-DQ8; (c) DR4-DQ8 haplotype; (d) the simultaneous presence of the DR5-DQ7 and DR7-DQ2 haplotypes; and lastly (e) any combination A. J. Lucendo (&) S. González-Castillo L. C. De Rezende Department of Gastroenterology, Hospital General de Tomelloso, Vereda de Socuéllamos, s/n, 13700 Tomelloso, Ciudad Real, Spain e-mail: alucendo@vodafone.es Á. Arias Research Unit, Complejo Hospitalario La Mancha Centro, Alcázar de San Juan, Spain I. Pérez-Martı́nez L. Rodrigo Department of Gastroenterology, Hospital Central de Asturias, Oviedo, Spain A. López-Vázquez Department of Immunology, Hospital Central de Asturias, Oviedo, Spain J. Ontañón-Rodrı́guez Department of Immunology, Hospital General Universitario de Albacete, Albacete, Spain of haplotypes not conferring risk for the development of CD. Results The HLA DQ2 and DQ8 alleles were analyzed in 58 adult EoE patients from hospital #1 and in 20 patients from hospital #2, and they were compared to recorded HLA genotyping data from 298 and 123 healthy donors, respectively. No differences were found between the distribution of the HLA frequencies of the patients and controls at both hospitals and the data could be combined. EoE patients did not show increased frequencies of DQ2 and DQ8 alleles compared to controls. Conclusions Our work does not allow us to establish a common genetic basis for EoE and CD because an increased frequency of the HLA DQ2 and DQ8 alleles predisposing to CD was not observed in adult EoE patients compared to controls. Keywords Eosinophilic esophagitis Celiac disease HLA antigens DQ2 genotype DQ8 genotype Introduction Celiac disease (CD) and eosinophilic esophagitis (EoE) are two different well-known diseases which affect the gastrointestinal mucosa. The former is located in the small bowel and the latter in the esophagus. Both diseases are predominantly diagnosed in young patients, but can appear at any stage of life. EoE is an immunoallergic disease characterized by chronic, recurrent esophageal symptoms and dense esophageal eosinophilic infiltrate [1]. The pathogenesis of EoE includes a Th2-type immunological reaction, which is triggered by exposure to dietary allergens causing infiltration of the esophageal mucosa by T lymphocytes, mast 123 1108 cells and eosinophils [2]. The prevalence of EoE has risen over the last few decades to the same extent as other immunoallergic diseases. CD is an autoimmune disease triggered by the ingestion of food containing gluten and manifests in genetically susceptible individuals, giving rise to different types of lesions in the small bowel mucosa [3, 4]. CD affects between 1 and 3% of the European and US population at some stage in life [5]. CD has a higher class II MHC association than detected previously in many other autoimmune diseases [6]. Approximately 90% of celiac patients carry the HLA-DQ2 heterodimer which is encoded by the DQA105 and DQB102 genes carried either in cis position on the DR3-DQ2 haplotype, which is common to many autoimmune diseases, or in trans, where the a chain is encoded on the DR5-DQ7 (DRB111/12, DQA10505, DQB10301) haplotype on one chromosome and the b chain on the DR7-DQ2 (DRB107, DQA10201, DQB10202) haplotype on the other chromosome [7]. Most patients who are DQ2 negative carry the DQ8 genotype (DRB104, DQA10301, DQB1*0302). DQ2 and DQ8 molecules present gluten peptides or related antigens to disease-specific CD4? T cells. The relationship between EoE and CD was recently brought to our attention and both disorders could have various aspects in common. A number of case reports have been published in relation to children displaying signs of both diseases [8–11] and a true association is suggested to exist between them. However, so far, the relationship between EoE and CD has not been systematically studied. This article analyzes the presence of HLA DQ2 and HLA DQ8 conferring risk for CD in a large group of EoE patients at two Spanish hospitals. It compares the distribution of these alleles to the reference frequency observed in a wide group of healthy donors from our hospitals, representing the general Spanish population. Patients and Methods The presence of HLA alleles conferring risk for CD was assessed in all the adult EoE patients (C16 year old) who were attended at two general hospitals in different Spanish regions over the past 2 years (#1, Hospital General de Tomelloso and #2, Hospital Central de Asturias). EoE was diagnosed based on accepted criteria following the examination of esophageal, gastric and duodenal biopsies [1]. EoE was diagnosed by esophageal and/or upper gastrointestinal tract symptoms accompanied by C15 intraepithelial eosinophils/HPF in 1 or more biopsy specimens without pathologic GERD, as shown by normal pH monitoring of the distal esophagus or the lack of response to 123 Dig Dis Sci (2011) 56:1107–1111 high-dose proton pump inhibitor (PPI) medication. Gastric and duodenal biopsies were assessed in order to exclude eosinophilic gastroenteritis. Villous atrophy and lymphocytic infiltration were also excluded in duodenal samples. Four different haplotypes for CD were analyzed in all patients using the polymerase chain reaction single nucleotide polymorphism techniques (PCR-SNPs) performed on peripheral blood leukocytes. We specifically assessed: (a) the presence of the DR3-DQ2 haplotype with no distinction between homozygosis or heterozygosis with another nonrisk allele; (b) the presence of the DR3-DQ2—DR4-DQ8 combination; (c) the expression of DR4-DQ8 haplotype (with no distinction between homozygosis or heterozygosis with another non-risk allele); (d) the simultaneous presence in trans combination of the DR5-DQ7 and DR7-DQ2 haplotypes; and (e) any combination not conferring risk for the development of CD. The estimated distribution of the alleles in the reference populations was obtained from the registered data of 421 unrelated healthy individuals compiled in two databases, of which 298 were kidney and bone marrow donors from the area of hospital #1, and 123 were bone marrow donors from the area of hospital #2. The geographical origin of the numerous control groups enabled them to represent the distribution of the alleles in each reference population. We compared the relative frequencies (percentages) of the distribution of antigens between the patient and control groups using chi-square tests (Fisher’s exact test, where appropriate). Statistical analyses were performed using statistical analysis software PASW 18.0 (SPSS Inc). This research was carried out in accordance with the Helsinki Declaration and approved by the ethics committees in our institutions. Results HLA alleles predisposing to CD were analyzed in 58 adult EoE patients attended at hospital #1 (50 males, 8 females; mean age 32.2 years, standard deviation [SD] 12.7) and in 20 at hospital #2 (14 males, 6 females; mean age 36.55 years, SD 20.28) and were compared to registered HLA frequencies in the donor databases at each hospital, respectively. We did not observe any increased frequency of the DQ2 and DQ8 alleles in the EoE patients at each hospital compared to the respective control groups (Table 1). In statistical terms, no significant differences were observed regarding each HLA haplotype in the respective control and patient groups at each hospital (P [ 0.05), and the data could be combined. Overall, there were no differences in the distribution of the proportions of each analyzed haplotype between EoE Dig Dis Sci (2011) 56:1107–1111 1109 Table 1 Distribution and frequencies of the HLA DQ2 and DQ8 alleles conferring risk for celiac disease (CD) in two series of EoE patients at two Spanish hospitals compared to the frequencies of HLA CD-related HLA antigens Hospital #1 Number in patient group DR3 DQ2 DR3 DQ2/DR4 DQ8 DQ2 and DQ8 in the control groups comprising healthy kidney and bone marrow donors at each hospital Hospital #2 % Number in control group % P Number in patient group % Number in control group % P 10 17.24 76 25.5 0.18 5 25 28 22.7 0.78 3 5.17 6 2 0.17 1 5 4 3.2 0.54 DR4 DQ8 4 6.90 49 16.4 0.06 1 5 15 12.2 DR7 DQ2/DR5 DQ7 4 6.90 12 4 0.31 1 5 8 6.5 155 52 0.1 298 100 Haplotype not conferring risk 37 Total 58 63.79 100 12 60 68 20 100 123 55.3 0.7 1 0.69 100 The DR4-DQ8 haplotype was more frequent in the control groups than in the patient groups, which gained statistical significance following the inclusion of patient series from both hospitals patients and controls, except for the DR4-DQ8 haplotype, which was significantly increased in the control group compared to the EoE patients (P = 0.039). These data are shown in Table 2 and Fig. 1. Haplotype not conferring risk 52,97 62,82 4,75 DR7 DQ2 / DR5 DQ7 DR4 DQ8 15,20 2,38 p=0.25 DR3 DQ2 Discussion 24,70 p=0.29 19,23 0 The proportion of the DR4 DQ8 haplotype was significantly higher in the control groups than the EoE patients (P = 0.039) Controls Patients p=0.039 6,41 5,13 Table 2 Distribution and frequencies of the HLA DQ2 and DQ8 alleles in our series of EoE patients at two Spanish hospitals compared to the control groups p=0.57 6,41 DR3 DQ2 / DR4 DQ8 Our work analyzes the frequency of the well-known classII MHC antigens predisposing to CD—HLA DQ2 and DQ8—in a series of adult EoE patients at two Spanish hospitals. Both centers have genetically homogenous patients as no differences in allele distribution were observed between them. Given that the frequencies of haplotypes predisposing to CD were not increased in EoE patients compared to the control groups, we are unable to establish a true association between both disorders. Over the past few years, several studies have identified a potential relationship between both diseases based on certain clinical observations. In a case series of ten patients with EoE correlatively diagnosed in Australia, it was reported that eight out of them expressed the HLA-DQ2 haplotype (with a frequency affecting approximately 45% of the local population), and one more was DQ8? [12]. In an Italian pediatric case series of 16 EoE patients, six children were simultaneously diagnosed with CD based on p=0.11 10 20 30 40 50 60 70 % Fig. 1 Comparative representation of the frequencies of each HLA allele conferring risk for CD in our series of EoE patients and control groups from our study. No statistical differences were identified between the two study groups serological criteria and duodenal villous atrophy [10]. Another Italian case report presented three children with both EoE and CD who showed favorable clinical and histological esophageal evolution after they followed glutenfree diets [9]. Lastly, a recently published study aimed to show the relationship between both diseases by estimating the prevalence of EoE among children diagnosed with CD at an Australian institution over an 8-year period. The authors discovered that at least 4% of the children with CD also suffered from EoE, and they suggested that this percentage could be higher if esophageal biopsies had been systematically sampled in each patient undergoing an HLA antigens Number of patients DR3 DQ2 % Number of controls % P 15 19.23 104 24.70 DR3 DQ2/DR4 DQ8 4 5.13 10 2.38 0.25 DR4 DQ8 5 6.41 64 15.20 0.039 DR7 DQ2/DR5 DQ7 0.29 5 6.41 20 4.75 0.57 Haplotype not conferring risk 49 62.82 223 52.97 0.11 Total 78 100 421 100 123 1110 endoscopy for suspected CD [11]. In contrast to these findings, the majority of them referred to children; our study was unable to demonstrate a common genetic basis for EoE and CD in adults. A direct relationship between both diseases cannot be supported attending to the frequency of genetic susceptibility markers as opposed to in the case of certain autoimmune diseases [13], the prevalence of which is much higher in CD, especially type-1 diabetes mellitus and autoimmune thyroid disorders [14]. Some reasons could have led some authors to consider the possibility of a real relationship between EoE and CD. Both are mainly chronic inflammatory digestive diseases determined by exposure to certain dietary components. Both EoE and CD are frequently associated with a wide variety of extra intestinal manifestations and clinically respond to the elimination of specific foods from the diet or to treatment using steroidal anti-inflammatory drugs. Both diseases clearly run in families. However, CD is a genetically determined disorder affecting individuals who majorly express the HLA DQ2 and DQ8 haplotypes, which are considered necessary but are not responsible for the development of the disease alone. This genetic basis determines a frequent association of cases within the same family, reaching up to 20% in first degree relatives and up to 75% in monozygotic siblings [15]. Many cases of EoE have also been described to run in families [16–19]. This would indicate that the disease has a relatively strong genetic background and no differences in clinical, pathological or molecular characteristics have been observed between sporadic and family-related cases of EoE [20]. Furthermore, given the high prevalence of CD in Europe [5], this condition might simply overlap with EoE as a coincidence. This would explain some of the cases in the literature where the two diseases do clearly co-exist. Conversely, several epidemiological reasons distinguish CD from EoE, e.g. CD predominantly affects females at a ratio of 2:1 irrespective of race [15], and differences were observed in different geographical regions in accordance with dietary habits (i.e. more rice than wheat in the staple Asian diet). EoE predominates in males, with a 3:1 ratio to women, and has been mainly described in Caucasians [21, 22]. Although CD patients may present respiratory or skin symptoms [15], atopic manifestations are nearly always associated with EoE [23]. Our work shows that frequencies of risk alleles for CD are not increased in EoE adult patients compared to the control population. Consequently, we cannot establish a common genetic basis for both diseases because we lack sufficient evidence of HLA DQ2 and DQ8 predisposing to the development of EoE. We unexpectedly found a significantly higher frequency of the DR4-DQ8 haplotype in the control group compared to EoE patients (P = 0.039). Although the results were difficult to interpret, we assume 123 Dig Dis Sci (2011) 56:1107–1111 that EoE patients express the DR4-DQ8 allele with a reduced frequency, which is only present in about 10% of celiac patients. This data, which must be confirmed by further studies, encourages us to carry out further research on the genetic markers and on the putative role of different antigen-presenting molecules in the development of EoE. Furthermore, our results do not completely rule out the possible role of gluten exposure in a small number of EoE patients exhibiting HLA DQ2 and/or DQ8. Our study shows that these alleles are not present in EoE at a greater rate than in healthy controls, but it does not provide any detail into the reverse issue, i.e. if patients with celiac disease are more likely to have EoE, as suggested by a recent paper [11]. We should bear in mind that esophageal eosinophilic infiltration could be a gluten-associated manifestation, at least in a small subgroup of pediatric patients with CD, and could be caused by CD itself. HLA DQ2 and DQ8 are also genetic risk factors for other diseases which have an autoimmune basis [13] and are much higher in CD [14]. There is evidence, although somewhat controversial, that if CD is left untreated it could predispose to the development of other autoimmune diseases, including Sjögren’s syndrome, Addison’s disease, autoimmune liver disease, cardiomyopathies and neurological disorders. In this respect, some cases of EoE (an inflammatory disorder of immunological origin) could be triggered by CD, which has not yet been diagnosed. These matters require further research. Acknowledgments We would like to thank Dr Jose Marı́a Tenias Burillo for providing us with his methodological support. This work was funded by a grant (FISCAM AN-2008/21) awarded to Alfredo J. Lucendo. Conflict of interests None. References 1. Furuta GT, Liacouras CA, Collins MH, et al. Eosinophilic esophagitis in children and adults: a systematic review and consensus recommendations for diagnosis and treatment. Gastroenterology. 2007;133:1342–1363. 2. Lucendo AJ, Lucendo B. An update on the immunopathogenesis of eosinophilic esophagitis. Expert Rev Gastroenterol Hepatol. 2010;4:141–148. 3. Trier JS. Celiac sprue. N Engl J Med. 1991;325:1709–1719. 4. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (‘celiac sprue’). Gastroenterology. 1992;102:330–354. 5. Rewers M. Epidemiology of celiac disease: what are the prevalence, incidence, and progression of celiac disease? Gastroenterology. 2005;128:S47–S51. 6. Thorsby E, Lie BA. HLA associated genetic predisposition to autoimmune diseases: genes involved and possible mechanisms. Transpl Immunol. 2005;14:175–182. Dig Dis Sci (2011) 56:1107–1111 7. Sollid LM, Markussen G, Ek J, et al. Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med. 1989;169:345–350. 8. Kagalwalla AF, Shah A, Ritz S, et al. Cow’s milk protein— induced eosinophilic esophagitis in a child with gluten-sensitive enteropathy. J Pediatr Gastroenterol Nutr. 2007;44:386–388. 9. Verzegnassi F, Bua J, De Angelis P, et al. Eosinophilic oesophagitis and coeliac disease: is it just a casual association? Gut. 2007;56:1029–1030. 10. Quaglietta L, Coccorullo P, Miele E, et al. Eosinophilic oesophagitis and coeliac disease: is there an association? Aliment Pharmacol Ther. 2007;26:487–493. 11. Leslie C, Mews C, Charles A, et al. Celiac disease and eosinophilic esophagitis: a true association. J Pediatr Gastroenterol Nutr. 2010;50:397–399. 12. Lipschitz B. Eosinophilic oesophagitis: association with HLADQ2 and HLA-DQ8. Gastroenterology. 2007;132:A282. 13. Barker JM, Liu E. Celiac disease: pathophysiology, clinical manifestations and associated autoimmune conditions. Adv Pediatr. 2008;55:349–365. 14. Green PH, Jabri B. Coeliac disease. Lancet. 2003;362:383–391. 15. Rodrigo L. Celiac disease. World J Gastroenterol. 2006;12: 6585–6593. 1111 16. Martı́n-Muñoz MF, Lucendo AJ, Navarro M, et al. Food allergy and eosinophilic esophagitis: two cases studies. Digestion. 2006;74: 49–54. 17. Straumann A, Simon HU. Eosinophilic esophagitis: escalating epidemiology? J Allergy Clin Inmunol. 2005;115:418–419. 18. Patel SM, Falchuk KR. Three brothers with dysphagia caused by eosinophilic esophagitis. Gastrointest Endosc. 2005;61:165–167. 19. Weller PF. The immunobiology of eosinophils. N Engl J Med. 1991;324:1110–1118. 20. Collins MH, Blanchard C, Albonia JP, et al. Clinical, pathologic, and molecular characterization of familial eosinophilic esophagitis compared with sporadic cases. Ciln Gastroenterol Hepatol. 2008;6:621–629. 21. Kapel RC, Miller JK, Torres C, et al. Eosinophilic esophagitis: a prevalent disease in the United States that affects all age groups. Gastroenterology. 2008;134:1316–1321. 22. Katzka DA. Demographic data and symptoms of eosinophilic esophagitis in adults. Gastrointestinal Endosc Clin North Am. 2008;18:25–32. 23. Soma J, Brown-Whitehorn TA, Spergel JM. Association of eosinophilic gastrointestinal disorders with other atopic disorders. Immunol Allergy Clin N Am. 2009;29:85–97. 123
https://openalex.org/W2615461438	https://figshare.com/articles/journal_contribution/The_Road_Not_Taken_Understanding_Barriers_to_the_Development_of_Police_Intelligence_Practice/10206818/1/files/18399794.pdf	English	null	The Road Not Taken: Understanding Barriers to the Development of Police Intelligence Practice	The international journal of intelligence, security, and public affairs	2,017	cc-by	6,258	Abstract Abstract To better understand police intelligence practice, we examined practitioners’ views of their work and their relations with the wider law enforcement community. We surveyed intelligence staff (N=110), and interviewed a random sample of respondents (n=12). Our analysis suggested that traditionalism and the dominant action-oriented culture limit the organization’s understanding of intelligence practice. Largely, the focus in that context has been on street cops’ propensity to reject reflection in favour of action but intelligence practitioners need also look to themselves. Too often, the philosophy of ‘need to know’, is prioritized over its antithesis ‘dare to share’. Though perceived by practitioners as low-risk and consistent with organisational norms, we argue that inappropriately applied, ‘need to know’ is the enemy of efficiency and real accountability, offering low levels of reward and discouraging the kinds of partnership, reciprocity and multi-directional knowledge transfer that policing needs, to be successful in the information age. We reconceptualised an interactivity/isolationism continuum, used in the natural sciences, to help interpret that phenomenon. We argue that isolationism is but one factor in a complex organisational dynamic but it is a significant one because it can subtly limit the influence and reach of the intelligence milieu in previously unacknowledged ways. Keywords: knowledge transfer; intelligence practice; organisational culture The Road Not Taken: Understanding Barriers to the Development of Police Intelligence Practice Adrian James Mark Phythian Fiona Wade & Julian Richards Adrian James, Mark Phythian, Fiona Wade & Julian Richards Introduction: Police intelligence practice Police intelligence units’ raison d'être is to collect, analyse, and evaluate intelligence for incorporation into assessments that may inform the plans of others in the institution. Whether those in the operational world always appreciate their efforts, is moot. Despite the institution’s commitment to intelligence-led policing, overwhelmingly it remains action- oriented (Reiner, 2010). Arguably, that has limited the development of intelligence practice and marginalised intelligence staffs. Researchers consistently have found that some operational cops saw intelligence staffs’ efforts as ancillary to the ‘real’ business of policing (see for example; White, 1972; Amey et al, 1996; Cope, 2004; and Author 1, 2013). Rather than representing a pragmatic rejection of more thoughtful approaches to the problem 1 1 of crime as some (including Author 1, 2013 and 2016) have argued, the expression of these views may simply confirm ‘street cops’ rejection of increasing police managerialism (Reuss- Ianni, 1983), or reveal officers’ insecurities over the civilianization and diversification of police functions and roles (Crawford, 2008). Those processes have only accelerated over the last 20 years in all of the uniformed public services, in the armed forces and in other service industries, which have experienced the introduction of late modern, post-bureaucratic, organizational reforms, intended to deliver ‘models more suitable for volatile market conditions’ (Morris and Farrell, 2007 p.1576). Against that background, through the eyes of practitioners, we critically assess police intelligence practice and reflect upon practitioners’ relations with the wider law enforcement community. The study contributes to our understanding of the complex dynamics of that practice and its influence on the institution. Knowledge generation and intelligence practice The formal history of police intelligence practice in the UK began in 1883 with Scotland Yard’s establishment of the Special Irish Branch to combat Irish terrorism. It was only in the 1960s that the British police extended the intelligence function into the mainstream (Wilmer, 1970; Grieve, 2004). For many years, it considered intelligence solely a support function (ACPO, 1975 and 1986; HMIC, 1997). Though eventually, it endorsed reforms made in the Kent force and adapted them into a national strategy (Amey et al, 1996). Arguably, that strategy - the UK’s National Intelligence Model (NIM) - finally delivered the revolution in practice that insiders such as Flood (2003) and Phillips (cited in Rollington, 2013) had long advocated. NIM’s international influence has been considerable; it seems to have provided the intellectual foundation for the intelligence models of an increasing number of nations across the world (including: Sweden; Kosovo; Macedonia; Australia; and New Zealand) and to have reshaped standard structures and processes (see UNODC, 2006), even if its impact on the UK’s intelligence system and its intelligence staff is far less than its advocates would have us believe (see Author 1, 2013). Essentially, an elaboration of the standard intelligence cycle (see for example Ratcliffe, 2008; Phythian, 2015), NIM symbolized the police’s efforts to provide a business focus to its practices. Though some progress in that context may perhaps be inferred; it is difficult to attribute that to the NIM (Author 1, 2013). Arguably, the financial constraints on the public 2 2 policing institution in this age of austerity have had far greater influence on the reshaping of the institution. Often, assessments of relations between policing’s operational and intelligence worlds have focused on cop culture; street cops’ action-orientation and emphasis on pragmatism rather than on reflection (see for example Greenaway, 1999; Author 1, 2013). Researchers also look to the intelligence world better to explain those relationships. Treverton and Hebbard, (2008 p.xi) argue that the ‘tyranny of the immediate’, a constant in analysts’ work, limits deep reflection and real understanding of intelligence’s meaning. In their study of law enforcement practice in Western Australia; Joseph and Corkill (2011 p.100) found that analysts’ evaluations could be ‘superficial’ and therefore less readily accepted by their operational colleagues. Knowledge generation and intelligence practice Gill (1998, p.309) has argued that the influence of police intelligence units on the wider organization depends upon the extent to which they are able to encourage others to share their ‘definitions of policing problems and potential solutions’. That may speak to the importance of persuasion, negotiation and other ‘soft’ skills in these relationships. Table 1 - sample characteristics (job role and specialist/non-specialist) HERE We negotiated access to research respondents through the National Police Chiefs Council’s Intelligence Innovation Working Group. The group’s support for our endeavours, opened sufficient doors in a range of police forces and law enforcement agencies to enable us to collect the primary data we needed. Participants initially were recruited at a national intelligence conference. Subsequently, the snowball technique was used to reach respondents until saturation was reached. Respondents self-identified as specialists (employed in a headquarters role concerned with the investigation of terrorism or organised crime) or non- specialists (generalists employed in local policing units). The sample included members of 28 police and law enforcement bodies in England and Wales. Each respondent is referred to in this paper by role and by a number assigned by the researchers. 32 respondents were female, 73 were male, five did not declare their gender. The lottery system of random sampling was used to select respondents from this group for interview (n=12). In the social sciences, there is a long tradition of drawing on phenomena more often associated with the natural sciences. For example, in the first half of the twentieth century, the science of ecology provided many ideas that were adapted and used to explain the social world. One of the best-known exponents of the ecological approach was Ernest Burgess, a significant figure in a pioneering group of US researchers active in the inter-War years who came, collectively, to be known as the Chicago School of Sociology. Burgess was a key figure in the creation of a new sociological paradigm that adapted concepts from the fields of animal and plant ecology and then applied them to human behaviour (Park and Burgess, 1921). Methodology Substantially, our empirical data were collected in a case study of police intelligence practice in England and Wales. The research has multidisciplinary features (two researchers’ backgrounds are in national security and international relations; one identifies with criminology, the other with the social sciences more broadly) but we took an interdisciplinary approach to our data to answer the research question, ‘What do police intelligence staffs’ attitudes to their knowledge generation and transfer activities tell us about the influence and reach of intelligence practice in policing’? Secondary data were collected through systematic analyses of the scholarly literature, official reports, reviews and so on. Primary quantitative and qualitative data were collected from a survey of law enforcement intelligence staff. More qualitative data were collected in semi- structured interviews with randomly selected members of the larger sample. Respondents provided personal accounts of their professional experiences and assessed individual elements of their work. We used standard analytic tools to make sense of our data. A self- selected sample of intelligence staff was surveyed (N=110). The composition of the sample is shown at Table 1. 3 3 Figure 1 – The interactivity/isolationism continuum – HERE They are not necessarily generalizable to the wider law enforcement community though it is reasonable to suggest that the views expressed by respondents would be shared by others in that community in the UK and in similar communities elsewhere. Figure 1 – The interactivity/isolationism continuum – HERE We reconceptualised a continuum used in biology; often, in the study of aquatic parasites (see Poulin and Luque, 2004 and Stock and Holmes, 1988), to help us better understand the research situation and to convey understanding of our data. We acknowledge both the ethical and epistemological challenges inherent in borrowing conceptual frameworks from the natural sciences but we are satisfied that the complexity of the research situation merited our 4 attempt to follow in the footsteps of scholars like Burgess to make sense of the milieu. We claim only that the continuum is a useful heuristic that may aid understanding of an extremely complex set of phenomena. Our research focused on a particular policing sub-group, formed of those employed in the police intelligence milieu. We afford continuum its ordinary meaning as a range of entities that slightly differ from each other and that exist along a notional line between two poles. In ecological research, the term ‘cline’ is used to describe the infinite number of gradations theoretically possible from one pole to the other (see Blanckenhorn and Demont, 2004). In our version, interactivity is situated at one pole: isolationism, long recognised as a defining characteristic of police culture (see for example: Reiner, 2010; Skolnick, 1977; and Banton, 1964), is at the other. We explain the term interactive as the process by which the intelligence world interacts with other elements of the police service, with partners, and with communities for their mutual benefit. More than that, it implies a predisposition, to do so. We define the term isolationism as the deliberate act of keeping one’s affairs to oneself and the affairs of others at a distance. Again, we look beyond the instrumental and imply a reluctance to do so. The difference between each of the clines is imperceptible but at the poles they are quite distinct. In practical terms, we encountered few meaningful difficulties. Our findings are indicative of the views of, and generalizable to, the UK law enforcement community’s intelligence staffs. They are not necessarily generalizable to the wider law enforcement community though it is reasonable to suggest that the views expressed by respondents would be shared by others in that community in the UK and in similar communities elsewhere. In practical terms, we encountered few meaningful difficulties. Our findings are indicative of the views of, and generalizable to, the UK law enforcement community’s intelligence staffs. Research findings Respondents ranked the factors that were significant to their practice from a list provided by the researchers. As Table 2 shows, there was broad agreement on the importance of skilled staffs, information technology, and HUMINT. Interestingly; the NIM, advanced by the service as the means by which intelligence would take centre stage in policing, was ranked at or near the bottom of most respondents’ lists. Table 2 – Respondents’ ranking of elements of practice - HERE Table 2 – Respondents’ ranking of elements of practice - HERE 5 5 We collected qualitative data on respondents’ perceptions of their work. Our analysis sorted that data into three discrete themes: inside the bubble; capability and capacity; and advocacy and influence. We collected qualitative data on respondents’ perceptions of their work. Our analysis sorted that data into three discrete themes: inside the bubble; capability and capacity; and advocacy and influence. Inside the bubble Respondents reported on their own practice and the physical, philosophical, ideological, and cultural divisions between the intelligence and operational worlds. The physical barriers between the two were perhaps the most easily explained. The other divisions were inferred from respondents’ accounts which routinely were candid. Interestingly, respondents described the secret world as ‘mysterious’ (Intelligence manager N94), and ‘exclusive’ (Analyst N111). Invariably, intelligence units operate in a virtual bubble; close to (sometimes co-located with) operational units but separated – often physically but invariably in terms of information flows – from the latter in the causes of ‘need to know’ and of operational security more generally (Grieve). DOI N108 described a kind of ‘us and them’ world where intelligence staffs’ enthusiasm for collecting and managing data to help make sense of the world was not matched by others in the operational world. Naturally, respondents felt they had a better understanding of criminal intelligence work than their generalist colleagues; 88% of respondents considered themselves intelligence experts. Intelligence officer N01 said that their knowledge was ‘better than the average officer’. Officer N52 said that the work required the kinds of thought processes and problem solving skills not necessarily found elsewhere in the organisation. The policing world is more fragmented than it may appear to outsiders. Each of the 43 forces that make up the police service of England and Wales divides its intelligence assets between specialist units (dedicated to the investigation of serious and organised crime – usually serviced by force intelligence bureaux) and its local policing units. Regional intelligence units and a network of confidential units (responsible for managing the transmission of sensitive material between the police and national security and/or international agencies) complete the picture (Intelligence officer N86). Beyond that, the distribution of those resources was determined by executive decisions at the local level. Intelligence manager N74 said that in their force, everything was ‘very separated, very segregated, so that the Divisional Intelligence Bureaux (at LPUs) … are] owned and managed by their local management 6 6 teams. A negative was that the force had five different ways of doing everything ‘So every process … is done in five different ways, due to local idiosyncrasies or whatever’. teams. A negative was that the force had five different ways of doing everything ‘So every process … is done in five different ways, due to local idiosyncrasies or whatever’. 1 The Regulation of Investigatory Powers Act 2000 (RIPA) is the primary legislation covering covert policing activities. Inside the bubble Our data revealed something of an identity crisis in the secret world. Respondents were asked if they felt that intelligence work was a front-line or a support function. Of those who answered that question directly, 55 considered the work to be front-line’: 47 considered it ‘support’ to the front-line. Manager N104 said it was a frontline function the work required ‘proactive decisions around threat and risk’. Manager N106 said that on a day-off, they were: On the phone for four hours and made over 70 telephone calls regarding two unconnected operations … I spoke with six source handlers and two deputy controllers to task sources to identify key intelligence objectives laid down to help save a life – that is not back-office work. Manager N24 was one of nine respondents who talked about the limited appreciation in the wider organisation, of the realities of intelligence practice. The identification of intelligence work as a back-office function represented a ‘lack of understanding of the value that strong intelligence functions can provide to community safety and crime fighting’. Intelligence officers’ views on the subject were mixed. Officer N01 believed that the front line consisted solely of ‘response, neighbourhood and CID’. Officer N02 said intelligence work should complement front line response but was not in itself deserving of that label. Officer N05 agreed with that view. Officer N06 saw the work as both front line and back office, saying it was ‘front line… managing day to day risks [and] back office as regards longer term intelligence development and strategic work’. That point was developed by Officer N03. Agreeing with N06, they said: The best intelligence possible will always be via the eyes of a police officer or via a recording device which necessarily requires front line activity… in the modern world of computer systems and databases a lot of backroom activity is needed. 7 7 Officer N101 felt the work was a front line activity because it actively supported front line officers in their ongoing investigations. They said: Officer N101 felt the work was a front line activity because it actively supported front line officers in their ongoing investigations. They said: Assistance extends from basic help with RIPA applications, advice on directing investigations to achieve their objects through the most cost effective and proportionate means. As well as covert assistance, implementing… static and foot observations… dealing with the subsequent [court] issues, and sensitive disclosures.1 Capability and capacity Many respondents considered that the lack of formal training provided undermined intelligence staffs’ efforts. Though there was something of a disconnect between respondents’ perceptions of their own skills and abilities and their satisfaction with their training. Just 58% of respondents considered themselves adequately trained for their roles. A significant minority (greater than 30%) felt their training had been inadequate. These are interesting findings in their own right but they also may say something about the identity crisis we described earlier. The amount of training provided varied from force to force. The UK’s College of Policing provides a variety of courses for officers and staff but though many respondents had received training in some form, we found that significant dissatisfaction with it. Intelligence manager N37 said that he found much of the training, repetitive. DOI N114 said that training was too rudimentary. The dominant view was not that the training was deficient but that there should be more of it. DOI N108 said that in their force, sworn officers received very little training in intelligence work. While the force’s 300 special constables and 172 volunteers received no training at all. In some cases, ‘on-the-job’ training was the norm. Of course, as one intelligence manager commented that may have considerable value but we should be cognizant of the fact that ‘learning by mistakes’, which was described as a positive, also can have significant negative consequences for the individual, for the organization, and for communities. In an earlier piece 8 8 of research, a senior intelligence officer told this paper’s first author that the norm was ‘to sit around and wait for the wheels to fall off and then [when they fell off] to hide all the bits of paper that talked about wheels falling off’ (Author 1, 2013 p.89), which is the kind of behaviour that may be construed as providing even more evidence of the intelligence world’s isolationist tendencies. Several respondents made the link between capability and intelligence outputs. Officer N02 said there was a need to develop experience and skills so that ‘an ethos of pride in professionalism was encouraged’. Manager N10 summed up many respondents’ views on this subject when they said: Skilled intelligence staff are the most important part of good intelligence work. Capability and capacity Those members of staff that are interested in the work, who know where to look, know who to ask, know how to communicate and know how to write relevant and informative reports, are priceless. However, DOI N108 said that staff were neither ‘slick enough nor flexible enough’ for the service’s needs. Civilianization was a factor; the employment of civilian staff as intelligence officers was gathering momentum but it was starting from a very low base. There appeared to be less resistance to civilianization in principle (than, for example, was observed by Cope in 2004) but in practice – at least in one department - ‘recruiting the right calibre of personnel had not adequately compensated for the loss of sworn officers’ (Manager N24). In another, the too rapid replacement of sworn officers meant that many staff failed to understand ‘the power of intelligence’ (DOI N108). Advocacy and influence Some 12 years ago; John Grieve, the first appointee to the post of director of intelligence at Scotland Yard, called for police intelligence in Britain to be ‘reclaimed from the secret world, made less threatening to communities and used in their service’ (2004, p.26). Many respondents spoke about meeting that challenge but said that a significant factor in that context was the ‘need to know’ philosophy that undermined their efforts. Both manager N10 and officer N23 believed that their department’s insularity meant that the police were continually playing ‘catch-up’ with the criminal element in society. 9 9 Officer N61 said that the organisation was conflicted; managers were promoted into the department without an intelligence background, ‘making decisions based on what they think is the way to do things when in fact they create divisions and destroy good work’. The lack of effective leadership – also a factor in the capacity and capability of the units - limited the influence of intelligence staffs because when intelligence assessments reached the operational world, they were not able to compete with the pragmatic faith in experience and ‘common sense’ they encountered (Manager N10). Some respondents argued for greater openness about intelligence success but they represented a very small minority of the sample. Only four respondents were willing to discuss their successes and then only in the most basic of terms. Officer N03 defined success as ‘getting to the point where you have disrupted criminal activity or, even better, [provided] evidence to use in a prosecution’. N03 provided two examples. In the first case, human intelligence was developed to bring a prosecution against a man who was receiving stolen mobile phones. In the second, the deployment of a covert recording device not only confirmed the guilt of some suspects but also the innocence of others – an important factor in retaining the confidence and support of communities. Officer N05 described a case where human intelligence was developed to resolve a previously undetected shooting that had developed into a threat to life. Few respondents questioned the importance of effective engagement with partners and with communities. Manager N10 said community engagement was an important part of intelligence work. Patrol officers needed encouragement to build relationships. Officers N06 and N80 both said it was important to build trust with others. Manager N75 said it was ‘pivotal to providing a full intelligence picture. Advocacy and influence Especially when analysing the harm caused by organised crime groups’. DOI N14 said it was ‘essential to have support from the public in identifying observation points, CHIS, intelligence gathering and prevention of those joining gangs’. Manager N24 agreed with that sentiment. DOI N110 felt that more should be done to develop the institution’s external relationships. They said: DOI N110 felt that more should be done to develop the institution’s external relationships. They said: All too often we fail to engage with our diverse communities – many of whom do not have English as a first or second language. There is still much to be done in being more 10 creative and innovative. The processes we employ tend to be well used, and mildly successful but limited. Respondents said that there was greater tension between police and partners than between police and communities (Managers N10 and N24, officer N23). That perhaps reinforces the view (expressed for example by Noaks, 2008; and Gilling, 2002) that the challenges of working with communities, that may have very different expectations of police, or between agencies that may share a vision but have very different working practices, structures and cultures, should not be underestimated. Manager N75 said that effective communication/ relationship-building was a key element in the work of intelligence staff but highlighted that relationships in the intelligence milieu could be complicated by the need to protect sensitive intelligence. DOI N110 said that as things stood, ‘processes and protocols inhibited, rather than encouraged the free flow of information’. Applying the interactive/ isolationist continuum Borrowed from the field of ecological studies, the concept of the interactive/ isolationist continuum gave us new ways of thinking about long-recognised, intractable problems in that work. The linking of complexity with interdisciplinary research is no coincidence. Multi- faceted problems appear different when viewed from different angles. Practitioners, naturally, see their problems in instrumental terms; to be resolved with more training, more technology. Managers see them, fundamentally, as issues of resources, logistics, leadership and so on (the kinds of management and command phenomena with which they typically are most comfortable). The failure, substantially, to resolve these issues demands consideration of new approaches that draw on a wider range of insights. Reconceptualized for the intelligence milieu, our clines describe a drift from interactivity to isolationism, the relative ‘riskiness’ of the behaviour described and the extent to which that behaviour represents a consolidation of traditional practice or a shift towards creativity and innovation. In the context of the ‘need to know’ vs. ‘dare to share’ debate, we posit that the former is more closely related to isolationism, which we characterize as carrying low risk for the organisation; at least superficially. The latter is more obviously, a driver for interactivity, which may carry higher risk (of, for example, data being shared inappropriately) but promises higher reward. We conceive of concepts such as partnership, connectedness, and reciprocity 11 as being close to the interactive pole while protectionism, monopolization, and bounded knowledge are closer to the isolationist pole. Constructing meaning Our findings suggest that intelligence staffs are suffering something of an identity crisis. Identity crises usually arise in periods of confusion and uncertainty. They often follow changes in societal expectations. Our findings suggest that the essence of that phenomenon in this context is best captured in the long running debate over need to know and dare to share but the dispute over the status of the work as a frontline, a back office; a support or an operational function also is germane. The attraction of strict adherence to need to know in a professional environment may be obvious (see for example Heaton, 2011) but we argue that in that paradigm, expert knowledge inevitably is bounded and opportunities for interaction and partnership are limited. Of course, greater interactivity carries risks; information may be shared unlawfully or inappropriately but as Bichard, 2004 and Zelikow, 2011 found, failing to share can carry just as many risks. Given that intelligence practice is so often associated with failure (see Betts, 1978: Dahl, 2013; ISC, 2014), this research represented an opportunity for practitioners to redress the balance; to present their work in a more positive light. However, they were reticent about doing so (only four of 110 respondents were willing to share a ‘good news’ story). That reticence may be the product of cultural conditioning (another dimension of the need to know vs. dare to share debate) or it may simply be a manifestation of the police’s natural tendency to conceal covert methodologies. Either way, we view it as further evidence of the isolationism of the intelligence world, which largely operates in what two of our respondents described as ‘an air of mystery’. Reciprocity is the practice of exchanging things with others for mutual benefit. In this context, we infer that largely is the exchange of information and services. We recognise that intelligence staffs routinely share both with other law enforcement agencies and other partners. Naturally, the need to know paradigm dominates those exchanges; we recognise that operational security is a significant concern but – given respondents concerns about the quality of their training - we do wonder if that dynamic is understood well enough by practitioners. Arguably, the value of reciprocity is better appreciated by individuals; many of whom have proved adept at building their own, often cross-cultural, communication networks 12 and partnerships (Safjański, 2013 and Den Boer, 2015). Constructing meaning For them, national boundaries are no barrier to such exchanges but a negative dimension of that behaviour is that knowledge is shared only with other insiders and rarely lands in those central contact points where it can be analysed and evaluated more formally. Consequently, behaviour that on the face of it appears to demonstrate a degree of interactivity also may be understood as more evidence of isolationism. Conclusions We critically examined police intelligence staffs’ views on their own practice, to better understand their relations with the wider law enforcement community. We learned that intelligence practice is complex and multi-faceted. For all the emphasis the institution has put on models, structures, and processes in modernity it is fundamentally human activity - the interaction of individuals with each other, with operating processes and with management systems. Those interactions are influenced inter alia by political, organizational, and social norms and by institutional cultures. We found that the intelligence environment remains a closed; to many, a mysterious, world. We found little evidence that the concept of ‘need to know’ was any less entrenched in the psyches of police intelligence staffs than it ever has been. We posit that isolationism, of which need to know is emblematic, limits the influence and reach of the intelligence milieu. Moreover, that it disincentivises the kinds of partnership, reciprocity and multi-directional knowledge transfer consistent with the partnership and multi-agency working that the institution wants. To continue to hold a monopoly on relevant knowledge or to rely only on the knowledge it creates for itself, is incompatible with those aims. We wonder how significant the lack of training is in the persistence of ‘need to know’ as the standard operating model for information sharing. It can only be speculation, but as we suggest in Figure 1, on one level it may be perceived as the least risky option for an organisation which puts untrained and/or inexperienced staff into its intelligence units. To do otherwise; the institution would need expert staff that understand relevant law, the operating environment, and above all how to assess and manage risk, to a level above that which currently may be the norm. We present the interactivity/isolationism continuum as a medium for understanding the We present the interactivity/isolationism continuum as a medium for understanding the 13 information management dynamic. We eschew any suggestion that there are simple solutions to complex problems. The continuum is no more than a construct; potentially a useful heuristic. We make no claim for it beyond that. We recommend it to practitioners and researchers who share our commitment to transparency and accountability, to help them find new ways to think about the intelligence operating environment in policing. It is understood that the police institution rarely can solve policing problems on its own. Conclusions Notwithstanding oft- cited, and no doubt well-founded concerns about operational security, we believe that further reflection on the utility of ‘need to know’ is both desirable and necessary. Our research suggests that secrecy and ‘need to know’ invariably can be rationalized but we argue that they can be self-defeating, isolationist phenomena that may subtly undermine intelligence staffs’ efforts to persuade those outside the intelligence bubble that they can offer credible solutions to policing problems. Commonly, promoting interactivity generates creativity and new insights into old problems. Transparent connectedness encourages contributions from a wide range of disciplines. Ultimately, we hope that these will deliver more comprehensive but also more nuanced understandings of the milieu that in time will translate into organizational reforms, that deliver models more suitable for today’s market conditions. After 40 years of reviews, inquiries, and sub-optimal reforms, it surely is worth looking at the problem of intelligence from a different perspective, one perhaps that takes a different road to the one already so well-travelled. 14 14 References: ACPO (1975). Report of ACPO Sub-Committee on Intelligence (The Baumber Review). London: ACPO. ACPO (1986). Report of the ACPO Working Party on Operational Intelligence (The Ratcliffe Review). London: ACPO. ACPO (1986). Report of the ACPO Working Party on Operational Intelligence (The Ratcliffe Review). London: ACPO. Amey, P; Hale, C; and Uglow, S (1996). Development and Evaluation of a Crime Management Model. London: Home Office Police Research Group. Amey, P; Hale, C; and Uglow, S (1996). Development and Evaluation of a Crime Management Model. London: Home Office Police Research Group. Banton, M (1964). The policeman in the community. London: Tavistock. Banton, M (1964). The policeman in the community. London: Tavistock. Best, R (2011). Intelligence Information: Need-to-Know vs. Need-to-Share. US Congressional Research Service, 7-5700 www.crs.gov R41848. Betts, RK (1978). Analysis, war, and decision: Why intelligence failures are inevitable. World Politics, 31(01), pp.61-89. Bichard, M (2004). The Bichard Inquiry Report. London: HMSO. Blanckenhorn, WU and Demont, M (2004). Bergmann and converse Bergmann latitudinal clines in arthropods: Two ends of a continuum? Integrative and Comparative Biology, 44, pp.413-24. Cope, N (2004). Intelligence Led Policing or Policing Led Intelligence? Integrating Volume Crime Analysis into Policing. Br J Criminol (2004) 44 (2), pp.188-203. Crawford, A. (2008). Plural policing in the UK: Policing beyond the police. T. Newburn (Ed.) Handbook of policing. Willan: Cullompton, p.147. Crawford, A. (2008). Plural policing in the UK: Policing beyond the police. T. Newburn (Ed.) Handbook of policing. Willan: Cullompton, p.147. Dahl, E. J. (2013). Intelligence and surprise attack: Failure and success from Pearl Harbor to 9/11 and beyond. Washington, DC: Georgetown University Press. Dahl, E. J. (2013). Intelligence and surprise attack: Failure and success from Pearl Harbor to 9/11 and beyond. Washington, DC: Georgetown University Press. Den Boer, M (2015). Counter-Terrorism, Security and Intelligence in the EU: Governance Challenges for Collection, Exchange and Analysis, Intelligence and National Security, 30:2-3, pp.402-419. Den Boer, M (2015). Counter-Terrorism, Security and Intelligence in the EU: Governance Challenges for Collection, Exchange and Analysis, Intelligence and National Security, 30:2-3, pp.402-419. Flood, B (2003). Strategic aspects of the UK National Intelligence Model in J. Ratcliffe (Ed.), Strategic Thinking in Criminal Intelligence. Leichardt NSW: Federation Press, pp.37-51. Gill, P (1998). Making sense of police intelligence: A cybernetic model in analysing information and power in police intelligence processes. Policing and Society: 46 8(3): pp.289-314. Flood, B (2003). Strategic aspects of the UK National Intelligence Model in J. References: Ratcliffe (Ed.), Strategic Thinking in Criminal Intelligence. Leichardt NSW: Federation Press, pp.37-51. Gill, P (1998). Making sense of police intelligence: A cybernetic model in analysing information and power in police intelligence processes. Policing and Society: 46 8(3): pp.289-314. Gilling, D (2005). Partnership and Crime Prevention in N. Tilley (Ed) Handbook of Crime Prevention and Community Safety. Cullompton: Willan, pp,734-56. Greenaway, K (1999). Are the police now using their intelligence? Unpublished BSc di t ti I tit t f P li d C i i l i l St di U i it f P t th 15 15 Grieve, J (2004). Developments in UK criminal intelligence in J. Ratcliffe (ed.) Strategic Thinking in Criminal Intelligence (Annandale NSW: Federation Press), pp.25-36. Heaton, R (2011). We Could Be Criticized! Policing and Risk Aversion. Policing (2011) 5(1) pp.75-86. Thinking in Criminal Intelligence (Annandale NSW: Federation Press), pp.25-36. Heaton, R (2011). We Could Be Criticized! Policing and Risk Aversion. Policing (2011) 5(1) pp.75-86. HMIC (1997). Policing with Intelligence: Criminal Intelligence, HMIC Thematic Inspection Report on Good Practice. London: HMIC. ISC - Intelligence and Security Committee of Parliament (2014). Report on the intelligence relating to the murder of Fusilier Lee Rigby. London: UK HMSO. Joseph, J and Corkill, J (2011). Information evaluation: how one group of intelligence analysts go about the task. Proceedings of the Australian Security and Intelligence Conference 2011, pp.97-103. Noaks, L (2008). Private and public policing in the UK: a citizen perspective on partnership. Policing & Society, 18(2), pp.156-68. Park, RE and Burgess EW (1921). Introduction to the Science of Sociology. Chicago, IL: University of Chicago Press. Poulin, R and Luque, J (2003). A general test of the interactive-isolationist continuum in gastrointestinal parasite communities of fish. International Journal for Parasitology 67 DOI: 10.1016/j.ijpara.2003.09.005. Phythian, M (Ed.) (2015). Understanding the intelligence cycle. Abingdon: Routledge. Ratcliffe, J (2008). Intelligence-Led Policing. Cullompton: Willan. Reuss-Ianni, E (1983). Two Cultures of Policing. New Brunswick, NJ: Transaction Books. R lli A (2013) S I ll f h 21 C Th M M h d ss-Ianni, E (1983). Two Cultures of Policing. New Brunswick, NJ: Transaction Books. Reuss-Ianni, E (1983). Two Cultures of Policing. New Brunswick, NJ: Transaction Books. Rollington, A (2013). Strategic Intelligence for the 21st Century: The Mosaic Method. Oxford: OUP. Rollington, A (2013). Strategic Intelligence for the 21st Century: The Mosaic Method. Oxford: OUP. Safjański, T (2013). Barriers to the Operational Effectiveness of Europol. References: Internal Security, January–June 2013 Vol. 5 Issue 1, pp.53-69. kolnick, JH (1997). Sketch of the Policeman's Working Personality in GF. Cole and MG ertz (Eds ) Criminal Justice System: Politics and Policies Seventh Edition pp 116 33 Skolnick, JH (1997). Sketch of the Policeman's Working Personality in GF. Cole and MG. Gertz, (Eds.) Criminal Justice System: Politics and Policies, Seventh Edition, pp.116-33. Stock, TM and JC Holmes (1988). Functional relationships and microhabitat distributions of enteric helminths of grebes (Podicipedidae): The evidence for interactive communities. Journal of Parasitology 74 pp.214–27. Treverton and Hebbard (2008). Assessing the Tradecraft of Intelligence Analysis. Santa Monica, CA: RAND. Treverton and Hebbard (2008). Assessing the Tradecraft of Intelligence Analysis. Santa Monica, CA: RAND. UNODC (2006). Police Intelligence and Information Systems. Vienna: UNODC. UNODC (2006). Police Intelligence and Information Systems. Vienna: UNODC. 16 White, SO (1972). A perspective on police professionalism. Law and Society Review, Vol. 7, pp.61-85. Wilmer M (1970). Crime and Information Theory. Edinburgh: Edinburgh University Press. Wilmer M (1970). Crime and Information Theory. Edinburgh: Edinburgh University Press. 17 17 Table 1 18 Figure 1 Figure 1 19 19 Table 2 Table 2 20
https://openalex.org/W4322710937	https://ojs.uma.ac.id/index.php/bisman/article/download/7915/4565	English	null	Quality of Work Life as a Mediator on the Impact of Work-Life Balance on Job Satisfaction	JKBM (Jurnal Konsep Bisnis dan Manajemen)	2,022	cc-by	6,645	g Abstrak Work-life Balance (WLB) dapat mempengaruhi sikap karyawan di perusahaan, seperti kepuasan kerja (JS) dan juga berpengaruh pada kualitas kehidupan kerja (QWL). Penelitian ini bertujuan untuk menguji dan menganalisis signifikansi pengaruh langsung dan tidak langsung WLB terhadap JS melalui QWL sebagai mediator, dengan mengambil objek karyawan PT KAI (Persero) Daop 7 Madiun. Pendekatan penelitian adalah kuantitatif. Data primer yang digunakan dalam penelitian diperoleh dari kuesioner variabel penelitian yang disebarkan kepada 115 karyawan PT KAI (Persero) Daop 7 Madiun sebagai sampel penelitian. Teknik pengambilan sampel yang digunakan adalah proportional stratified random sampling. Analisis data menggunakan analisis jalur, diolah dengan software SPSS dan Sobel Test. Hasil studi empiris menunjukkan bahwa: 1) WLB dapat meningkatkan JS secara signifikan; 2) WLB dapat meningkatkan QWL secara signifikan; 3) QWL dapat meningkatkan JS secara signifikan; 4) QWL memediasi sebagian efek WLB pada JS. Kata kunci: Sikap Karyawan; QWL; WLB. JKBM (Jurnal Konsep Bisnis dan Manajemen), 9 (1) November 2022 JKBM (Jurnal Konsep Bisnis dan Manajemen), 9 (1) November 2022 JKBM (JURNAL KONSEP BISNIS DAN MANAJEMEN) ISSN 2407-2648 (Print) 2407-263X (Online), DOI 10.31289/jkbm.v9i1.7915 Available online http://ojs.uma.ac.id/index.php/bisman Quality of Work Life sebagai Mediator Pengaruh Work-Life Balance terhadap Kepuasan Kerja Quality of Work Life as a Mediator on the Impact of Work- Life Balance on Job Satisfaction Vano Halal Marga Pratama1) Veronika Agustini Srimulyani1)* 1) Management Study Program (Madiun City Campus), Faculty of Business, Widya Mandala Surabaya Catholic University, Surabaya, Indonesia Submitted: 21-08-2022; Reviewed: 26-11-2022; Accepted: 27-11-2022 *Coresponding Email: veronika.agustini.s@ukwms.ac.id Abstrak Submitted: 21-08-2022; Reviewed: 26-11-2022; Accepted: 27-11-2022 Abstract Work-life Balance (WLB) can affect employee attitudes in the company, such as job satisfaction (JS) and also have an effect on the quality of work-life (QWL). This study aims to test and analyze the significance of the direct and indirect influence of WLB on JS through QWL as a mediator, by taking the object of PT KAI (Persero) Daop 7 Madiun employees. The research approach is quantitative. The primary data used in the study were obtained from a questionnaire on research variables distributed to 115 employees of PT KAI (Persero) Daop 7 Madiun as a research sample. The sampling technique used is proportionate stratified random sampling. Data analysis using path analysis, processed with SPSS and Sobel Test software. The results of empirical studies show that: 1) WLB can significantly increase JS; 2) WLB can significantly improve the QWL; 3) QWL can significantly increase JS; 4) QWL partially mediates the effect of WLB on JS. J Keywords: Employee Attitude; QWL; WLB. Keywords: Employee Attitude; QWL; WLB. How to Cite: Pratama, V.H.M. & Srimulyani, V.A. (2022). Quality of Work Life as a Mediator on the Impact of Work- Life Balance on Job Satisfaction. JKBM (Jurnal Konsep Bisnis dan Manajemen). 9 (1): 14-27 How to Cite: Pratama, V.H.M. & Srimulyani, V.A. (2022). Quality of Work Life as a Mediator on the Impact of Work- Life Balance on Job Satisfaction. JKBM (Jurnal Konsep Bisnis dan Manajemen). 9 (1): 14-27 INTRODUCTION In the era of the Industrial Revolution (I.R.) 4.0, competition in the business world is increasingly highly competitive. Every company management must prepare a surefire strategy to face this competition, so that the managed business can continue to develop or at least survive. One of the strategies that can be practiced is to maximize the potential of human resources (HR) in the company. Human resources have an important role in carrying out the company's business activities or can be said to be a determinant of the achievement of company goals. To achieve a goal desired by the company, it is necessary to have superior, reliable, and capable HR in solving the tasks given by the company. Qualified HR will help the company in achieving a company's goals. Every company must ensure that all its employees have high motivation and work productivity, in order to produce good performance and achieve good goals for the company. To maintain opti- mal employee performance, employee job satisfaction needs to be considered by the company. By achieving work satisfaction, employees will be encouraged to complete their job tasks with all their abilities. Thus, employee productivity and performance will increase optimally along with high job satisfaction (JS). The high JS in the company is inseparable from the high level of quality of work life (QWL) of the employees. High QWL can improve JS in terms of decision making and em- ployee opportunities to grow (Putra et al., 2021). QWL can be described as the quality of the relationship between employees and the work environment which includes, adequ- ate & fair compensation, rewards & recognition, safe & sound work, opportunities to use & develop the capacity of HR owned, career development opportunities, social integra- tion in employment, WLB, participatory management (Nair & Subash, 2019). The QWL can affect the JS of an employee, since his rights when working are already fulfilled. For example, the fulfillment of the right to welfare, the right to health, and occupational safety, as well as other rights to which he is entitled as an employee. Results of previous empirical studies by Nair & Subash (2019); Putra et al. (2021); shows that the QWL has a significant positive impact on attitude behavior. That is, the higher the QWL level, the higher the JS level. 14 Jurnal Konsep Bisnis dan Manajemen, 9 (1) November 2022: 14-27 INTRODUCTION This indicates that QWL is very important for corporate management to pay attention to so that employees can achieve satisfaction at work. In the midst of work from home (WFH) conditions, the balance between WLB is a critical component. This is due to the absence of a boundary between working hours and the hours during which individuals carry out domestic duties within the family. If certain 15 Pratama, V.H.M. & Srimulyani, V.A. Quality of Work Life as a Mediator on the Impact of Work-Life Balance on Job … conditions are met, WFH will be recommended, this happens because working from ho- me can affect satisfaction in working. The study Srimulyani & Budi Hermanto (2022), showed a significant increase in the WLB of lecturers during the work from home period. One of the shapers of the QWL is the achievement of WLB. On the other hand, WLB can also reduce JS. Empirical studies on nurses at Qutor General Hospital show that WLB pos-itively affects the QWL and life satisfaction of nurses (Mohammed El-Demerdash, 2019). WLB is positive and significant impact on JS (Nurhasanah M et al., 2019). Findings in the field by Maccabees et al. (2015) also showed that WLB status and the impact of worklife imbalance on JS. The study of Alfatihah et al. (2021) by taking a sample of employees working in companies in Indonesia, obtained results that WLB and work mo- tivation (WM) affect JS, and WLB can mediate the effect of WM on JS. Field study of Efendi et al. (2022) showed that WLB had a significant impact on JS and JS mediating the influence of WLB on the employee performance. Meanwhile, Arul- doss et al (2020) examined the relationship of QWL with WLB in the cosmopolitan city of India, showing the result that job stress was negatively related to WLB; JS is positively related to WLB; work commitments are positively impact to WLB; and there is partial mediation of work stress, JS, and work commitment in the impact QWL on WLB. WLB can be seen from two different sides, namely from the employee side is the choice to manage work and obligations, while from the corporate's side it is a challenge to realize an organizational culture that supports employees so that employees can focus while working. INTRODUCTION Therefore, to achieve job satisfaction, employees must have the ability to manage time in order to achieve WLB, while for companies they must pay more attention to WLB so that JS is maintained. Empirical studies on the impact of WLB on JS were conducted on different types of organizations. Fatmawati & Irbayuni (2021) in an empirical study on employees at the Bhakti Loyal Women's Cooperative Surabaya showed that a well-managed WLB can in- crease JS; Likewise, good compensation can increase JS. Sitorus et al. (2018) proved that WLB can significantly improve JS. This indicates that JS will increase if employees can achieve an increasing WLB as well. Taking into account some of the results of the empirical studies that have been des- cribed, the purpose of this study is to explore the role of QWL as a mediator of the influ- ence of WLB on JS during the Covid-19 pandemic, which has not been studied by many researchers before. The object of the research taken was employees at one of the compa- 16 Jurnal Konsep Bisnis dan Manajemen, 9 (1) November 2022: 14-27 nies that violated public transportation, which during the pandemic was also affected by the economy, namely PT Kereta Api Indonesia (Persero) or abbreviated as PT KAI. PT KAI provides services including passenger and freight transportation. PT KAI is head- quartered on. Perintis Kemerdekaan No.1 street, Bandung. PT KAI has nine Operating Areas (DAOP), one of which is the VII Madiun Operational Area or abbreviated as Daop 7 Madiun. As a company engaged in rail transportation services, Daop 7 Madiun is required to always provide good service quality for its customers. The existence of good service quality will also increase customer satisfaction and maintain customer retention. Daop 7 Madiun certainly expects JS for each employee to always provide the best service to cust- omers. Similarly, JS can also improve employee commitment and performance. The QWL and WLB program implemented by Daop 7 Madiun as a form of the company's comm- itment to increasing the JS of its employees, including: the availability of a health clinic (Mediska) that operates for 24 hours to improve health to its employees, a vaccination program that is attended by hundreds of workers and subsidiaries to provide health pro- taction during the Covid-19. INTRODUCTION From the previous description, it is known that WLB and QWL have a strategic role in improving JS in an organization, as well as on employees of PT KAI (Persero) Daop 7 Madiun Region, so this research was conducted as an effort to measure the direct and indirect impact of WLB on JS through QWL on these corporate employees. JS is the main component in influencing the life satisfaction of each employee, this is due to the level of job satisfaction that employees have which will form a liking and dis- like attitude towards work. JS refers to pleasant or unpleasant feelings and emotions ass- ociated with employees in assessing their work (Nair & Subash, 2019). JS can be inter- preted as an employee's response to how well the work done gives something that is considered important. QWL can affect the survival of the organization, because QWL is considered to be able to increase employee engagement with the organization in assisting in achieving go- als. WLB plays a role in increasing employee motivation at work and employee commit- ment to work (Alfatihah et al., 2021). QWL program initially emphasized the needs of female employees, then expanded to all employees (Putra et al., 2021). Herrick and Mac- coby (https://www.yourarticlelibrary.com/) there are four basic principles of QWL, na- mely: a) Security Principles: Working conditions must be safe and there is no fear of eco- nomic conditions, where employees do not feel anxious, and are not afraid of losing their 17 ama, V.H.M. & Srimulyani, V.A. Quality of Work Life as a Mediator on the Impact of Work-Life Balance on Job jobs. b) The Principle of Equity, implies a fair appreciation of the efforts made by each who works. c) Principle of Individualization: recognizes that each employee is different in terms of attitude, skills, potential, etc., so that each individual should be given the opp- ortunity to develop both from the aspect of his personality and his potential. d) Demo- cratic Principles, implying the right to personal privacy, freedom of speech, and fair treatment. Providing opportunities for employees to participate in the decision-making process can improve QWL. WLB is a condition that leads to low levels of stress and high levels of well-being felt by employees (Bhende et al., 2020). INTRODUCTION WLB is very important and should be applied by every individual in the world of work, this is because WLB has benefits, including: increa- sing productivity, preventing stress, better relationships, having time to do activities outside of work, and others. The higher the WLB a person feels, the more able one is to balance the two roles between work and personal life that must be fulfilled. WLB refers to the actions of individuals in balancing three dimensions, namely life in organizations, life in person, and social life (Sitorus et al., 2018). This means that the idea of WLB has to do with the actions of individuals in balancing three dimensions namely organizational life, personal life, and social life. JS can be caused by WLB, since WLB includes organizational policies in terms of flexible working hours and benefits that the company provides to employees. The imp- act of WLB on JS refers to the two-factor theory from Herzberg, where job satisfaction is mo-tivated by several factors, both intrinsic and extrinsic factors that can create a WLB. WLB is defined as the ability that a person has in meeting the demands of work, commitment to family, and other responsibilities outside of work. A person's ability to balance roles in work and personal life contributes positively to his work, these include; increased JS, high organizational commitment (OC), and a low desire to leave the orga- nization (Sitorus et al., 2018). In an empirical study Mani et al. (2020)showed that WLB can increase JS in service companies in Kualalumpur, Malaysia. The higher the WLB, the higher the level of JS with employees. This is supported by the results of previous re- search by Sitorus et al. (2018); Fatmawati & Irbayuni (2021) which proves that WLB can significantly increase JS. Based on studies and empirical evidence in previous studies, in this study it is expected that WLB has a significant positive impact on JS, so the formulation of the first hypo-thesis is as follows: H1: Achieving WLB in employees can significantly increase JS. H1: Achieving WLB in employees can significantly increase JS. 18 Jurnal Konsep Bisnis dan Manajemen, 9 (1) November 2022: 14-27 WLB is critical to improving employee QWL. WLB is a necessity for every employee to create a meaningful and quality life. The reason a person works is to achieve a good le- vel of quality of life (well-being). The achievement of WLB can improve life satisfaction (LS), QWL, general welfare, and reduce work-life conflicts (El-Demerdash, 2019). An em- ployee who feels that he has a high WLB usually tends to have a minimal level of stress, has good work motivation, and has a close relationship with fellow colleagues and super- iorsThis condition shows the employee's ability to balance work and personal life affairs well. Work-family and QWL conflicts correlate significantly in employees studying part- time job enrichment (Talip et al., 2020). In the study Leitão et al. (2019) WLB is one of the shapers of QWL. In this study, it is hoped that it can provide evidence of the positive infl- uence of WLB on QWL, so that the second hypothesis is formulated as follows: H2: Achieving WLB in employees can significantly increase QWL. JS and QWL employees need to be managed properly so that employees remain passionate in contributing to the growth and effectiveness of the organization (Nair & Subash, 2019). JS is an important component that every employee should have. If the em- ployee's level of JS is high, then the employee can do his job happily without feeling bur- dened by the work he does. To improve employee JS, one of the factors that need to be considered by the company is QWL. The implementation of the QWL program can affect the JS of employees in the company. This is in accordance with the two-factor theory from Herzberg, that there are two factors, namely: 1) The satisfiers factor is also called the intrinsic aspect, which is the aspect that comes from within (job content); 2) Dissa- tisfiers factors are also called extrinsic aspects, that is, aspects around the work envi- ronment (job context), which can affect a person's job satisfaction. The better the company in creating programs to improve the QWL, the higher the level of JS in emplo- yees. This is in line with empirical studies Bekti (2018); Nair & Subash (2019); Putra et al. (2021) who proved that the QWL has a significant positive impact on JS. Based on studies and empirical evidence by previous studies, in this study it is hoped that QWL can significantly improve JS, so the third hypothesis is formulated as follows: H3: Achieving QWL in employees can significantly increase JS H3: Achieving QWL in employees can significantly increase JS. WLB can also indirectly affect JS which is basically an individual thing, through the QWL that employees feel. QWL is a form of organizational response to employee needs through the mechanism of employee involvement in designing a good work life so as to create pleasant working conditions, support, and increase JS through rewarding, job secu- 19 ama, V.H.M. & Srimulyani, V.A. Quality of Work Life as a Mediator on the Impact of Work-Life Balance on Job rity, and opportunities for employees to develop. The application of QWL in an enter- prise is to improve JS. QWL can improve JS significantly (Ruhana et al., 2019; Setyaning- rum & Ekhsan, 2021). The better the employee QWL, the more JS will increase. JS is an absolute thing that every employee must achieve at work. To achieve JS, it is necessary for individuals to balance the demands of work and personal life, and with the level of WLB achieved by employees can encourage the high QWL. The level of QWL can impact on JS. A person who works for a supportive and family-friendly organization will be more satisfied with his work. In this study, we tried to test the effect of the indirect impact of WLB on JS with QWL as a mediator, so that the fourth hypothesis was formulated as follows: RESEARCH METHODS This research approach is quantitative with a type of causality research. This study used questionnaires as a data collection technique. The total population in the study was 162 employees of PT KAI (Persero) Daop 7 Madiun. Determination of the number of sam- ples using the Slovin formula, obtained the number of samples of 115 people. The sam- pling technique used is proportionate stratified random sampling method based on the field of work or position at PT KAI (Persero) Daop 7 Madiun. Measurements of research variables are as follows: 1) WLB measured by indica- tors: a) time balance; b) involvement balance; c) satisfaction balance; 2) QWL measured by indicators: a) employee participation; b) equitable compensation; c) pride; d) save en- vironment; e) career development; f) conflict resolution; g) communication; 3) JS mea- sured by indicators: a) psychological; b) social; c) physical; d) financial. The measure- ment of the three research variables used a Likert scale of 1-5 points, namely: strongly disagree (1); disagree (2); neutral (3); agree (4); and strongly agree (5). Data analysis techniques include: 1) data quality tests (validity tests and reliability tests); 2) descriptive statistical; 3) classical assumption test; 4) hypothesis test (simple and multi- ple linear regression analysis, t test, and Sobel test). Data analysis techniques were per- formed using the SPSS version 22 program and the Sobel test online. H4: QWL mediates the impact of WLB on JS. The following research model (figure 1) used in the study, in which describing WLB and QWL is predicted to positively impact on JS; and it is also predicted that the QWL mediates the influence of WLB on JS. Figure1. Research Model Figure1. Research Model Figure1. Research Model The regression equation developed for path analysis (Fig. 1) is as follows: QWL = α + β WLB + e (path a) JS = α + β WLB + e (path c) JS = α + β WLB + β QWL + e (path b and path c’) Description of notation: a =raw (unstandardized) coefficient regression on the influence of WLB (exogeno- us variable) on QWL (mediator). b =raw (unstandardized) coefficient regression on the influence of the mediator (QWL) on the endogenous variables (JS); when the WLB is also a predictor of the JS. c = raw (non-standardized) coefficient regression of WLB influence on JS directly c = raw (non-standardized) coefficient regression of WLB influence on JS directly 20 Jurnal Konsep Bisnis dan Manajemen, 9 (1) November 2022: 14-27 c’=raw (unstandardized) coefficient regression of WLB influence on JS by controll- ing QWL c’=raw (unstandardized) coefficient regression of WLB influence on JS by controll- ing QWL c’=raw (unstandardized) coefficient regression of WLB influence on JS by controll- ing QWL α =constant β = raw (unstandardized) coefficient e = errors α =constant β = raw (unstandardized) coefficient e = errors e = errors e = errors Result The validity test results on 12 WLB measurement items were declared valid, for the vali- dity test results of 18 items of QWL statements there were 2 invalid statement items, so that the measurement of QWL used 16 valid statement items from 14 items of JS statements there were 2 invalid statement items, so that the measurement of JS used 12 items of statements that were declared valid. Reliability tests were carried out by comparing the values of Cronbach's Alpha 21 ama, V.H.M. & Srimulyani, V.A. Quality of Work Life as a Mediator on the Impact of Work-Life Balance on Job (α). Variables are declared reliable if (α) > 0.60. Table 1 shows that the statement items for the variables WLB, QWL, and JS are stated to be reliable. Table 1. Reliability Test Results Variables Alpha Count Critical alpha Result WLB 0.818 0.60 R QWL 0.665 0.60 R JS 0.641 0.60 R Source: author's calculation results (2022) Description of notation: R = Reliable. To describe the high and low average of respondents' answers, a measuring scale range of 0.8 was made, which was obtained from the following calculation= (5-1/5 = 5/4=0.8). The average respondents' responses to the variables WLB, QWL, and JS are presented in Table 2 below: Table 2. Mean Value of Research Variables Variables Mean Criterion WLB 4.09 H QWL 3.96 H JS 3.90 H Source: author's calculation results (2022) Description of notation: H = high; VH= very high Table 2. Mean Value of Research Variables Variables Mean Criterion WLB 4.09 H QWL 3.96 H JS 3.90 H Source: author's calculation results (2022) Description of notation: H = high; VH= very high Based on table 2 shows that the WLB variable is measured from three aspects or indica- tors: time balance, engagement balance, and satisfaction balance; getting an average score of 4.09 (high). Table 2 shows that the variables of QWL are measured from nine aspects or indi- cators: employee engagement, balanced compensation, a sense of pride in the company (work-place company), a sense of security towards work, safety of the work environment, well-being, career development, problem solving, and communication; got an average score of 3.96 (high). In Table 2 it can be seen that the variables of JS are measured from four aspects or indicators: psychological, social, physical, and financial; getting an average score of 3.90 (high). Source: author's calculation results (2022) Result The resu-lts of hypothesis testing are shown in Table 3 below: Table 3. Path Analysis Path Unstandardized Coefficients Standardized Coefficients t count Sig Result B SE B 1 Constant 2.318 .344 6.747 .000 WLB→ JS (path c) .388 .084 .474 4.637 .000 Significant H1 accepted 2 Constant 2.434 .302 8.058 .000 WLB→ QWL (path a) .357 .074 .491 4.853 .000 Significant H2 accepted 3 Constant 1.604 .458 3.504 .001 QWL→ JS (path b) .283 .094 .347 3.032 .003 Significant H3 accepted WLB→ JS (path c’) .293 .129 .260 2.279 .026 Significant Source: author's calculation results (2022) 22 Jurnal Konsep Bisnis dan Manajemen, 9 (1) November 2022: 14-27 The test results shown in table 3 show that H1, H2, and H3 are accepted. Testing the role of QWL mediation on the effect of WLB on JS was performed using the Sobel test (Table 4). Table 4. Sobel Test Results Path t test SE Sig Result WLB→QWL→JS 3.336 0.041 0.000 Significant H4 accepted Source: author's calculation results (2022) Table 4. Sobel Test Results Path t test SE Sig Result WLB→QWL→JS 3.336 0.041 0.000 Significant H4 accepted Source: author's calculation results (2022) Results of the mediating test with the Sobel test (table 4) the results were obtained that H4 was accepted. The role of mediation of the QWL in employees of PT KAI (Pesero) Daop 7 Madiun is partial mediation, this is shown in the influence of WLB on JS before and after controlling the QWL, remains significant. Discussion H1 test results, showed that WLB was able to significantly improve JS. This indicat- es that the higher the WLB felt by employees will be able to improve the QWL of emplo- yees. These empirical results support previous empirical studies, such Fatmawati & Irba- yuni (2021) which proves the positive and significant influence of WLB on JS. Based on the overall average value, WLB gets a relatively high average value of 4.09. Likewise, the average value of JS has an average value of 3.90 (high category). This indicates that the policies implemented by PT KAI (Persero) Daop 7 Madiun in striving for employees to feel that the WLB is good, so that employees feel a high WLB, so that it has a dominant impact on increasing JS of employees. H2 testing proved that WLB significantly improves QWL in employees. This indicat- es that the higher the WLB felt by employees, the higher the QWL felt by employees. In principle, the QWL is the existence of a balance between work and non-employment of the employees. The company's efforts in increasing employee involvement can be in the form of providing opportunities to engage in the decision-making process both in regular meetings and in virtual forums. Empirical results are the development of the results of studies from Talip et al. (2020) which showed the existence of a significant correlation between the WLB in QWL of employees who study part-time. The results of the H3 test showed that the QWL has a significant influence on JS. That is, the increase in QWL, will improve JS significantly. These findings are consistent with the findings of several previous studies, such as Bekti (2018); Nair & Subash (2019); 23 ama, V.H.M. & Srimulyani, V.A. Quality of Work Life as a Mediator on the Impact of Work-Life Balance on Job Putra et al. (2021) who showed that improving the QWL can increase JS. Based on the overall average value, the QWL got a relatively high average score of 3.96. This indicates that the QWL felt by employees of PT KAI (Persero) Daop 7 Madiun is in the high cate- gory, so that employees feel high JS as well. H4 testing shows that QWL acts as a partial mediation on the influence of WLB on JS. Discussion This means that the level of WLB that employees have can impact the QWL which can further impact JS, but the WLB felt by employees can also affect employee JS directly. The company's goal of offering family-friendly benefits programs that employees need, thro- ugh flextime, job sharing and others is as an effort by company management to create a WLB for employees. In this study, it was shown that WLB impact positively significantly the QWL of employees. This shows that companies can implement various HR programs that support WLB so that it can help employees to be able to further balance their work roles and family roles, get improved welfare and provide organizational benefits such as through the implementation of flexible working hours, telecommuting or job-sharing. The QWL is seen as a set of employee perceptions regarding a sense of security at work, job satisfaction, as well as conditions for development aimed at improving emp- loyee dignity. The basic concept of QWL is to demonstrate the attention and respect of the organization's management over HR in the organizational environment for QWL, as a management effort to create a conducive work climate, so that the organization technica- lly and humanely leads to a better QWL of employees. WLB was found to partially medi- ate the influence of QWL on JS (Table 4). In other conditions, JS can be formed directly due to the high WLB of employees (Table 3). This indicates that individuals who are able to manage their careers and personal lives well, will feel more comfortable at work, so that in some individuals can increase JS; however, in some other individuals it is shown that the impact of WLB on JS through the QWL. This makes them more comfortable at work which will ultimately encourage JS. JS is an assessment of the difference between what an employee expects from the work being performed and what the organization gives to the employee. JS is an illustration of employee happiness with components of the work being carried out, supervisors, and the overall state of the work environment. Referring to Hudson (2005) there are three determinants of WLB, namely time bal- ance (TB), involvement balance (IB), and satisfaction balance (SB). TB refers to the equa- lity between a person's time for a career and time for family or other aspects of life such as recreation, gathering with friends and family. Discussion IB refers to the balance of employees in 24 Jurnal Konsep Bisnis dan Manajemen, 9 (1) November 2022: 14-27 psychological involvement in meeting the demands of work and family roles, which can involve stress levels and employee involvement in work and in the personal life of empl- oyees. SB is the level of satisfaction at work and outside of work, for example the level of comfort of employees in work roles and personal roles of employees. These three aspects of WLB are individual, each employee has a different ability to achieve WLB, and based on empirical studies on employees of PT KAI (Persero) Daop 7 Madiun, the level of WLB achieved by some employees can directly increase JS as a whole, but in some employees it can indirectly increase JS felt through QWL which is felt. CONCLUSION The conclusions obtained from the results of the study are as follows: 1) An increa- se in WLB can have an impact on increasing JS significantly, meaning that if the WLB felt by employees increases, the more JS felt by employees increases.; 2) The increase in WLB can have an impact on significantly improving the QWL, meaning that if the WLB felt by employees increases, the QWL felt by employees will also increase; 3) Improving the QWL can have an impact on significantly increasing JS, meaning that if the QWL of empl- oyees in an organization increases, the more JS felt by employees increases; 4) The QWL acts as a partial mediator of the impact of WLB on JS. This result implies that the comp- any's efforts in realizing WLB need to be strengthened by a QWL program for employees in order to be able to maximize employee job satisfaction. This research emphasizes the importance of WLB aspects and the QWL as determinants of JS, especially for transport- ation service managers, such as PT KAI (Persero). This research can be an illustration related to aspects of WLB and what QWL needs to be fulfilled and improved for the ma- nagement of PT KAI (Persero). There are new findings that can be used as a reference in the development of sub- sequent research, where in previous studies it is still rarely carried out, namely the im- pact of WLB on QWL and the role of QWL partial mediation on the influence of WLB on JS. For example, 1) further research could explore the role of work culture as a mediation of the influence of WLB on employee well-being at work (Stankevičienė et al., 2021); 2) Further research can explore the role of QWL in mediating WLB compliance in work eng- agement (Rebecca et al., 2020). Further research can examine the influence of WLB and JS on employee performance (Abdirahman et al., 2018); (Thakur & Sharma, 2019); or assessing the impact of QWL on organizational excellence (Dayana & Nadarajan, 2019), 25 ama, V.H.M. & Srimulyani, V.A. Quality of Work Life as a Mediator on the Impact of Work-Life Balance on Job organizational commitment (Risla & Ithress, 2018), and organizational performance (Al- Shawabkeh & Hijjawi, 2018]. REFERENCES Abdirahman, H.I.H., Najeemdeen, I.S., Abidemi, B.T., Binti Ahmad, R. (2018). The Relationship between Job Satisfaction, Work-Life Balance and Organizational Commitment on Employee Performance. IOSR Journal of Business and Management (IOSR-JBM), 20 (5), 76-8i. www.iosrjournals.org. Alfatihah, I., Nugroho, A. S., Haessel, E., & Maharani, A. (2021). The Influence of Work-Life Balance with Work Motivation as Mediating Factor on Job Satisfaction A Prediction toward Transition to New Normal Situation. The Management Journal of Binaniaga, 6(1), 79. https://doi.org/10.33062/mjb.v6i1.431 g J f g ( ) p // g/ / j Al-Shawabkeh, K.M., & Hijjawi, G.S. (2018). Impact of Quality of Work-Life (QWL) on Organizational Performance: An Empirical Study in the Private Jordanian Universities. Asian Social Science, 14 (6),145- 156. Aruldoss, A., Kowalski, K.B. and Parayitam, S. (2021). The relationship between quality of work life and work-life-balance mediating role of job stress, job satisfaction and job commitment: evidence from India. Journal of Advances in Management Research, 18 (1), 36-62. https://doi.org/10.1108/JAMR-05- 2020-0082 Bekti, R. R. (2018). Pengaruh Kualitas Kehidupan Kerja Terhadap Kepuasan Kerja Karyawan Rumah Sakit Ibu Dan Anak X Surabaya. Jurnal Administrasi Kesehatan Indonesia, 6(2), 156. https://doi.org/10.20473/jaki.v6i2.2018.156-163 p // g/ /j Bhende, P., Mekoth, N., Ingalhalli, V., & Reddy, Y. V. (2020). Quality of Work Life and Work–Life Balance. Journal of Human Values, 26(3), 256–265. https://doi.org/10.1177/0971685820939380 Dayana, M.C., & Nadarajan, S. (2019). The Impact of Quality of Work Life Factors on Organisational Excellence Among Employees in Msmes-A Pragmatic Analysis. Journal of Management, 4(2),81–86 Ef di S P t A & S i E (2022) Th Eff t f W k f H W k Lif B l d W k Dayana, M.C., & Nadarajan, S. (2019). The Impact of Quality of Work Life Factors on Organisational Excellence Among Employees in Msmes-A Pragmatic Analysis. Journal of Management, 4(2),81–86 Efendi, S., Purwanto, A., & Sugiono, E. (2022). The Effect of Work from Home, Work Life Balance, and Work Motivation on Job Satisfaction and Their Impact on the Performance of Non-Lecturer Education Efendi, S., Purwanto, A., & Sugiono, E. (2022). The Effect of Work from Home, Work Life Balance, and Work Motivation on Job Satisfaction and Their Impact on the Performance of Non-Lecturer Education Personnel at Pertamina University Jakarta. Budapest International Research and Critics Institute-Journal (BIRCI-Journal), 5 (1), 5382-5397. DOI: https://doi.org/10.33258/birci.v5i1.4244. ( J ) ( ) p // g/ / Fatmawati, S., & Irbayuni, S. (2021). CONCLUSION As a suggestion for further research, it is hoped that it can conduct research using PT KAI (Persero) employees in other operational areas, as a sam- ple that allows researchers to get a larger number, considering that this study only uses a sample of PT KAI (Persero) Daop 7 Madiun employees, so that the research results can be more generalized to PT KAI (Persero). g , ( ), p // g/ / N. T. D., Ardika, I. W., Antara, M., Idrus, S., & Hulfa, I. (2021). The Effects of Quality of Work Life on REFERENCES Effect of Work Life Balance and Compensation on Job Satisfaction At Koperasi Setia Bhakti Wanita Surabaya. Jurnal Ekonomi Balance, 17(1), 90–101. https://doi.org/10.26618/jeb.v17i2.6115 Herrick and Maccoby. 4 Basic Principles of Humanisation of Work as. Accesse https://www.yourarticlelibrary.com. Â Leitão, J., Pereira, D., & Gonçalves, Â. (2019). Quality of work life and organizational performance: workers’ feelings of contributing, or not, to the organization’s productivity. International Journal of Environmental Research and Public Health, 16(20), 1–18. https://doi.org/10.3390/ijerph16203803 Makabe, S., Takagai, J., Asanuma, Y., Ohtomo, K., & Kimura, Y. (2015). Impact of work-life imbalance on job satisfaction and quality of life among hospital nurses in Japan. Industrial Health, 53(2), 152–159. https://doi.org/10.2486/indhealth.2014-0141 p // g/ / Mani, V. A., Geetha, S., & Al-Khaled, A. A. S. (2020). Factors of Work Life Balance and its Influence on Job Satisfaction in the Service Industry. International Journal of Academic Research in Business and Social Sciences, 10(7), 571–589. https://doi.org/10.6007/ijarbss/v10-i7/7472 Mohammed El-Demerdash, S. (2019). The Influence of Work life Balance on Quality Work Life and Life Satisfaction among Head Nurses. International Journal of Novel Research in Healthcare and Nursing, 6(2), 1155–1174. www.noveltyjournals.com Nair, P. R., & Subash, T. (2019). Quality of Work Life and Job Satisfaction Questionnaire . 8(02), 15–21. Satisfaction among Head Nurses. International Journal of Novel Research in Healthcare and Nursing, 6(2 1155–1174. www.noveltyjournals.com Nair, P. R., & Subash, T. (2019). Quality of Work Life and Job Satisfaction Questionnaire . 8(02), 15–21. Nair, P. R., & Subash, T. (2019). Quality of Work Life and Job Satisfaction Questionnaire . 8(02), 15–21. Nurhasanah M, W. O., Kalimin, L. O., & Syaifuddin, D. T. (2019). The effect of work-life balance on job satisfaction and female employee performance in commercial bank in Kendari City. IOSR Journal of Business and Management, 21(5 Ser. II), 1–7. https://doi.org/10.9790/487X-2105020107 g ( ) p // g/ / Putra, I. N. T. D., Ardika, I. W., Antara, M., Idrus, S., & Hulfa, I. (2021). The Effects of Quality of Work Life on 26 Jurnal Konsep Bisnis dan Manajemen, 9 (1) November 2022: 14-27 Job Performance, Work Motivation, Work Ethics, Job Satisfaction, and Self-efficacy of Hotel Employees in Lombok. Asia-Pacific Journal of Innovation in Hospitality and Tourism, 10(3), 19–37. Rebecca, Sarinah, & Putra, A. I. D. (2020). Hubungan antara Work Life Balance dengan Employee Engagement Pada Bank Sinarmas KC Medan The Relationship between Work Life Balance with Employee Engagement at Bank Sinarmas Medan Branch. stein, S. H. (2008). How To Practice The Art Of Life Balance. E-Book. Copyright: Stacey Weckst ffer, November, 1–107. REFERENCES Jurnal Penelitian Pendidikan, Psikologi Dan Kesehatan (J-P3K), 1(1), 44–49. Risla, M.K.F, & Ithrees, A.G.I.M. (2018) The Impact of Quality of Work Life on Organizational Commitment with Special Reference to Department of Community based Corrections. Global Journal of Management and Business Research: G Interdisciplinary, 18 (1). p y ( ) Ruhana, I., Astuti, E. S., Utami, H. N., & Afrianti, T. W. (2019). The Effect of Quality of Work Life (QWL) on Job Satisfaction and Organization Citizenship Behavior (OCB) (A Study of Nurse at Numerous Hospitals In Malang, Indonesia). Journal of Public Administration Studies Corporate Values, 4(2), 51–58. Setyaningrum, R. P., & Ekhsan, M. (2021). the Role of Job Satisfaction in Mediating the Effect of Quality of Work on Employee Performance. Management Research Studies Journal, II(1), 44–54. https://doi.org/10.31967/mba.v3i2.361 p // g/ / Sitorus, D. R. H., Raharjo, K., & Kusumawati, A. (2018). The Influence of Work-Life Balance on Job Satisfaction, Organizational Commitment, and Turnover Intention. Wacana, 21(4), 181–187. https://doi.org/10.34283/ksgs.2018.12.2.15 p // g/ / g Srimulyani, V. A., & Hermanto, Y.B. (2022). Work-Life Balance Before and During Work from Home in a Covid-19 Pandemic Situation. Jurnal Manajemen Indonesia, 22(1), 31. https://doi.org/10.25124/jmi.v22i1.2915 p // g/ /j Stankevičienė, A., Tamaševičius, V., Diskienė, D., Grakauskas, Ž., & Rudinskaja, L. (2021). The mediating effect of work-life balance on the relationship between work culture and employee well-being. Journal of Business Economics and Management, 22(4), 988–1007. https://doi.org/10.3846/jbem.2021.14729 Talip, D. S. N. A., Hassan, Z., Kasa, M., Sabil, S., & Ibrahim, D. K. A. (2020). The Relationship of Work Life Balance and the Quality of Life among Employees Studying Part Time. International Journal of Academic Research in Business and Social Sciences, 11(14), 270–284. https://doi.org/10.6007/ijarbss/v11- i14/8573 Thakur, R. & Sharma, D. (2019). A Study of Impact of Quality of Work Life on Work Performance Sage Journal, 44 (3). https://doi.org/10.1177/0258042X19851912. Weckstein, S. H. (2008). How To Practice The Art Of Life Balance. E-Book. Copyright: Stacey Weckstein Hoffer, November, 1–107. 27
https://openalex.org/W3171896043	https://geusbulletin.org/index.php/rapggu/article/download/7764/13634	English	null	The un-named Silurian(?) dolomite formation, Børglum Elv, central Peary Land	Rapport	1,981	cc-by	2,095	Rapp. Grønlands geol. Unders. 106, 29-34 (1981) Rapp. Grønlands geol. Unders. 106, 29-34 (1981) THE UN-NAMED SILURIAN(?) DOLOMITE FORMATION, BØRGLUM ELV, CENTRAL PEARY LAND H. A. Armstrong and P. D. Lane Fig. 9. Index map showing section locality. Unit 1 (c. 6 m) The basal 1 m of this unit consists of very finely crystalline, almost porcellanous, pale-weathering dolomite which is finely laminated towards the top. Above, 3 m of medium grey crystalline dolomite show planar and trough cross-bedding. No clear current directions are exhibited by the smaller sets, but the larger ones indicate currents from the southern quadrant. Some bedding planes show scours and faint ripples. Above this, finely crystalline thin-bedded dolomite passes up into wavy bedded, faintly laminated dolomite which is terminated by a 0.5 m brecciated horizon. This 'breccia' consists of fragments of finely crystalline, paie dolomite about 10 mm thick and a few tens of millimetres long, set in a darker, more coarsely crystalline matrix. Commonly fragments have suffered only minimal displacement or rotation; they are closely aligned, one with the next, and fine laminae can be traced easily from one to its neighbours over distances of a few metres. Un-named Silurian(?) dolomite formation (c. 95 m) The formation can be divided into four distinct units on the basis of lithology and weath- ering characteristics. The lowest three units are equivalent to the 'lower member' of Christie & Peel (1977), and the uppermost to their 'upper member'. Børglum River Formation The upper 10 m of this unit were examined. The rocks here are typical of the formation, being massive, dark-weathering Iimey dolomites with abundant silicified horizontal burrows, Maclurites and actinoceratid cephalopods. Although predominantly dark in colour, the weathered rock is usually mottled in appearance which, perhaps, reflects dolomitization centred on the extensively burrowed original sediment. Unit 2 (c. 28 m) Dark-weathering, massive dolomite with burrow-centred mottling predominates in this unit, although at about halt thickness a thin-bedded unit of finely crystalline dolomite occurs. In the massive dolomite, vugs are common and are usually filled with buff dolomite or, rarely, with calcite. Lithostratigraphy Lithostratigraphic divisions are taken as in Christie & Peel (1977). H. A. Armstrong and P. D. Lane The un-named Silurian(?) dolomite formation, described originally from the upper reaches af the valley of Børglum Elv (fig. 9) by Christie & Peel (1977, figs. 3,4) and widely distributed in Peary Land, has received little detailed attention in any of its outcrops. Although these outcrops were visited by R. L. Christie and J. S. Peel in 1974 (Peel & Christie 1975, Christie & Peel 1977), P. D. Lane and A. T. Thomas in 1978 and J. E. Mabillard in 1979 (the reports of the last two parties held as unpublished field accounts by GGU), no detailed log af the whole of this formation has been previously published. The section (fig. 9) was chosen 112 km south of that measured by Christie & Peel (1977, fig. 3H) in order to include the base af the formation. Fig. 9. Index map showing section locality. 82·30' o, 10 km , I.- Kap Harald Moltke Independence Fjord 30 ~ Ql C. 4 c. ::l 2! E .2o "1j ..... c: Ol .;: ::J Ul "1j Ql E 3 Olc: c: ::l .......... Brachiopod coquina 'If~ Mudcracks ./.../ Cross bedding ~ø Cephalopods + Gastropods ~~~ Bioturbation ~ RippIe @ Stromatoporoids h,-J--t::;==:;::~-... <-l- • : •••••• - IPfiiiIl ., '-lb .. '.' .... -(.. tf-" ........... -@J B Dolomite ~ Limestone a Limey dolomite I:f> :':'1 Massively bedded .... with vugs ~ Thin/bedded ~. Wavy thin bedded 1=,::::::;:=1 'Breccia' 5 B 'Ilc: O 7 Cii Ql E I ··.··.··9·:·:. -lIl :.:~: :":": -~l ~=~F..~C?~'C:::>~~~. -{l SI ... ' 1'-7----L-,I . . ... .. .: = ~Sl..fSS 1'-7----L-,I' .' • _ fll .Q.~ ...~. .......... -/./ /,./ 1'-7-.....<-,1 ...<J . : . ~.....<-,I.'. ···.-RI ....'. f-r-L.-J '. '. c) . f-r-L.-J : '. '. . .'. - ~\I !'-7-.....<-,I.:Q .. : ... - ~l 2 ~ 'Il ~o ...J Fig. la. Lithologicallog of the un-named Silurian(?) dolomite formation. Scale bar c. 14 m. 5 Dolomite Fig. la. Lithologicallog of the un-named Silurian(?) dolomite formation. Scale bar c. 14 m. We present here a complete log from the upper portion of the Børglum River Formation into the basal part of the un-named Silurian limestone formation, which Iie respectively below and above the un-named Silurian(?) dolomite formation (fig. 10). 31 31 Unit 3 (c. 10 m) Characteristic of this unit is pale-weathering, porcellanous and finely crystalline dolomite with scattered, randomly oriented, thin-shelled, pentamerid brachiopods, pelmatozoan fragments and smooth-valved ostracods, similar in character to the upper part of Unit 1. 32 '... , ' " ••• • . ,'. .; .- • , .. " -: \' f. , .. ....., "." , II ., • " , Fig. Il. -8reccialed" appcar..nce of dolomile$ al lhe lop af uml 5 uf .hc un-namcd Silurian lime· Slone formalion. Fig. Il. -8reccialed" appcar..nce of dolomile$ al lhe lop af uml 5 uf .hc un-namcd Silurian lime· Slone formalion. Unit 4 (c. 53 m) This unit consisIs ;\Jmost cnlircly af massive, d;lrk-wc:llhcring, dark grey monlcd dolo- mile. It has fine IO medium crystallinilY wilh li slrong biluminous smell whcn frcshly broken. A fe..... scaltcrcd calcilc-fillcd vugs arc present. and Ihe general aspecl af this unit is likc Unil2. U'I-1ItI1l1ed SilllriOll limeslOflt! [ormmiofl (member A) Fig. Il. -8reccialed" appcar..nce of dolomile$ al lhe lop af uml 5 uf .hc un-namcd Silurian lime· Slone formalion. Unit 6 (c. S.S m) Very dark, lhinly-bedded limestone characterizcs this unit. The 10P few thin beds displllY well-developed polygomll cracks whosc morphology is consistent with an origin duc \O desiceation (fig. 12). ThTec umts (here numbcrcd 5-7) arc rccognized In mcmbcr A. ThTec umts (here numbcrcd 5-7) arc rccognized In mcmbcr A. Unn 5 (c. I·U m) Thmly bcdded, fine grained. dark ItmeslOnc and hme) dolomltc wllh abundanl, smooth oslracods form Ihe basal 6 m af Ihis unil. Upward Ihe rocks becomc laminated. al firsl planar. then wavy and finally brcccialcd (fig. II) to resemble lhose at the 10p of Unit I (see below). In SOffie places localized breccialion, morphologieally similar IO tepee Slructure (Shinn. 1969; Evamy, 1973). can bc seen. bul on a mueh smaller sealc. Unit 6 (c. S.S m) This unit is composed of massive, blue-grey, medium-grained, bioclaslic limestone con- taining abundant parliaIly silicified brachiopods (mainly Ihick-shcllcd pentamerids), gas- Iropods, cephalopods. tabulate corals and stromatoporoids. Above Unit 7, a I m bed of medium grained calcarenitc with vel)' abundanllhkk-shcllcd pentamerids, but almosl no other fossils, marks the base of Membcr B of the un-namcd Silurian limestone formalion. Sedimenhlry environrncnls Rocks wilh the lilhological characters of the Børglum Ri'"cr Formalion arc common!y formed in a 10w energ)'. shelterecl. probabty sublidal cn\irOnmcnl as indlcalcd by recent wQrk (Kendall. 1977; Morro..... 1978: Jones. Oldershaw &. Narbonne. 1979) on similar carbonatc facies. Howc...cr. duc IO the n3IUfC of the rocks of thc un-namcd Silurian(?) dolomilC fonnalion. and pcrhaps p3rticularly to Ihe dolomilizalion. only a few features arc prcservcd Wllh which a dClailed analysis of thc cn\'ironmenlS of dcposition ma) be attcmp- lcd. AI thc base of the formatIon, howc\cr, the cross-bcddcd unit indlcalcs highcr cncrgy conditions for Ihis interval compared wilh lhose under which Ihe Børglum Ri\er Formallan below accumulaled. Shallower waler condllions may IllUS bc indicaled for Ihe base of lhe un-namcd Silurian(?) dolomile formalion Ihan for Ihe underlying bed.). Furlhcr, Ihe pam, rcslricled la monospccificbrachiopod, pelmalozoan oroSlraeod faunas foundsparsdy lhrough Ihc un-named Silurian(?) dolornile formalion indicale Ihal deposilion in an opcn. fully marine cnvironment is unlikcly. Fursich & Hursl (1980) indced, have reeently postulalcd Ihal the characler of Ihesc faunas, along .....ilh regional and stratigraphie consideralions. indicaie deposilion in an "al Ieasl 51igh1ly hypersaline em'ironmenf' (p. 306), perhaps lagoonaUinlcrlida!. In membcr A of Ihe un-namcd Silurian limcslone formalion (our unil 5) possible small-scale lepec slruelures are present. Although original1y described as Iypical of suprali- dal deposils (Shinn, 1969; Evamy. 1973) Ihey havc since been reeognizcd in submarine environmcnts in Recenl deposils (Till, 1978). so Ihal no spccific deplh of dcposilion is necessarily indiealed. The eXlensivcly devclopcd mudcracks highcr in the scqucncc (our unit 6) afford evidence for al [east a shorl period af emergenee IO a supralidal environmenl. Unit 7 (c. IS m) This unit is composed of massive, blue-grey, medium-grained, bioclaslic limestone con- taining abundant parliaIly silicified brachiopods (mainly Ihick-shcllcd pentamerids), gas- Iropods, cephalopods. tabulate corals and stromatoporoids. Above Unit 7, a I m bed of medium grained calcarenitc with vel)' abundanllhkk-shcllcd pentamerids, but almosl no other fossils, marks the base of Membcr B of the un-namcd Silurian limestone formalion. 33 Fig. 12. Bedding surfacc In lhe lime) dolomIIcs of Mcmber A (Jf thc un-namcd Sllurian IImC'!;lonc formalion, showing mud cracks. Fig. 12. Bedding surfacc In lhe lime) dolomIIcs of Mcmber A (Jf thc un-namcd Sllurian IImC'!;lonc formalion, showing mud cracks. J IIoI'J'Or1 nI I(lf, The age of the un·named Silurian(?) dolomite formation Christie & Peel (1977, p. 23) considered Ihalthe lopof the Børglum Ri\'er Formalion was of carly Lale Ordovician (Edcn+Maysville, 'Red Ri\'er') age. The un-named Silurian(?) dolomile formal ion itself has yielded poor braehiopods giving a possible carly IO middle L1andovery age IO ils upper pari (Chrislie & Peel. 1977, p. 24), while Peel (1980, p. 68) 34 collected Ordovician fossils from basal beds of the formation in Kronprins Christian Land. Preliminary conodont identifications from the lower part of this formation in Peary Land by R. J. Aldridge and one of us (HAA) has revealed a probable late Ordovician age for the lower 40 m. This evidence suggests that the un-named Silurian(?) dolomite formation con- tains the Ordovician-Silurian boundary. Conc1usions The un-named Silurian(?) dolomite formation is in part of Ordovician and in part of Silurian age. Although it is a shallow marine sequence showing evidence of some shaIlowing, there is no unequivocal evidence of major relative depth changes in the rocks represented. Brenchley & Newall (1980) have postulated a marine regression of worldwide extent which occurred at the end of the Ordovician. This regression, it is argued, was represented by a sea level fall of a minimum of 40 m and a maximum of 170 m, which, it would be expected would produce features in all shallow marine sequences of the relevant age. No evidence of a major regression is seen in the rocks of the section described. The possibie regression indicated by the mudcracks in member A of the un-named Silurian limestone formation is probably of middle Llandovery or younger age, on the evidence of brachiopods from the upper part of the member. References Brenehley, P. J. & Newall, G. 1980: A facies analysis of Upper Ordovieian sequenees in the Oslo Region, Norway - a record of glaeio-eustatie ehanges. Palaeogeogr. Palaeoclim. Palaeoecol. 31, 1-38. Christie, R. L. & Peel, J. S. 1977: Cambrian-Silurian stratigraphy of Børglum Elv, Peary Land, eastern North Greenland. Rapp. Grønlands geol. Unders. 82, 48 pp. my, B. D. 1973: The preeipitation of aragonite and its alteration to ealcite on the Trucial Coast o Evamy, B. D. 1973: The preeipitation of aragonite and its alteration to ealcite on the Trucial Coast of the Persian GulL In Purser, B. H. (edit.) The Persian Gulf, 329-342. Berlin: Springer Verlag. my, B. D. 1973: The preeipitation of aragonite and its alteration to ealcite on the Trucial Coast o Persian GulL In Purser, B. H. (edit.) The Persian Gulf, 329-342. Berlin: Springer Verlag. rsian GulL In Purser, B. H. (edit.) The Persian Gulf, 329-342. Berlin: Springer Verlag. Fiirsieh, F. T. & Hurst, J. M. 1980: Euryhalinity of Palaeozoie artieulate braehiopods. Lethaia 13, 303-312. Jones, B., Oldershaw, A. E. & Narbonne, G. M. 1979: Nature and origin of rubbly limestone in the Upper Silurian Read Bay Formation of Arctic Canada. Sedim. Geol. 24, 227-252. Kendall, A. C. 1977: Origin of dolomite mottling in Ordovieian limestones from Saskatehewan and Manitoba. Bull. Can. Petroleum Geology 24, 480-504. ow, D. W. 1978: Dolomitization of Lower Paleozoic burrow-fillings. J. sedim. Petrol. 48,295-306 Peel, J. S. 1980: Geological reeonnaissance in the Caledonian foreland of eastern North Greenland, with comments on the Centrum Limestone. Rapp. Grønlands geol. Unders. 99,61-72. Peel, J. S. & Christie, R. L. 1975: Lower Palaeozoic stratigraphy GreenJand. Rapp. Grønlands Geol. Unders. 75,21-25. Peel, J. S. & Christie, R. L. 1975: Lower Palaeozoic stratigraphy of southem Peary Land, eastern North GreenJand. Rapp. Grønlands Geol. Unders. 75,21-25. Shinn, E. A. 1969: Submarine lithifieation of HoJoeene earbonate sediments in the Persian Gulf. Sedimentology 12, 108-144. Till, R. 1978: Arid shorelines and evaporites.ln Reading, H. G. (edit.) Sedimentary environments and facies, 178-206. Oxford: BJaekweIl.
https://openalex.org/W2907837217	https://hal.inria.fr/hal-02279546/file/477597_1_En_13_Chapter.pdf	English	null	A Deep Learning Approach for Network Anomaly Detection Based on AMF-LSTM	Lecture notes in computer science	2,018	cc-by	2,021	To cite this version: Mingyi Zhu, Kejiang Ye, Yang Wang, Cheng-Zhong Xu. A Deep Learning Approach for Network Anomaly Detection Based on AMF-LSTM. 15th IFIP International Conference on Network and Par- allel Computing (NPC), Nov 2018, Muroran, Japan. pp.137-141, 10.1007/978-3-030-05677-3_13. hal-02279546 Distributed under a Creative Commons Attribution 4.0 International License A Deep Learning Approach for Network Anomaly Detection based on AMF-LSTM Mingyi Zhu, Kejiang Ye⋆, Yang Wang, Cheng-Zhong Xu Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences {my.zhu, kj.ye, yang.wang1, cz.xu}@siat.ac.cn Abstract. The Internet and computer networks are currently suﬀering from diﬀerent security threats. This paper presents a new method called AMF-LSTM for abnormal traﬃc detection by using deep learning model. We use the statistical features of multi-ﬂows rather than a single ﬂow or the features extracted from log as the input to obtain temporal cor- relation between ﬂows, and add an attention mechanism to the original LSTM to help the model learn which traﬃc ﬂow has more contributions to the ﬁnal results. Experiments show AMF-LSTM method has high accuracy and recall in anomaly type identiﬁcation. ⋆Corresponding author HAL Id: hal-02279546 https://inria.hal.science/hal-02279546v1 Submitted on 5 Sep 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 2 AMF-LSTM Model We proposed an AMF-LSTM (Attention-base Multi-Flow LSTM) model for network anomaly detection. Attention means that our model is based on the attention mechanism [6]. Multi-Flow means that we not only use the character- istics of the current ﬂow itself to detect the anomalies, but also use the previous traﬃc ﬂows with temporal correlation to assist in detecting abnormal traﬃc. LSTM means the main body of our network is based on the long short-term memory networks [7]. Fig. 1 shows the structure of AMF-LSTM model. LSTM F1 h1 LSTM F2 h2 LSTM F3 h3 LSTM F4 h3 LSTM F5 h4 ATTENTION FULL CONNECTION Fig. 1. Structure of AMF-LSTM ATTENTION Fig. 1. Structure of AMF-LSTM 1 Introduction The Internet and computer networks are currently suﬀering from diﬀerent security threats [1]. The Global State of Information Security Survey 2015 [2] found there is a great increase in security incidents during the last several years. Network anomalies stand for a large fraction of the Internet traﬃc and compro- mise the performance of the network resources [1, 3]. With the growing network scale, the traditional methods face two problems: i) the processing speed is too slow, unable to cope with the massive network traﬃc data in today’s Internet en- vironments; ii) it may invade the user’s privacy. This situation can be alleviated by using machine learning methods, which are successfully used in many other areas. However, most of the traditional machine learning methods always focus on the traﬃc itself and extract their own characteristics to detect the potential anomalies. As we know the data transmitted in network is in the form of ﬂows. There is always a temporal correlation between ﬂows, which is also true for abnormal traﬃc in the network. In previous work, researchers focus on the characteristics of traﬃc itself, but ignore that many network anomalies have potential temporal correlation. RNN (Recurrent Neural Networks) is widely used in the ﬁelds that are time series related. Recently, there are some works using RNN and LSTM (Long-Short Term Memory) to detect abnormal traﬃc [4, 5], but they only use a single ﬂow to repeat multiple times, which can only learn the relationship between themselves and cannot learn the relationship between diﬀerent ﬂows. This paper presents an anomaly detection method using deep learning model based on AMF-LSTM. The proposed method has three important features: i) use the previous traﬃc ﬂows as auxiliary features of the traﬃc to be detected; ii) use LSTM to ﬁnd the hidden temporal correlation between these ﬂows, and iii) use the attention mechanism to make model focus on the traﬃc and features that are useful for the results. 3 Experiment We use CICIDS2017 [8] as the experimental dataset. The dataset contains benign and the most up-to-date common attacks, which resembles the true real- world data. The implemented attacks includes the most common attacks based on the 2016 McAfee report [9]. Our experiment mainly has the following hyperparameters: n, the number of ﬂows are selected to detect the traﬃc; the learning rate, which is the step size of neural network for each learning; and the number of LSTM hidden nodes, which is the number of nodes that LSTM uses to learn. Naïve Bayes Adaboost SVM MLP LSTM AMF-LSTM Accuracy 82% 71% 83% 77% 82% 91% Recall 7% 83% 65% 75% 77% 91% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Fig. 2. Accuracy and Recall of 8-Category Classiﬁcation Naïve Bayes Adaboost SVM MLP LSTM AMF-LSTM Accuracy 82% 71% 83% 77% 82% 91% Recall 7% 83% 65% 75% 77% 91% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Fig. 2. Accuracy and Recall of 8-Category Classiﬁcation Fig. 2. Accuracy and Recall of 8-Category Classiﬁcation We ﬁrst study the eﬀect of learning rate and hidden nodes on the model accuracy, and ﬁnd the best value of hidden nodes is 256, and the optimal learning rate is 0.0001. Then, we perform two sets of experiments with n = 10 and n = 20. The experimental results are similar, probably because the attention can focus on where it is needed. We compare the performance of our model with several classic machine learning algorithms, such as Naive Bayes, SVM, AdaBoost, MLP, and the original LSTM. The results of accuracy and recall comparison are shown in the Fig. 2. We can see that our model is signiﬁcantly better than other machine learning algorithms, both in accuracy or recall. We further conduct a deeper study on model with n = 10, lr = 0.0001, node num = 256, which achieves the best performance. The evaluation metrics are shown in the Table 1. As we know, it is far more harmful for a system to judge abnormal traﬃc as normal traﬃc than to judge normal traﬃc as abnormal traﬃc. Therefore, we pay more attention to the value of recall. According to the table, our model can identify most of the anomalies correctly. Table 1. The Results of Diﬀerent Evaluation Metrics Table 1. The Results of Diﬀerent Evaluation Metrics Precision Recall F1-score Flows Normal 0.98 0.91 0.94 348631 DDoS 0.83 0.98 0.90 25606 PortScan 0.82 0.99 0.90 31786 BOT 0.05 0.75 0.10 394 Inﬁltration 0.00 0.75 0.01 8 Web Attack 0.04 0.81 0.07 436 Patator 0.38 0.53 0.44 2767 DoS 0.87 0.88 0.87 50532 5 Conclusion This paper presents a method for abnormal traﬃc detection in the Inter- net by using deep learning model based on AMF-LSTM. We use the statistical features of multi-ﬂows rather than a single ﬂow as the input to obtain temporal correlation between ﬂows, and add an attention mechanism to the original LSTM to help the model learn which traﬃc ﬂow has more contributions to the result. Compared with other classic machine learning algorithms, our model achieves about 10% improvement in accuracy and recall on the multi-classiﬁcation prob- lems. 4 Related Work In prior studies, a number of approaches have been proposed for network anomaly detection. Sun et al. [10] present a survey of intrusion detection tech- niques for mobile ad-hoc networks (MANET) and wireless sensor networks (WSN). Sperotto et al. [11] explain the concepts of ﬂow and classiﬁed attacks, and pro- vide a detailed discussion of detection techniques. Abbes et al. [12] introduce an approach that uses decision trees with protocol analysis for eﬀective intrusion detection. Khan et al. [13] use genetic algorithms to develop rules for network intrusion detection. Tthere are also large number of methods using Neural Net- work. An example of ANN-based IDS is RT-UNNID [14]. Thilina et al. [15] pro- pose a novel framework to perform intruder detection and analysis using deep learning nets and association rule mining. Yuan et al. [16] use the LSTM-CNN framework to ﬁnd users anomalous behavior. Most recently, Zhu et al. [4] use CNN model for network anomaly detection and identiﬁcation and achieve bet- ter performance than traditional machine learning algorithms. Although RNN [5] and LSTM [17] have been used to detect abnormal traﬃc before, they only use a single ﬂow as the input of RNN and recurrent itself multiple times. In our opinion, they can only learn the relation in the traﬃc itself, and can not fully utilize the characteristics of RNN, which can learn the potential relations between diﬀerent traﬃc. Acknowledgment This work is supported by the National Key R&D Program of China (No. 2018YFB1004804), National Natural Science Foundation of China (No. 61702492, U1401258), and Shenzhen Basic Research Program (No. JCYJ20170818153016513, JCYJ20170307164747920). [1] P. Garcia-Teodoro, J. Diaz-Verdejo, G. Maci´a-Fern´andez, and E. V´azquez, “Anomaly-based network intrusion detection: Techniques, systems and chal- lenges,” computers & security, vol. 28, no. 1-2, pp. 18–28, 2009. References [1] P. Garcia-Teodoro, J. Diaz-Verdejo, G. Maci´a-Fern´andez, and E. V´azquez, “Anomaly-based network intrusion detection: Techniques, systems and chal- lenges,” computers & security, vol. 28, no. 1-2, pp. 18–28, 2009. [2] M. Ahmed, A. N. Mahmood, and J. Hu, “A survey of network anomaly detection techniques,” Journal of Network and Computer Applications, vol. 60, pp. 19–31, 2016. [3] T. Benson, A. Akella, and D. A. Maltz, “Network traﬃc characteristics of data centers in the wild,” in Proceedings of the 10th ACM SIGCOMM conference on Internet measurement, 2010, pp. 267–280. [4] M. Zhu, K. Ye, and C.-Z. Xu, “Network anomaly detection and identiﬁcation based on deep learning methods,” in International Conference on Cloud Comput- ing, 2018, pp. 219–234. [5] C. Yin, Y. Zhu, J. Fei, and X. He, “A deep learning approach for intrusion de- tection using recurrent neural networks,” IEEE Access, vol. 5, pp. 21 954–21 961, 2017. [6] J. K. Chorowski, D. Bahdanau, D. Serdyuk, K. Cho, and Y. Bengio, “Attention- based models for speech recognition,” in Advances in neural information process- ing systems, 2015, pp. 577–585. [7] S. Hochreiter and J. Schmidhuber, “Long short-term memory,” Neural computa- tion, vol. 9, no. 8, pp. 1735–1780, 1997. [8] “Intrusion detection evaluation dataset (cicids2017),” 2018. [Online]. Available: ”http://www.unb.ca/cic/datasets/ids-2017.html” [9] “Mcafee labs threats report,” 2018. [Online]. Avail- able: ”https://www.mcafee.com/cn/security-awareness/articles/mcafee-labs- threats-report-mar-2016.aspx” [10] B. Sun, L. Osborne, Y. Xiao, and S. Guizani, “Intrusion detection techniques in mobile ad hoc and wireless sensor networks,” IEEE Wireless Communications, vol. 14, no. 5, 2007. [11] A. Sperotto, G. Schaﬀrath, R. Sadre, C. Morariu, A. Pras, and B. Stiller, “An overview of ip ﬂow-based intrusion detection.” IEEE Communications Surveys and Tutorials, vol. 12, no. 3, pp. 343–356, 2010. [12] T. Abbes, A. Bouhoula, and M. Rusinowitch, “Eﬃcient decision tree for protocol analysis in intrusion detection,” International Journal of Security and Networks, vol. 5, no. 4, pp. 220–235, 2010. [13] M. S. A. Khan, “Rule based network intrusion detection using genetic algorithm,” International Journal of Computer Applications, vol. 18, no. 8, pp. 26–29, 2011. [14] M. Amini, R. Jalili, and H. R. Shahriari, “Rt-unnid: A practical solution to real- time network-based intrusion detection using unsupervised neural networks,” com- puters & security, vol. 25, no. 6, pp. 459–468, 2006. [15] A. Thilina, S. Attanayake, S. Samarakoon, D. Nawodya, L. Rupasinghe, N. Pathi- rage, T. Edirisinghe, and K. References Krishnadeva, “Intruder detection using deep learning and association rule mining,” in Computer and Information Technology (CIT), 2016 IEEE International Conference on, 2016, pp. 615–620. [16] F. Yuan, Y. Cao, Y. Shang, Y. Liu, J. Tan, and B. Fang, “Insider threat detec- tion with deep neural network,” in International Conference on Computational Science, 2018, pp. 43–54. [17] J. Kim, J. Kim, H. L. T. Thu, and H. Kim, “Long short term memory recur- rent neural network classiﬁer for intrusion detection,” in Platform Technology and Service (PlatCon), 2016 International Conference on, 2016, pp. 1–5.
https://openalex.org/W2175707540	https://ejtr.vumk.eu/index.php/about/article/download/53/54	English	null	Lines in the Sand: An Anthropological Discourse on Wildlife Tourism	European journal of tourism research	2,010	cc-by	2,149	© 2010 International University College. All rights reserved © 2010 International University College. All rights reserved Citation: Burns, G.L. (2010) Lines in the Sand: An Anthropological Discourse on Wildlife Tourism. Doctoral dissertation summary. European Journal of Tourism Research 3(2), pp. 119-122 Citation: Burns, G.L. (2010) Lines in the Sand: An Anthropological Discourse on Wildlife Tourism. Doctoral dissertation summary. European Journal of Tourism Research 3(2), pp. 119-122 Received: 21/08/2010 Institution awarding the Ph. D. Degree: Murdoch University, Western Australia Goal and objectives of the dissertation Goal between wildlife and people in wildlife tourism settings? This exploration is an interdiscip- linary journey drawing on understandings, methods and theories from anthropology, tourism and environmental science. It promotes an engagement between anthropology and the scholarly discourses of tourism and environmental science, and a broad approach to defining and understanding the wildlife tourism community. The management of wildlife tourism has been dominated by ideologies informed by western colonialism and its values of nature. These ideologies, made transparent through communicative and interpretative discourses, influence the way management policies and practices are devised and enacted. The inherent scientific and utilitarian views are supported by a doctrine of separation. This is apparent in the dualism posed, and enacted, between nature and culture that sees humans as being the sole carriers of culture that separates them from the uncultured and uncivilised world of nature into which all other animals, and certainly untamed wildlife, belong. It justifies the use of non-humans for human purposes and continues to allow us to treat non-human animals and other forms of nature in often abominable ways. Results Th The case studies and theories explored in this thesis highlight the way wildlife tourism is managed. The dominant constructions under- lying management policies and practices are exposed, and challenged, and alternatives posed. The application of an anthropological lens to the study of tourism and environment, particularly to the subfield of wildlife tourism, has some sound benefits for enhancing understanding and assessing management practices and implications. For wildlife tourism scholars, researchers, planners and mana- gers the significance of this thesis lies in its contribution to a greater understanding of the wildlife tourism community, as well as its exposure of the dominant ideologies governing management and the challenges they pose for alternative ways forward. An important outcome from this research is the insights it provides into how wildlife-based tourism in Australia can continue in a sustainable framework that is suitable for the wildlife, the tourists, other stakeholders, and the managers of this complex phenomenon. I demonstrate throughout that anthropology, in its incarnation as environmental anthropology and as a team player in a necessarily interdisciplinary approach to understanding and resolving environmental issues, has much to offer. This engagement has the potential to enhance not only the sustainable future of nature-based activities like wildlife tourism but also the relevance of anthropology in the postcolonial contem- porary world. Thus the thesis contributes to the body of literature on the anthropology of tourism, environmental anthropology and wildlife tourism. Objectives Objectives Approaches to wildlife tourism management are needed which will optimise stakeholder satisfaction with wildlife tourism, within the constraints of ensuring sustainability of the wildlife product. This thesis is designed around a series of objectives aimed at understanding these constraints and finding new approaches. Guiding the research that informs this thesis are questions that ask why management of the human-wildlife interface occurs in the way it does, and what values underpin the constructions of wildlife that, in turn, enable and legitimise the management. This thesis explores one of the central dilemmas facing wildlife tourism: what are the implications of the separation constructed DOCTORAL DISSERTATION 119 119 Lines in the Sand: An Anthropological Discourse on Wildlife Tourism. Doctoral dissertation summary. understand, and then manage, in the best interests of all parties. Methodology This thesis investigates two situations in which wildlife tourism occurs in Australia. Fraser Island and Penguin Island are two wildlife tourism destinations on opposite sides of the continent with very different wildlife but some very similar issues. From these two contexts data was collected through inter- views, focus groups, participant observation, and from literary and documentary sources. Understanding the empirical data collected from these case studies is facilitated through a social constructionist view of discourse analysis that allows an unpacking of the messages and a stance from which to challenge the dominant ideologies that frame management and interaction. The argument is made for a rethinking of the way wildlife tourism interactions are managed in some settings. The ideology of separation, enacted both conceptually and physically to create maintain boundaries, is demonstrated through the two case studies and the ways in which interactions between humans and wildlife are currently managed. An alternative is posed, that by reconstructing management in settings where wildlife tourists may be more accepting of their own responsibility towards nature, a model can be developed that allows people and wildlife to co-exist without ‘killing’ the natural instincts of either. Practical application of the dissertation The thesis proposes ways of reconstructing the management of wildlife tourism that have practical applications for wildlife tourism managers. Theoretical conclusions The need for a holistic framework encompassing all the stakeholders in any wildlife tourism venture is proposed. This approach to wildlife tourism is best serviced by examining perspectives, values and concerns of all members of the wildlife tourism community at any given destination. It is only through this type of holistic and situated focus that we can hope to effectively Abstract of chapter three Abstract of chapter three Chapter Three explains the rationale of the design behind collection methods and how data were collected during the research. Topics covered include the combined research strategies of ethnography and case studies, as well as a description of each of the data collection methods used (sampling, interviews, literature and documentary sources, and participant observation). The ethical considerations pertinent to the collection of data and how they were dealt with are also discussed here. Finally, the chapter turns to an explanation of the data analysis and theorisation. Discourse analysis is presented as a guiding analytical and theoretical tool for collecting, collating and understanding the data. An overview of social constructionism explains how this perspective is used as a context for the study and understanding of nature and the environment in this thesis. Abstract of chapter six Chapter Six examines the links between wildlife tourism, conservation and sustain- ability, arguing that conservation practices are informed by the dominant, Eurocentric, ideology of nature that is a legacy of colonisation. The relationship between nature and culture explored in this chapter provides a framework that is later applied to the separation of wildlife and people in wildlife tourism settings by management authorities. The use, or denial, of anthropomorphism is discussed as an example of a discourse that influences the construction of wildlife tourism and interactions between people and wildlife. Abstract of chapter seven Chapter Seven focuses on the Fraser Island case study as an example of wildlife management in a tourism context. It illustrates the dilemma of deciding between prioritising of management of wildlife for people or management of people for wildlife. The first, and only, human death from a dingo attack on Fraser Island occurred in April 2001, and was immediately followed by a government- ordered cull of dingoes. This chapter explores issues surrounding both this decision and the management strategies implemented after- wards. Based on interviews with a variety of Abstract of chapter four In Chapter Four I discuss where, and why, data from the case studies fits into bodies of literature on anthropology of tourism and environmental anthropology and why this literature provides both a sound, and unique, framework for the thesis. This includes exploration of the historical relationship between anthropology and tourism. The history of environmental anthropology is also discussed, along with its recent rise and importance in Australia. Content of the dissertation Abstract of chapter one p Chapter One provides an overview of the thesis, explaining the aim of the research and discussing its significance. Because an understanding of terminology is important, some of the key terms used throughout the thesis are introduced in this first chapter 120 Burns, G.L. (2010) / European Journal of Tourism Research 3(2), pp. 119-122 the field of environmental science are outlined. which concludes with an outline of, and rationale for, the structure of the thesis. Abstract of chapter five Chapter Five discusses literature on the contested notion of community as it is relevant to wildlife tourism. The wildlife tourism community referred to throughout the thesis is made up of many stakeholder groups and individuals with interests in wildlife tourism that are often diverse and conflicting, and the importance of inclusion of these stakeholders in decisions about wildlife tourism is made implicit. Some of the issues addressed include involvement of stake- holders, community attitudes and values, and their impact on people, wildlife and the sustainability of tourism. The importance of considering all these issues when planning for, and managing, wildlife tourism, is discussed. Abstract of chapter two The two case studies, from which empirical evidence was collected for this thesis, are introduced in Chapter Two. Fraser Island, off the Queensland coast, and Penguin Island, off the Western Australian coast, are both destinations for wildlife tourism. This chapter discusses the location of these two case studies, the wildlife they contain, and their wildlife tourism community (managers, tourists, and other stakeholders). Abstract of chapter three Opportunities and challenges for increasing both the academic and applied engagement of anthropology with 121 Lines in the Sand: An Anthropological Discourse on Wildlife Tourism. Doctoral dissertation summary of wildlife change, management of interactions must also change. stakeholders in the wildlife tourism community, many conflicting perspectives on human-wildlife interaction as a component of tourism are identified. The conclusion is drawn that while strategies for managing dingoes are essential, if such attacks are a consequence of humans feeding wildlife and resultant wildlife habituation, then strategies for successfully managing people are also necessary for this type of wildlife tourism to be sustainable. Abstract of chapter ten In Chapter Ten the physical and conceptual separation of people and wildlife in the two wildlife tourism settings is explored and its implications interrogated and challenged. The construction of boundaries between people and wildlife are analysed in terms of the ideologies and values that underpin them. The role of anthropomorphism in creating and maintaining these boundaries is investigated and the argument made that anthro- pomorphising penguins may assist their conservation while the ‘anthropodenial’ of dingoes hinders it. The chapter calls for a reconstruction of people and wildlife, and a reconstruction of the management of their interactions. Embracing the ideals of eco- tourism, and empowering of wildlife tourists to take responsibility for their own actions is suggested as a way forward. Abstract of chapter eight Chapter Eight uses a framework of social constructionism to analyse the Fraser Island Dingo Management Strategy (FIDMS). To ensure the validity and effectiveness of wildlife management, it is necessary to consider the assumptions underlying wildlife management and perpetuated through management discourses. This chapter deconstructs dingo management on Fraser Island. It argues that the process of construction in defining environmental problems and possible solutions needs to be recognised and the nature of the ideologies underpinning environmental management acknowledged. Management practices need to be consistent with discourses, reflecting priorities, addressing the problem and facilitating the fulfilment of expectations. Ultimately, entrenched ideologies and assumptions need to be challenged to create new possibilities for action. Abstract of chapter eleven Chapter Eleven comments on the value and utility of the notion of community for understanding, and ultimately managing sustainably, wildlife tourism. Abstract of chapter three The chapter advocates the need to challenge construc- tions underpinning wildlife management and the accepted interactions between wildlife and people, arguing for recognition of humans as part of the natural system and a relaxing of management strategies that focus on the separation of people and nature. It also encourages further research in the interdisciplinary overlap between anthro- pology, environmental science, and tourism: the anthropology of environmental tourism. The combination of perspectives within this area offers crucial insights that can lead to positive outcomes for the environmental and social challenges faced by wildlife, and for the sustainability of wildlife tourism in Australia. Abstract of chapter nine Chapter Nine offers a comparison of the similarities and differences in wildlife tourism on Fraser Island and Penguin Island. This chapter examines the roles of key stake- holders in the wildlife tourism community, focusing on the values placed on wildlife, as well as the nature of human-wildlife interactions and management policies and practices in the two wildlife tourism settings. The relationships between people and dingoes, and between people and penguins, are explored and shown to have shifted over time, and be subject to different controls in each location. Data show that as perceptions Available: This thesis is available on Murdoch’s Open Repository at http://researchrepository.murdoch.edu.au/714 Available: This thesis is available on Murdoch’s Open Repository at http://researchrepository.murdoch.edu.au/714 122
https://openalex.org/W3163054779	https://jmedicalcasereports.biomedcentral.com/track/pdf/10.1186/s13256-021-02855-w	English	null	Outcomes of vitrectomy for retinal detachment in a patient with Ehlers–Danlos syndrome type IV: a case report	Journal of medical case reports	2,021	cc-by	4,268	© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: The Ehlers–Danlos syndrome (EDS) is a group of connective tissue disorders characterized by fragile blood vessels and an increased tendency for bleeding and scarring. Here, we report the outcome of a pars plana vitrectomy for the treatment of rhegmatogenous retinal detachment in a patient with EDS type IV (vascular type). Case presentation: A 40-year-old Slovenian man with high myopia, unilateral bullous retinal detachment, and vitre- ous hemorrhage was referred for surgery. The patient had a history of colon perforation, muscle and arterial rupture in both lower limbs, and recurrent shoulder joint luxations. Genetic testing revealed a pathogenic mutation in the COL3A1 gene. The patient underwent a 25-gauge three-port pars plana vitrectomy. The tendency for bleeding during surgery was prevented by endodiathermy applied to the edges of the retinal breaks. Endolaser photocoagulation was performed under air. The surgical procedure was completed with the injection of gas tamponade, followed by the patient remaining for a few days in a face-down position. Mild postoperative vitreous hemorrhage was resorbed in first week after the surgery. Postoperative extensive pigment dispersion on the posterior lens face persisted for several weeks. After the gas tamponade had resorbed, the retina was flat and remained attached during the follow-up period. Eight months after the surgery, visual acuity continued to improve from a preoperative 6/38 to 6/6.6 (Snellen chart) at the last checkup. Conclusion: A small-gauge pars plana vitrectomy with gas tamponade and laser photocoagulation under air may successfully achieve reattachment of the retina in patients with high myopia with EDS type IV and restore visual acuity. Keywords: Ehlers–Danlos syndrome, Vascular type, Rhegmatogenous retinal detachment, Pars plana vitrectomy, Case report Keywords: Ehlers–Danlos syndrome, Vascular type, Rhegmatogenous retinal detachment, Pars p Case report Keywords: Ehlers–Danlos syndrome, Vascular type, Rhegmatogenous retinal detachment, Pars plana vitrectomy, Case report Case report scarring [1]. According to the Villefranche nosology, EDS is classified into six major groups [2]. Lumi et al. J Med Case Reports (2021) 15:249 https://doi.org/10.1186/s13256-021-02855-w Open Access Xhevat Lumi1, Gaber Bergant2, Anila Lumi1 and Mina Mahnic1 Xhevat Lumi1, Gaber Bergant2, Anila Lumi1 and Mina Mahnic1 Background Ehlers–Danlos syndrome (EDS) is a heritable heteroge- neous group of connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tis- sue fragility leading to significant bruising and atrophic i EDS type IV is characterized by changes in the vascu- lar structure. These patients are prone to arterial, diges- tive, and obstetrical complications. Congenitally thin and fragile arterial walls lead to arterial dissection and tears, bleeding, and hematomas [3]. Although this tendency is higher in large- and medium-sized arteries, all ana- tomical areas can be affected [3]. It is therefore assumed that surgery for rhegmatogenous retinal detachment (RRD) in patients with EDS type IV has the potential *Correspondence: xhlumi@hotmail.com 1 Eye Hospital, University Medical Centre Ljubljana, Grablovičeva ulica 46, 1000 Ljubljana, Slovenia Full list of author information is available at the end of the article Lumi et al. J Med Case Reports (2021) 15:249 Page 2 of 6 to be complicated by arterial bleeding and consecutive scarring. retinal detachment superiorly with two U-shaped retinal tears (Fig. 1). Very few reports have been published on the occur- rence of retinal detachment in patients with EDS [4–7], and only two of these describe a clinical case of RRD that was managed by a pars plana vitrectomy (PPV) [6, 7]. In both of the cases described, the patients underwent several surgeries due to redetachment with proliferative vitreoretinopathy, ultimately ending up with silicone oil tamponade and worse visual acuity [6, 7].hf The patient had high myopia and had undergone pho- torefractive keratectomy on both eyes 9 years previously. His previous refraction was − 9.50 Dsph (diopter sphere) in the right eye and − 5.50 Dsph in the left eye. Ophthal- moscopy of the left eye showed multiple degenerative changes on the retinal periphery. The medical history of the patient included colon per- foration, muscle and arterial rupture in both lower limbs, and recurrent shoulder joint luxation, as well as sus- pected EDS. His family history for connective tissue dis- eases was negative. The patient underwent genetic testing for EDS, which showed a pathogenic mutation in the COL3A1 gene that is associated with EDS type IV (vas- cular type). The inheritance pattern of this type of EDS is autosomal dominant and there is typically a heterozygous mutation in the COL3A1 gene that encodes type III col- lagen [1]. There are different types of surgical approaches for RRD, including pneumatic retinopexy, scleral buckling, and PPV [8]. The type of surgery chosen depends on the complexity of the detachment, age of the patient, and the surgeon’s preference. Primary PPV is indicated in com- plex retinal detachments [8]. Here, we report the case of a bullous subtotal retinal detachment and a vitreous hemorrhage managed by a small-gauge PPV in a patient with EDS type IV. Variant description and interpretation Fourth, with the gene also demonstrating evolutionary intolerance for missense variants (PP2) [11], multiple algorithms for theoretical pathogenicity predictions, including MutationTaster2 and MetaSVM, provide a unanimously pathogenic pre- diction of the effects of the variant on protein function, which is further supported by the location of the variant affecting an evolutionary conserved amino acid (PP3) [12, 13]. In accordance with ACMG/AMP standards and guidelines for the interpretation of sequence variants, this variant was therefore classified as a class 5–patho- genic variant [14]. A 25-gauge pars plana vitrectomy was performed. After cleaning the vitreous hemorrhage, eight more reti- nal breaks were found positioned in all four quadrants, which could not be seen preoperatively due to the vitre- ous hemorrhage (Fig. 2). During the surgery, bleeding was prevented by endo- diathermy applied at the edges of the retinal breaks. After shaving the vitreous periphery, fluid/air exchange was done. Laser photocoagulation around the retinal tears was performed under air. The endotamponade at the end was 10% perfluoropropane (C3F8) gas. The patient was instructed to maintain a face-down position for a few days after the surgery. Immediately after the surgery, we noted both a mild vitreous hemorrhage which resorbed within 1 week and extensive pigment dispersion on the posterior lens face which persisted for several weeks (Fig. 3). Otherwise, there were no other complications.h well-established mechanism of pathogenicity in collagen- opathies (PM1) [10]. Third, the variant was not present among approximately 141,000 controls in the gnomAD project v2.1.1 (PM2) [11]. These findings indicate a mis- sense variant in a gene for which missense variants are a common mechanism of disease. Fourth, with the gene also demonstrating evolutionary intolerance for missense variants (PP2) [11], multiple algorithms for theoretical pathogenicity predictions, including MutationTaster2 and MetaSVM, provide a unanimously pathogenic pre- diction of the effects of the variant on protein function, which is further supported by the location of the variant affecting an evolutionary conserved amino acid (PP3) [12, 13]. In accordance with ACMG/AMP standards and guidelines for the interpretation of sequence variants, this variant was therefore classified as a class 5–patho- genic variant [14]. The patient underwent several postoperative examina- tions. At the last checkup, 8 months after the surgery, the retina remained attached. Optical coherence tomography (OCT) of the macula lutea showed normal retinal struc- ture with distinctive layers (Fig. 4).The slit-lamp examina- tion of the anterior segment revealed an initial posterior subcapsular cataract. Variant description and interpretation A 40-year-old Slovenian man with high myopia was referred to the Eye Hospital, University Medical Centre Ljubljana, Slovenia complaining of a 2-day deterioration in the visual acuity of his right eye (OD). He described the presentation as a curtain-like dark zone in the infe- rior part of the visual field that progressively enlarged superiorly. He denied any eye injury. Whole-exome sequencing indicated the presence of a pathogenic heterozygous missense variant in the COL3A1 gene. Heterozygous variant c.1052G>T (LRG_3t1) in the COL3A1 gene causes substitution of the amino acid glycine with valine at position 351 in the amino acid sequence coded by COL3A1. The variant was previously established as pathogenic, in line with the correspond- ing American College of Medical Genetics and Genom- ics/Association for Molecular Pathology (ACMG/AMP) criteria for interpretation of sequence variants, based on the following. First, the variant was reported to be path- ogenic in a patient with vascular EDS (PS4_MOD) [9]. Second, the variant affects a glycine residue in a Gly-X- Y tripeptide repeat of the triple-helical region, which is a On presentation, the patient’s best corrected visual acuity (BCVA) in the right eye was 6/38 (Snellen chart) and the intraocular pressure was 10 mmHg. Slit-lamp examination of the anterior segment revealed pigment dispersion on the corneal endothelium; otherwise, the status was normal. Indirect ophthalmoscopy showed a vitreous hemorrhage and a macula-off subtotal bullous Fig. 1 Left: Color fundus photograph of the right eye shows bullous retinal detachment with mild vitreous hemorrhage. Right: Optical coherence tomography (OCT) scan demonstrates macula-off retinal detachment Fig. 1 Left: Color fundus photograph of the right eye shows bullous retinal detachment with mild vitreous hemorrhage. Right: Optical coherence tomography (OCT) scan demonstrates macula-off retinal detachment Lumi et al. J Med Case Reports (2021) 15:249 Lumi et al. J Med Case Reports (2021) 15:249 Lumi et al. J Med Case Reports (2021) 15:249 Page 3 of 6 well-established mechanism of pathogenicity in collagen- opathies (PM1) [10]. Third, the variant was not present among approximately 141,000 controls in the gnomAD project v2.1.1 (PM2) [11]. These findings indicate a mis- sense variant in a gene for which missense variants are a common mechanism of disease. Variant description and interpretation Since the patient was satisfied with the functional outcome, he was not scheduled for cata- ract surgery. The BCVA of the right eye was 6/6.6 and the intraocular pressure was 9 mmHg. A 25-gauge pars plana vitrectomy was performed. After cleaning the vitreous hemorrhage, eight more reti- nal breaks were found positioned in all four quadrants, which could not be seen preoperatively due to the vitre- ous hemorrhage (Fig. 2). Fig. 2 Screenshots of the intraoperative video taken during the vitrectomy of the right eye, demonstrating several retinal breaks in four quadrants at the retinal periphery. a Two breaks at 12 o’clock, b breaks at 9 o’clock, c breaks at 3 o’clock, d breaks at 7 o’clock ig. 2 Screenshots of the intraoperative video taken during the vitrectomy of the right eye, demonstrating several retinal brea t the retinal periphery. a Two breaks at 12 o’clock, b breaks at 9 o’clock, c breaks at 3 o’clock, d breaks at 7 o’clock Fig. 2 Screenshots of the intraoperative video taken during the vitrectomy of the right eye, demonstrating several retinal breaks in four quadrants at the retinal periphery. a Two breaks at 12 o’clock, b breaks at 9 o’clock, c breaks at 3 o’clock, d breaks at 7 o’clock Fig. 2 Lumi et al. J Med Case Reports (2021) 15:249 Page 4 of 6 Fig. 3 Postoperative slit lamp photograph of the anterior segment of the right eye showing extensive pigment dispersion on the posterior lens face ive slit lamp photograph of the anterior segment of the right eye showing extensive pigment dispersion on the posterior len Fig. 3 Postoperative slit lamp photograph of the anterior segment of the right eye showing extensive pigment dispersion face Fig. 4 Left: postoperative color fundus photograph of the right eye showing flat retina; right: optical coherence tomography scan of the macular area demonstrating restored retinal layers Fig. 4 Left: postoperative color fundus photograph of the right eye showing flat retina; right: optical coherence tomography scan of the macular area demonstrating restored retinal layers Discussion variability, and clinical overlap of the different subtypes of Ehlers–Danlos syndromes, a definitive diagnosis is reached on the basis of molecular confirmation of a causative genetic variant. Molecular diagnosis provides information on the inheritance pattern, recurrence risk, and prognosis [1]. The 2017 international classification of EDS recognizes 13 subtypes which are defined by major (high diagnos- tic specificity) ± minor (lesser diagnostic specificity) clinical criteria that are suggestive of a specific subtype [1]. Due to the vast genetic heterogeneity, phenotypic Lumi et al. J Med Case Reports (2021) 15:249 Lumi et al. J Med Case Reports (2021) 15:249 Page 5 of 6 Page 5 of 6 Many ophthalmological changes have been described in EDS, such as convergence insufficiency, blue sclera, dry eye syndrome, keratoglobus, high myopia, and retinal tears [15]. Other studies have described angioid streaks, macro- and microstructural changes to the cornea, microcornea, spontaneous corneal rupture or, following minor trauma, corneal hydrops, severe conjunctivocha- lasis, fragility of blood vessels with recurrent vitreous hemorrhage, scleral rupture, and dislocation of the lens [16–21].hf quadrants. Since this type of EDS has a higher ten- dency for bleeding, we prevented intraoperative bleed- ing by coagulation to the edges of the retinal breaks by endodiathermy. By performing laser photocoagulation under air, the energy needed for photocoagulation was lower since the retina is closely attached to the retinal pigment epithelium, which reduces cell dispersion and the possible occurrence of scarification or postopera- tive proliferative vitreoretinopathy. Eight months after the surgery the retina remained attached. An OCT scan of the macula did not show structural changes. Apart from incipient lens opacifica- tion, there were no further changes during the follow- up period. The patient was satisfied with the functional result of the surgery and was not scheduled further for cataract surgery. The worst affection of the eyes has been described in brittle cornea syndrome, which is characterized by very thin corneas, early keratoconus, progressive keratoglo- bus, blue sclera, high myopia, retinal detachment, and a higher rate of enucleation due to the changes after a globe rupture, in addition to a wide variety of extra-ocu- lar manifestations [22]. In this single case of EDS type IV, we were able to reattach the retina and restore visual function. Type IV EDS is typically caused by a mutation in the COL3A1 gene, which encodes an important component of collagen type III. Funding g The authors declare no source of funding for this study. Authors’ contributions MM and AL prepared the manuscript. XL and MM were involved in the man- agement of the patient. GB performed genetic diagnostics, interpretation, and description. XL did a major scientific revision of the manuscript. All authors have read and approved the final manuscript. A similar course of the disease was reported by Whit- low et al. [7]. Their patient with EDS type VIa and bul- lous retinal detachment required several surgeries with scleral patch grafts and 20-gauge PPVs because of reti- nal detachment with proliferative vitreoretinopathy. The patient ended up with a worse anatomical and functional outcome, namely, a visual acuity 1/60 [7]. Availability of data and materialsi y The data that support the findings of this study are available from the medi- cal records. The authors confirm that all data underlying this case are fully available without restriction and can be accessed by contacting Xhevat Lumi (xhlumi@hotmail.com). Our patient with EDS type IV presented with an acute worsening of visual acuity in a high myopic eye that has already undergone corneal refractive surgery. The detachment was bullous in superior quadrants accompanied by a vitreous hemorrhage. We decided to perform a small gauge PPV (25-gauge) with the aim of reducing intraoperative trauma. Intraoperatively, we found a total of ten retinal breaks localized in all four Conclusions S ll Small-gauge vitrectomy can reduce intraoperative trauma and avoid intensive bleeding. Performing endo- diathermy to the edges of the retinal breaks and laser photocoagulation under air reduces the tendency for bleeding and cell dispersion. Therefore, we conclude that small-gauge PPV can produce good outcomes in high myopia retinal detachment in patients with EDS type IV. Apart from several reports on structural changes, there are very few publiations on retinal detachment and its management in EDS. Bodanowitz et al. reported a case of a patient with RRD in EDS type VI managed by PPV [6]. These authors described several complications during the surgery, such as choroidal detachment and intensive bleeding. The closure of the sclerotomies was also diffi- cult due to the very thin sclera. Because of redetachment with proliferative vitreoretinopathy, the patient needed two revitrectomies. The final visual acuity after the last surgical procedure with silicon oil tamponade was 6/60 [6]. Discussion Collagen type III is found in tissues of the walls of blood vessels, intestinal walls, lungs, and skin. Since corneal collagen fibrils are composed of col- lagen types I and V and the vitreous body collagen fibrils are types II, IX, V/XI, and VI collagens, the main affected structure in EDS type IV can be sclera, which is com- posed of collagen I and III [23–25]. bb e at o s EDS: Ehlers–Danlos syndrome; BCVA: Best corrected visual acuity; OCT: Optical coherence tomography; PPV: Pars plana vitrectomy; RRD: Rhegmatogenous retinal detachment. Abbreviations EDS Ehl D EDS: Ehlers–Danlos syndrome; BCVA: Best corrected visual acuity; OCT: Optical coherence tomography; PPV: Pars plana vitrectomy; RRD: Rhegmatogenous retinal detachment. References yp y changes. Invest Ophthalmol Vis Sci. 2013;54(13):8062–8. 1. Malfait F, Francomano C, Byers P, Belmont J, Berglund B, Black J, et al. The 2017 international classification of the Ehlers–Danlos syndromes. Am J Med Genet C Semin Med Genet. 2017;175(1):8–26. 1. Malfait F, Francomano C, Byers P, Belmont J, Berglund B, Black J, et al. The 2017 international classification of the Ehlers–Danlos syndromes. Am J Med Genet C Semin Med Genet. 2017;175(1):8–26. 18. Cameron JA. Corneal abnormalities in Ehlers–Danlos syndrome type VI. Cornea. 1993;12(1):54–9. 19. Whitaker JK, Alexander P, Chau DY, Tint NL. Severe conjunctivochalasis in association with classic type Ehlers–Danlos syndrome. BMC Ophthalmol. 2012;3(12):47. 2. Parapia LA, Jackson C. Ehlers–Danlos syndrome—a historical review. Br J Haematol. 2008;141(1):32–5. https://doi.org/10.1111/j.1365-2141.2008. 06994.x. 2. Parapia LA, Jackson C. Ehlers–Danlos syndrome—a historical review. Br J Haematol. 2008;141(1):32–5. https://doi.org/10.1111/j.1365-2141.2008. 06994.x. 20. Chikamoto N, Teranishi S, Chikama T, Nishida T, Ohshima K, Hatsukawa Y. Abnormal retinal blood vessels in Ehlers–Danlos syndrome type VI. Jpn J Ophthalmol. 2007;51(6):453–5. 3. Germain DP. Ehlers–Danlos syndrome type IV. Orphanet J Rare Dis. 2007;2:32. 3. Germain DP. Ehlers–Danlos syndrome type IV. Orphanet J Rare Dis. 2007;2:32. 4. Bossu A, Lambrechts J. Manifestations oculaires du syndrome d’Ehlers– Danlos. Ann Oculist (Paris). 1954;187:227–36. 4. Bossu A, Lambrechts J. Manifestations oculaires du syndrome d’Ehlers– Danlos. Ann Oculist (Paris). 1954;187:227–36. 4. Bossu A, Lambrechts J. Manifestations oculaires du syndrome d Ehlers Danlos. Ann Oculist (Paris). 1954;187:227–36. 5. Beighton P. Serious ophthalmological complications in the Ehlers–Danlos syndrome. Br J Ophthalmol. 1970;54(4):263–8. 21. Sharma Y, Sudan R, Gaur A. Post traumatic subconjunctival dislocation of lens in Ehlers–Danlos syndrome. Indian J Ophthalmol. 2003;51(2):185–6. 21. Sharma Y, Sudan R, Gaur A. Post traumatic subconjunctival dislocation of lens in Ehlers–Danlos syndrome. Indian J Ophthalmol. 2003;51(2):185–6. 5. Beighton P. Serious ophthalmological complications in the Ehlers–Danlos syndrome. Br J Ophthalmol. 1970;54(4):263–8. 22. Burkitt Wright EM, Porter LF, Spencer HL, Clayton-Smith J, Au L, Munier FL, et al.. Brittle cornea syndrome: recognition, molecular diagnosis and management. Orphanet J Rare Dis. 2013;8:68. https://doi.org/10.1186/ 1750-1172-8-68. 6. Bodanowitz S, Hesse L, Pöstgens H, Kroll P. Retinal detachment in Ehlers– Danlos syndrome. Treatment by pars plana vitrectomy. Ophthalmologe. 1997;94(9):634–7. 23. Holmes D, Gilpin C, Baldock C, Ziese U, Koster A, Kadler K. Corneal col- lagen fibril structure in three dimensions: structural insights into fibril assembly, mechanical properties, and tissue organization. Proc Natl Acad Sci USA. 2001;98:7307–12. https://doi.org/10.1073/pnas.111150598.). 7. Whitlow S, Idrees Z. References RD repair using 360-degree scleral graft for extensive scleral ectasia in a patient with Ehlers–Danlos syndrome. Am J Oph- thalmol Case Rep. 2019;17:100554. https://doi.org/10.1016/j.ajoc.2019. 100554. 7. Whitlow S, Idrees Z. RD repair using 360-degree scleral graft for extensive scleral ectasia in a patient with Ehlers–Danlos syndrome. Am J Oph- thalmol Case Rep. 2019;17:100554. https://doi.org/10.1016/j.ajoc.2019. 100554. 24. Lumi X, Hawlina M, Glavač D, Facskó A, Moe MC, Kaarniranta K, et al. Ageing of the vitreous: From acute onset floaters and flashes to retinal detachment. Ageing Res Rev. 2015;21:71–7. https://doi.org/10.1016/j.arr. 2015.03.006. 8. Lumi X, Lužnik Z, Petrovski G, Petrovski BE, Hawlina M. Anatomical success rate of pars plana vitrectomy for treatment of complex rhegmatogenous retinal detachment. BMC Ophthalmol. 2016;16:216. 9. Pepin MG, Schwarze U, Rice KM, Liu M, Leistritz D, Byers PH. Survival is affected by mutation type and molecular mechanism in vascular Ehlers– Danlos syndrome (EDS type IV). Genet Med. 2014;16(12):881–8. 25. Watson PG, Young RD. Scleral structure, organisation and disease. A review. Exp Eye Res. 2004;78(3):609–23. 10. Mizuno K, Boudko S, Engel J, Bächinger HP. Vascular Ehlers–Danlos syndrome mutations in type III collagen differently stall the triple helical folding. J Biol Chem. 2013;288(26):19166–76. 10. Mizuno K, Boudko S, Engel J, Bächinger HP. Vascular Ehlers–Danlos syndrome mutations in type III collagen differently stall the triple helical folding. J Biol Chem. 2013;288(26):19166–76. Consent for publication spectrum of loss-of-function intolerance across human protein-coding genes. bioRxiv. 2019. https://doi.org/10.1101/531210. spectrum of loss-of-function intolerance across human protein-coding genes. bioRxiv. 2019. https://doi.org/10.1101/531210. p Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the consent is avail- able for review by the Editor-in-Chief of this journal. 12. Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods. 2014;11(4):361–2. Ethics approval and consent to participate Ethics approval and consent to participate Ethics approval was obtained and written informed consent was obtained from the patient through the official hospital forms. Page 6 of 6 Lumi et al. J Med Case Reports (2021) 15:249 Lumi et al. J Med Case Reports Author details 1 1 Eye Hospital, University Medical Centre Ljubljana, Grablovičeva ulica 46, 1000 Ljubljana, Slovenia. 2 Clinical Institute of Genomic Medicine, University Medical Centre Ljubljana, Ljubljana, Slovenia. 15. Perez-Roustit S, Nguyen DT, Xerri O, Robert M, De Vergnes N, Mincheva Z, et al. Ocular manifestations in Ehlers–Danlos Syndromes: clinical study of 21 patients. J Fr Ophtalmol. 2019;42(7):722–9. (In French). Received: 9 April 2020 Accepted: 7 April 2021 Received: 9 April 2020 Accepted: 7 April 2021 16. Singman EL, Doyle JJ. Angioid streaks are not a common feature of Ehlers–Danlos syndrome. JAMA Ophthalmol. 2019;137(3):239. Ehlers–Danlos syndrome. JAMA Ophthalmol. 2019;137(3):239. 17. Villani E, Garoli E, Bassotti A, Magnani F, Tresoldi L, Nucci P, et al. The cor- nea in classic type Ehlers–Danlos syndrome: macro- and microstructural changes. Invest Ophthalmol Vis Sci. 2013;54(13):8062–8. Competing interests h h d l h 13. Kim S, Jhong JH, Lee J, Koo JY. Erratum to: Meta-analytic support vector machine for integrating multiple omics data. BioData Min. 2017;10:8. The authors declare that they have no competing interests. 14. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint con- sensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405–24. 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. g 11. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. Variation across 141,456 human exomes and genomes reveals the 11. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. Variation across 141,456 human exomes and genomes reveals the • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? 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https://openalex.org/W2811024719	https://zenodo.org/record/4286371/files/Hesslerova_et_al_2018.pdf	English	null	The effect of forest disturbance on landscape temperature	Ecological engineering	2,018	cc-by	9,943	1. Introduction studies: deforestation was always immediately followed by a period of water yield increase and the subsequent period of recovery (forest re- growth) may or may not be characterized by a decrease in water yield. 1.1. Opinions on the role of forests in water balance/hydrology and climate WeForest (2015) and Ellison et al. (2017), in a thorough review, state the following ﬁve forest processes as more important than pre- viously thought. Management to support them can result in short and long-term beneﬁts for water availability and climate: The debate on the role of forests in the hydrological cycle can be traced back in history – Antiquity, Middle Age and up to the present. Man tried to understand the impact of forests on the water cycle and was aware of the serious consequences deforestation had on hydrology, soil, climate, precipitation, and temperature. The role of forest stands and the consequences of their damage were based on long-term ob- servations and personal experience; scientiﬁc measurements and the eﬀort to describe the whole system in scientiﬁc terms appear in the 19th century. Adult forests dampen the extremes of climate; this is the ex- perience of both historical civilizations and generations of landscape managers, which was reﬂected in the Forest Law from the 18th century in the Austrian Monarchy. Over a long period of time a series of sci- entiﬁc papers and books were published on damping of temperature extremes (Geiger, 1957; Geiger et al., 2003, 2009). Forest practitioners wrote comprehensive books based on long- term experiences explaining the irreplaceable hydrological role of forests (Marsh, 1864; Úlehla, 1947). 1. Forests promote precipitation. 2. Trees and forests are natural cooling systems. 3. Forests generate air and moisture ﬂows. 4. Trees and forests can improve groundwater recharge 5. Forests can moderate ﬂooding. The authors bring evidence both from ecophysiological studies and from evaluation of how large forest complexes function. They empha- size the direct role of forests in the distribution of solar energy, cooling, water cycle, and local climate. The IPCC (Intergovernmental Panel on Climate Change) and main- stream science focus on the role of forests and wetlands in global cli- mate change in terms of the greenhouse eﬀect: forests aﬀect climate by serving as a sink/source for carbon dioxide and other greenhouse gases (GHGs). Contents lists available at ScienceDirect Contents lists available at ScienceDirect A R T I C L E I N F O Keywords: Deforestation Surface and air temperature Snow melting Landsat Ground thermal measurement Evapotranspiration Bark beetle Landscape drying Since the 1990s, the territory of the Šumava National Park (Czech Republic) has faced signiﬁcant changes in land cover, especially deforestation, in conjunction with several bark beetle disturbances and hurricane Kyrill in 2007. The aim of the study is to review the hydrological and climatic function of the forest and deforestation impacts on the landscape temperature. As a case study, surface temperature changes of the selected area of Šumava National Park from the satellite Landsat thermal data is presented from 1991 to 2016. At the sites with decayed forest, the surface temperature increased by 2–4 °C. Images from ground temperature measurements illustrate extreme temperature diﬀerences (∼35 °C) at locations where dead wood has not been removed; in the live forest, they are around 5 °C. Further, we show the increase in air temperature is associated with the decay of forest stands, including snow melting. The duration of the permanent snow cover on the mountaintops with the growing forest in the last four years is, on average, 11 days longer than the areas with decayed forest. The results show that the increase in surface temperature in the large area causes changes in the local climate and hy- drological regime. These changes may have a negative impact on the surrounding ecosystems, including the Šumava wetlands and peat bogs belonging to the Ramsar sites. https://doi.org/10.1016/j.ecoleng.2018.06.011 Received 31 March 2018; Received in revised form 5 June 2018; Accepted 11 June 2018 0925-8574/ © 2018 Elsevier B.V. All rights reserved. Corresponding author. E-mail addresses: hesslerova@enki.cz (P. Hesslerová), huryna@nh.cas.cz (H. Huryna), pokorny@enki.cz (J. Pokorný), prochaz@zf.jcu.cz (J. Procházka). ⁎ Corresponding author. E-mail addresses: hesslerova@enki.cz (P. Hesslerová), huryna@nh.cas.cz (H. Huryna), pokorny@enki.cz (J. Pokorný), prochaz@zf.jcu.cz (J. Procházka). Ecological Engineering 120 (2018) 345–354 Ecological Engineering 120 (2018) 345–354 1.2. Forests attract water from oceans several thousand km deep in continents The Šumava National Park (NP) was proclaimed in 1991 with an area of 680.6 km2. The subjects of protection are forests, peat bogs and other wetlands, glacial relief, and cultural non-forested areas. Forest stands occupy 80% of the area, and the most valuable are mountain spruces reaching the forest boundary (1100–1300 m above sea level). In these plots, a non-intervention strategy was introduced in the 1990s, with the assumption of no bark beetle propagation. Ecological experts, unlike foresters, claim that these areas are resistant primeval forests, where the bark beetle is a natural part of the entomofauna and its overgrowth is impossible because of the presence of natural predators. However, bark beetle reproduction signiﬁcantly increased from 2007 to 2011 after hurricane Kyrill. As a result of the non- intervention strategy, 17,000 ha of forest were lost in the Šumava NP (ÚHUL1) and 6500 ha of forest were lost in the neighbouring Bavarian NP, which is an estimated 8 million trees. Despite these events, the expansion of the non-inter- vention areas continues to be promoted, with the aim of achieving 51% wilderness cover in the NP territory. Evapotranspiration from forests plays an extensive role in the transport of moisture from oceans to continents. It is evident that an- nual precipitation is high in continents with large and continuous tracts of forest that extend from the coast to the continental interior (West Africa, Amazonia). The biotic pump theory (Makarieva and Gorshkov, 2007, Makarieva et al., 2009) suggests the atmospheric circulation that brings rainfall to continental interiors is driven and maintained by large, continuous areas of forest beginning from coasts. The theory explains that through transpiration and condensation, forests actively create low-pressure regions that draw in moist air from the oceans, thereby generating prevailing winds capable of carrying moisture and sustaining rainfall far within continents. The biotic pump concept explains why moist winds blow readily from oceans to well- forested land. This ﬂow can decline and reverse when forest cover is absent or depleted. Deforestation reduces this pressure diﬀerence, weakening or removing the coast-to- interior moisture transport. Reliable rainfall in the continental interiors of Africa, South America, and elsewhere (EuroAsia) may thus be depen- dent on maintaining relatively intact and continuous forest cover from the coast. 1. Introduction Forests aﬀect the climate positively through carbon dioxide Andréassian (2004) gives a historical evolution of ideas on the role of forests in hydrology and shows results of 137 paired watershed P. Hesslerová et al. Ecological Engineering 120 (2018) 345–354 with Corine Land Cover data. sequestration. On the other hand, a forest is dark (low albedo) and absorbs solar radiation, which contributes to planet warming. According to Bala et al. (2007), burning of boreal forest would con- tribute to planet cooling because the warming caused by the release of carbon dioxide is lower than the cooling eﬀect of increased albedo (the reﬂection of solar radiation) of burned forest. This thesis considers the forest as a passive subject of global warming driven by an increasing concentration of GHGs. According to the IPCC, the radiative forcing has increased since 1750 by 1–3 W·m−2 and resulted in higher evapo- transpiration and water losses from the landscape by evaporation. The IPCC (2013, p. 666) claims that land cover does not aﬀect the amount of water in the atmosphere; it is global warming which accelerates evaporation. Therefore, the only way to mitigate global warming and climate change is to reduce GHG production. Adaptation is based on the cultivation of species that tolerate lack of water and higher tempera- tures. with Corine Land Cover data. – For detailed evaluation of surface temperature diﬀerences between living and dead adult forest, ground thermal pictures were made in situ. – The long-term trends of air temperature in the period 1988–2017 were evaluated for the meteorological station located in the mountain area where adult spruce forest declined and compared with the data of other meteorological stations located in undamaged areas. – The long-term trends of air temperature in the period 1988–2017 were evaluated for the meteorological station located in the mountain area where adult spruce forest declined and compared with the data of other meteorological stations located in undamaged areas. – Time periods of snow cover duration in forested areas and in dead adult forest were evaluated. – Time periods of snow cover duration in forested areas and in dead adult forest were evaluated. – The data and results are discussed in terms of distribution of solar radiation (latent heat, sensible heat), evaporation and water cycle, and tree ecophysiology. Eﬀects of deforested areas on valuable peat land mountain ecosystem are considered. 1 ÚHUL – Forest Management Institute. – Assessment of long-term changes in surface temperature of the de- forested areas, thermal data of Landsat satellites were evaluated for August of 1991, 1998, 2004, 2005, 2009, and 2016 and compared 1.2. Forests attract water from oceans several thousand km deep in continents The area of interest extends on the subset of Landsat satellite scene covering 544 km2 (27.66 × 19.65 km), with the coordinates 49°05′53″ N, 13°19′34″ E and 48°55′34″ N, 13°42′34″ E. It is the transboundary area (Bavaria), however, only the Czech part was assessed (383 km2). From the Landsat data archive (https://earthexplorer.usgs.gov/), six free-clouds scenes (number 192-026) were selected from the fol- lowing years: The Šumava (Czech Republic) and Böhmerwald (Germany – Bavaria and partly Upper Austria) Mountains represent the largest forest com- plex in Central Europe on the boundary between Atlantic and con- tinental climates. The Šumava National Park was declared in 1991 on 68,060 ha (ha), of which 55,000 ha are forested. An unmanaged regime for wilderness development was adopted and up until 2017 up to 17,000 ha of adult spruce forest declined or had to be cut due to bark beetle infestation, which represented roughly 6 million dead adult trees (note: as a large calamity is considered more than million m3 of dry wood; one dead tree represents approx. 1 m3 of wood) The expansion of the unmanaged zones continues to be promoted, with the aim of achieving a wilderness cover of 51% in the NP territory. Over the last several decades, a heated discussion has taken place among the media, Parliament, local communities, and among scientists on the eﬀects of declining adult forests on hydrology and regional climate. The admin- istration of the Šumava National Park, nature conservationists, and scientists advocate the thesis that hydrological function of a declined adult forest is compensated for by the ground vegetation. Foresters, most of the local people, communal politicians, and some scientists warn against the long-term slow process of drying linked with mountain deforestation. In order to assess eﬀects of the decline of adult forest on regional climate following has been done: Satellite Landsat 5: 7. 8. 1991, 10. 8. 1998, 10. 8. 2004, 29. 8. 2005, 24. 8. 2009 Landsat 8: 27. 8. 2016 Satellite Landsat 5: 7. 8. 1991, 10. 8. 1998, 10. 8. 2004, 29. 8. 2005, 24. 8. 2009 Landsat 8: 27. 8. 2016 The scanning time was 9:40 GMT (Landsat 5) and 9:57 GMT (Landsat 8). Landsat satellites scan the electromagnetic radiation in the thermal portion of the spectrum in the channels: Landsat 5-channel B6, wavelength 10.4–12.5 μm. Landsat 8 – B10 channels (10.6–11.2 μm) and B11 (11.5–12.5 μm). The spatial resolution of thermal data (that is, the size of the area capturing one pixel) for Landsat 5 is 120 m; while Landsat 8 is 100 m. The data is suitable for analysis of larger territorial units. However, they capture vast territories of several hundred square kilometres at one point in time. The surface temperature is averaged for the smallest pixel from several surface types, in the case of very heterogeneous coverage. Using the intensity of the radiation that is recorded in the thermal channels, it is possible to calculate the surface temperature by means of – Assessment of long-term changes in surface temperature of the de- forested areas, thermal data of Landsat satellites were evaluated for August of 1991, 1998, 2004, 2005, 2009, and 2016 and compared 346 P. Hesslerová et al. Ecological Engineering 120 (2018) 345–354 Ecological Engineering 120 (2018) 345–354 algorithms. To calculate surface temperature values for Landsat 5, the so-called single-channel algorithm was used (Chander and Markham (2003); Sobrino et al. 2005; Jiménez-Muñoz et al. 2014). The Landsat 8 satellite enables more accurate temperature calculation (based on the split window algorithm) because it has two thermal channels. However, in order to ensure the same processing method, the single channel al- gorithm was also applied on Landsat 8 data. The temperature calcula- tion is based on the following calculations: The land cover masks were created based on the vector data; con- sequently, they were used to calculate the relative temperature of the land cover category. The relative temperature characteristics of land cover classes are shown in Figs. 1 and 2 for 1991 and 2016. Assessment of surface temperature change mainly focused on the evergreen forest category. 1991 was used as the reference year for temperature and forest occurrence. Images for the year of interest were compared with images from 1991 to assess temperature change in areas where coniferous forest remained and areas where it has decayed. 3.1. Surface temperature assessment based on Landsat satellite data Evergreen and mixed, and deciduous forests belong to the coldest categories of landscape cover (Fig. 1) with the typical values between −1.6 and 1.6. Conversely, ﬁelds and meadows, and transitional woodland shrubs are not able to lower the surface temperature due to water scarcity and these landscapes reach the highest temperature va- lues 0–2.5). The transitional woodland shrub area was characterized by low temperatures in 1991 (−0.4; at that time, it was represented only by grassland and shrubs). However, during the 25-year period the transitional woodland shrub area expanded into the area of decayed forest with dead trunks. This was accompanied by a temperature rise of nearly 1 °C in the transitional woodland shrub area (Fig. 2). Number of pixels (frequency) in Fig. 1 also shows the signiﬁcant decrease of forest area accompanied by the increase of transitional woodland shrubs territory. A speciﬁc case of landscape cover is peat bogs. If their surface layer (acrotelm) is adequately supplied with water and does not dry out, it is one of the coldest types of landscape cover due to the eva- poration of water (evapotranspiration) (Hesslerová et al. 2013, Huryna et al. 2014). However, if the surface layer is dry, the dry vegetation acts as a bare surface and the peat bogs appear warm on the thermal images. The algorithm (1) can also be corrected for emissivity or even at- mospheric correction. No topography was taken into account when calculating the absolute values. The study evaluates the temperature of the landscape coverage in six diﬀerent years. Although all images were taken in the same month of August, it is not objective to compare absolute surface temperature values with each other because data would be obtained under diﬀerent environmental conditions (atmospheric, seasonal, etc.). When ana- lysing time changes, we can only compare the relative temperature values obtained by standardization. The z-scores method was used to standardize the data. The method uses an approach introduced by (Brom et al., 2012). Eq. (3) was used for data standardization: = − T T T SD s i (3) = − T T T SD s i (3) where Ti is land surface temperature of pixel i, T is mean image tem- perature, and SD is standard deviation. Vector information on land cover was obtained from the CORINE Land Cover database for 1990, 2000, 2006 and 2012. 3.1. Surface temperature assessment based on Landsat satellite data This European classiﬁcation database is based on the visual interpretation of the IRS-6 and SPOT 5 satellite data. As complementary data, the LPIS database, topographic maps and aerial orthophotomaps were used. The basic map layers are available at a scale of 1:100,000. The data was used only for the territory of the Czech Republic. For the purpose of the study, the original 11 classes of land cover were grouped into 5 categories – coniferous forest, mixed and deciduous forests, ﬁelds and meadows, transitional woodland-shrub, and peat bogs. The transitional woodland- shrub is the area where the coniferous forests died after the bark beetle attack. The area of merged Corine classes is shown in Table 1. Changing land cover is accompanied by surface temperature changes (Fig. 3). When the mountain spruce ecosystem collapses, the surface temperature increases, as conﬁrmed by Hojdová et al. (2005) and Hais and Kučera (2008, 2009). In 1991, mountain spruce forest was the dominant land cover category, covering nearly 257 km2. Healthy forest stands can cool the landscape due to evapotranspiration, which is reﬂected in low surface temperatures. The relative temperature values ranged from −2.1 to 3.9, with the lowest at the southern border ridge area (around −1.8). Between 1995 and 1998 the ﬁrst extreme bark beetle attack occurred in the southern part of the study area (around the borderline Grosser Rachel – Lusen – Černá hora Mountain). The forest area decreased by 9 km2, which was accompanied by a temperature increase of 1–2 °C; however, in the damaged forest the temperature increased by nearly 4 °C. Consequently, the area of transitional wood- land shrubs increased by 8 km2, extending into the area of decayed forests, which was reﬂected by a temperature increase of less than 1 °C. When the spruce forest collapses, there is a gradual change in the vertical structure of the forest as the trees begin to die. However, the dead forest has a certain amount of longevity with which it is able to maintain the temperature-humidity conditions of the habitat. The rapid rise in temperature occurs with a certain delay, which is evident in 2004, 2005, and 2009. There was an abrupt temperature increase in the area of Grosser Rachel – Lusen – Černá hora by at least 4 °C. In January 2007, the Czech Republic was aﬀected by hurricane Kyrill that resulted in an extreme number of windfalls and windthrows. Satellite Landsat 5: 7. 8. 1991, 10. 8. 1998, 10. 8. 2004, 29. 8. 2005, 24. 8. 2009 Landsat 8: 27. 8. 2016 The results are presented mainly in graphical form, which allows for spatial visualization of temperature changes in relation to land cover. Fig. 3 shows land cover and the relative surface temperature change. For the detailed temperature change assessment, two sites aﬀected by bark beetles in diﬀerent years were selected (Figs. 4 and 5). The ground thermal images complete the temperature comparison between green and decayed mountain spruce forest (Fig. 6). = + ( ) T K ln 1 K L 2 λ 1 (1) (1) where T is land surface temperature (K), K1 and K2 are calibration constants for Landsat, Lλ is spectral radiance at the thermal channel in W/(m2.sr.µm), calculated as formula (2) ⎜ ⎟ = ⎛ ⎝ − ⎞ ⎠ + + L LMAX LMIN Q Q LMIN λ λ λ max λ (2) (2) 3. Results where LMAXλ and LMINλ are maximum and minimum spectral ra- diance in W/(m2·sr·µm) that is scaled to Qmax/min, Q is quantiﬁed as calibrated pixel value in DNs. 3.1. Surface temperature assessment based on Landsat satellite data The hurricane hit the top of the Šumava NP; in particular, the border ridges from Plesná Table 1 Changes in landcover classes area between 1990 and 2012 in the model area. Year (area in hectares) Land cover (Numbers of Corine classes) 1990 (ha) 2000 (ha) 2006 (ha) 2012 (ha) Urban areas (112) 103,8 103,8 135,5 135,5 Evergreen forest (312) 25 633,30 24 777 24 872,20 18 084,40 Mixed and deciduous forest (311, 313) 1 454,2 1 507 1 642,4 2 370 Fields and meadows (211, 231, 243, 321) 5696,4 5662,3 5302,4 5281 Transitional woodland-shrub (324) 4 517 5 360,8 5 195 11 272,40 Peatbogs (411, 412) 9 17,5 9 11,7 1 181,5 1 181,5 Table 1 Changes in landcover classes area between 1990 and 2012 in the model area. Locality 1 – Poledník mountain The area of interest (553 ha) is deﬁned by the coordinates 49°03′58″ N, 13°22′36″ E and 49°02′47″ N, 13°24′41″ E. In 2007, it was aﬀected by Hurricane Kyrill, which caused extensive windfalls. The temperature development is shown in Fig. 4. From 1991 to 2005 no change in temperature was apparent in this locality. In 2009, because of large windfalls and consequent bark beetle calamity and total decay of forest, the temperature increased. The temperature increase between 1991 (green forest) and 2016 (grassland, dead trunks) was 2.3 °C. Fig. 2. Modus values of relative surface temperature for land cover categories. in the western part of the park, Ždánidla and Poledník, then towards Modrava, in the southern part of the National Park, and the peaks in the vicinity of České Žleby and Trojmezná Mountains (out of model area). About 1 million fallen trees were removed and about 217,000 trees were left in the forests without processing. Unprocessed windfalls be- came the source of bark beetle propagation in the surrounding area. With regard to the number of destroyed trees, the greatest development of the insects in the modern history of the Šumava region occurred after 2007. The harvesting of aﬀected trees from 2008 to 2010 exceeded the historical maxima. In the forests left for spontaneous development, hundreds of thousands of adult spruces died again. In the model area the mountain spruce forest occupied 180 km2 in 2012 (in 1991 it was 256 km2) and transitional woodland shrubs area signiﬁcantly increased (from 45 km2 in 1991 to 113 km2 in 2012), which involved the areas of windfalls. This signiﬁcant land cover change was accompanied by a temperature increase of 2–4 °C. Like transitional woodland shrubs, non- forest vegetation, such as meadows and pastures, are warmer surfaces. In the event that the original spruce is replaced by grassland after its decay, it is not possible to assume a signiﬁcant decrease in the surface temperature to the level before the decay. in the western part of the park, Ždánidla and Poledník, then towards Modrava, in the southern part of the National Park, and the peaks in the vicinity of České Žleby and Trojmezná Mountains (out of model area). About 1 million fallen trees were removed and about 217,000 trees were left in the forests without processing. Unprocessed windfalls be- came the source of bark beetle propagation in the surrounding area. Locality 2 – Blatný vrch – Špičník Mountains Š The area Blatný vrch – Špičník (325 ha) is deﬁned by the co- ordinates 48°58′14″ N, 13°26′18″ E and 48°57′39″ N, 13°28′39″ E. In the mid-1990s there was extensive bark beetle calamity and decay of the forest stand. Presently, the dry torso of the trunks prevails with the Calamagrostis sp and the blueberry (Vaccinium myrtillus L.). Fig. 5 shows the relative temperature development. In 1991, the values were lowest (−1.8); with the progressive decay of forest stands the tem- perature increased until 2009 (1.2). Gradual grassing of the locality resulted in slight temperature decrease to 0.6 in 2016. Table 1 347 Ecological Engineering 120 (2018) 345–354 P. Hesslerová et al. Fig. 1. Relative surface t Fig. 2. Modus values of relative surface temperature for land cover categories. Fig. 1. Relative surface temperature of land cover. Fig. 1. Relative surface temperature of land cover. Fig. 2. Modus values of relative surface temperature for land cover categories. resulted in a temperature decrease of 0.3 °C. However, after hurricane Kyrill the temperature began to rise again. The temperature diﬀerence was around 0.7 °C by 2016. Detailed temperature assessment Detailed temperature assessment For the detailed development of the relative temperature in relation to change in land cover, two localities (see Fig. 3) – the area around the Poledník Mountain – locality 1 (1315 m a.s.l.) and the Špičník Moun- tain – locality 2 (1351 m a.s.l.) – were selected. Locality 1 – Poledník mountain With regard to the number of destroyed trees, the greatest development of the insects in the modern history of the Šumava region occurred after 2007. The harvesting of aﬀected trees from 2008 to 2010 exceeded the historical maxima. In the forests left for spontaneous development, hundreds of thousands of adult spruces died again. In the model area the mountain spruce forest occupied 180 km2 in 2012 (in 1991 it was 256 km2) and transitional woodland shrubs area signiﬁcantly increased (from 45 km2 in 1991 to 113 km2 in 2012), which involved the areas of windfalls. This signiﬁcant land cover change was accompanied by a temperature increase of 2–4 °C. Like transitional woodland shrubs, non- forest vegetation, such as meadows and pastures, are warmer surfaces. In the event that the original spruce is replaced by grassland after its decay, it is not possible to assume a signiﬁcant decrease in the surface temperature to the level before the decay. Locality 2 – Blatný vrch – Špičník Mountains Š Locality 2 – Blatný vrch – Špičník Mountains Š Locality 2 – Blatný vrch – Špičník Mountains Š 3.2. Ground thermal measurements Diﬀerences between landscape surface temperatures captured on the satellite images (Fig. 3) are likely much higher. This is due to the spatial resolution of the data. In order to provide detailed temperature measurements, ground thermal measurements are taken by a thermal camera (FLIR ThermaCamS65 HS). Images taken by the Thermovision Camera in the Třístoličník area in August 2016 captured detailed temperature distribution (Fig. 6). In the clearings, where fallen stems lie, temperatures ranged from 22 to 60 °C while the temperature of trunks was around 40 °C. The temperature in the forest was around 22 °C, with temperature inversion showing slightly higher temperatures The change in surface temperature between the reference year 1991 and others is following: The slight temperature increase (0.2 °C) was observed from 1991 to 1998. However, over the following 6 years, the temperature increased sharply and the diﬀerence reached more than 0.9 °C in comparison with the initial state. From 2004 to 2009, the dead trees were removed and replaced by transitional woodland. This 348 Ecological Engineering 120 (2018) 345–354 P. Hesslerová et al. Fig. 3. The mosaic of images shows the land cover changes in the years 1990, 2000, 2009 and 2012 (based on Corine Land Cover database); The surface temperature changes of coniferous forest are based on comparison temperature diﬀerence between reference year 1991 and following years 1998, 2004, 2005, 2009 and 2016. P. Hesslerová et al. Ecological Engineering 120 (2018) 345–354 Fig. 3. The mosaic of images shows the land cover changes in the years 1990, 2000, 2009 and 2012 (based on Corine Land Cover database); The surface temperature changes of coniferous forest are based on comparison temperature diﬀerence between reference year 1991 and following years 1998, 2004, 2005, 2009 and 2016. Fig. 3. The mosaic of images shows the land cover changes in the years 1990, 2000, 2009 and 2012 (based on Corine Land changes of coniferous forest are based on comparison temperature diﬀerence between reference year 1991 and following nd cover changes in the years 1990, 2000, 2009 and 2012 (based on Corine Land Cover database); The surface temperature comparison temperature diﬀerence between reference year 1991 and following years 1998, 2004, 2005, 2009 and 2016. Fig. 4. Relative surface temperature changes in Poledník Mountain area. in the canopy (23 °C) and one degree less in the undergrowth. Forest grasslands reached 27 °C. 4. Discussion Areas with disturbed forest experienced faster snowmelt and in- creased spring runoﬀ. These areas included the entire narrow border zone on the Bohemian side of the Šumava Mountain at the highest elevations (about 1250–1378 m a.s.l.), the mountain ranges from Jezerní hora and Svaroh in the west, to the Trojmezná ridge with the highest peak, and Plechý in the southeast. One of the few border peaks where mature forest is still present is the Smrčina region at about 1330 m a.s.l. (out of model area). From here, it is possible to compare snowmelt and snow accumulation at the highest elevations of the Šumava where adult living forests are still present with places where the forest has decayed, in this case, the area around the peak of Poledník (about 1310 m a.s.l.). Healthy forest stands (deciduous and mixed, coniferous) are sur- faces that cool the landscape due to evapotranspiration, which is re- ﬂected in low surface temperatures. The category of transitional woodland-shrub partly replaced the coniferous forest. It is character- ized by high surface temperatures and temperature increase, on average, by 4 °C. Hais and Kučera (2008) compared surface temperature of living spruce forest with decayed forest (standing dry trees) and clear-cuts in the similar model area from Landsat data. The results show an average increase of surface temperature by 3.5 °C and 5.2 °C, re- spectively. Non-forest vegetation such as meadows, pastures, and ﬁelds also belong to the warmest areas. Therefore, grassland vegetation that ap- pears in deforested localities as a compensation for the forest, cannot signiﬁcantly decrease the surface temperature. A speciﬁc case is peat bogs. If acrotelm has an adequate water supply, it is one of the coldest types of land cover due to evapotranspiration (Huryna et al., 2014). If the surface layer is dry, dry vegetation acts as a bare surface and peat bogs appear warm on the thermal images (Hesslerová et al., 2013). When the spruces break down, the surface temperature increases. This fact is evident in the entire border area (Fig. 3). The diﬀerence between forested and deforested localities can reach 10 °C (ground measure- ments). Forest disturbance aﬀects not only surface and air temperatures, but also inﬂuences timing and rate of snowmelt and runoﬀof water from the river basin. 3.3. Long term trends in air temperature he Thermovision Camera in the Třístoličník area in August 2016. Surface temperature diﬀerences in forest reach 4 °C, while in ly 35 °C. Fig. 6. Examples of images taken by the Thermovision Camera in the Třístoličník area in August 2016. Surface temperature diﬀerences in forest reach 4 °C, while in clearings with dead trunks reach nearly 35 °C. Fig. 6. Examples of images taken by the Thermovision Camera in the Třístoličník area in August 2016. Surface temperatu clearings with dead trunks reach nearly 35 °C. 3.3. Long term trends in air temperature In addition to surface temperatures, the long-term trends of air temperature from 1988 to 2017 were evaluated at three meteorological stations. Březník is located in the heart of mountain spruce forests da- maged by the disturbance on the tree ﬂoor. In the shallow valley, an amateur meteorological station has been in operation since 1987, which supplies data to the climatological network of the Czech Hydrometeorological Institute (CHMI). Therefore, it is possible to monitor the air temperature before the bark beetle calamity occurred, during the calamity, and at the current stage of gradual forest re- generation. To better document the described changes, a comparison was made between the meteorological stations. The second station was Churáňov (1118 m a.s.l., station of the CHMI), which is at a similar elevation, at the top of the hill 15 km NE from Březník outside the core area aﬀected by bark beetle. The third station was the amateur station Horská Kvilda (1055 m a.s.l.) lying similarly to Březník in shallow de- pression, but between wooded ﬂat ridges 11 km NNE from Březník. Fig. 4. Relative surface temperature changes in Poledník Mountain area. Fig. 5. Relative surface temperature changes in Blatný vrch – Špičník Mountains area. At these three stations, the average annual air temperatures were compared between 1988 and 2017. Compared to the Churáňov and Horská Kvilda stations, there was a signiﬁcant increase in the average annual air temperature at the Březník station in the last few years be- ginning in 2007–2008 (Fig. 7). If the diﬀerences between stations Churáňov – Horská Kvilda and Churáňov – Březník are compared, a similar trend will appear. The diﬀerence in average annual tempera- tures between Churáňov and Horská Kvilda remains roughly the same, between 1.5 and 1.8 °C, while the average diﬀerence between Churáňov and Březník changed. In the ﬁrst two decades of observation, this dif- ference was between 3 and 4 °C, but in the last decade there was a gradual decrease (Fig. 7). In the last ﬁve years, the average diﬀerence was 2 °C. Fig. 5. Relative surface temperature changes in Blatný vrch – Špičník Mountains area. 349 Ecological Engineering 120 (2018) 345–354 P. Hesslerová et al. Fig. 6. Examples of images taken by the Thermovision Camera in the Třístoličník area in August 2016. Surface temperature diﬀerences in forest reach 4 °C, while in clearings with dead trunks reach nearly 35 °C. 4. Discussion The climatic network of the CHMI is very sparse in the Šumava region, but in recent years, it has been possible to carry out regular monitoring of the height of the snow cover and air temperature in many places in the Šumava Mountains (Procházka et al., 2017). This monitoring, thanks to new technologies, also includes the highest ele- vations of the Šumava. Results from the Poledník and Smrčina summits during the last four winter periods show clear diﬀerences in spring snow ablation on bare surfaces after forest decay compared to forested areas that are positively aﬀected by the tree ﬂoor microclimate (Fig. 8). The accumulation of snow in early winter is always very similar. During the winter, due to the inﬂuence of known factors of the thaws (tem- perature, rain, wind) and solar radiation, the diﬀerences begin to show and peak in the spring season when snowmelt begins. The duration of permanent snow cover on forested peaks over the last four years was, on average, 11 days longer than on the peaks without trees. Landsat satellite acquires the images repeatedly approximately at 9:50 GTM (10:50 Central European Time) and therefore cannot capture afternoon maximums, yet the high temperature of the woodland- shrubs, as well as grasslands, is obvious. Diﬀerences in land cover temperatures as captured on the satellite images may actually be much higher. This is due to the spatial resolution of the data. For example, temperature information is the average pixel value for an area of 100 × 100 m in 2016, for others it is 120 × 120 m. If a given part of the territory is heterogeneous in terms of land cover, thermal information is 350 Ecological Engineering 120 (2018) 345–354 P. Hesslerová et al. Fig. 7. Average annual temperature at the meteorological stations of Březník (1137 m a.s.l.), Churáňov (1 118 m a.s.l.) and Horská Kvilda (1055 m a.s.l.). Air temperature diﬀerences (in °C) between Churáňov station x Březník and Churáňov × Horská Kvilda between 1988 and 2017 (Data source: CHMI and A. Vojvodík – operator of amateur meteorological stations). Fig. 7. Average annual temperature at the meteorological stations of Březník (1137 m a.s.l.), Churáňov (1 118 m a.s.l.) and Horská Kvilda (1055 m a.s.l.). Air temperature diﬀerences (in °C) between Churáňov station x Březník and Churáňov × Horská Kvilda between 1988 and 2017 (Data source: CHMI and A. Vojvodík – operator of amateur meteorological stations). 4. Discussion Reduced evapotranspiration from the dead stand is compensated for by increased evaporation from the surface enhanced by climate change. It is unlikely that the authors realized deforestation caused changes in evapotranspiration, resulting in a change in the dissipation of solar energy. Heat ﬂows are directed to sensible heat instead of the latent heat of the vapour, resulting in rising surface temperatures and further loss of water. The diﬀerence in latent and sensible heat dis- tribution of forest stands and in drained areas, on bright, hot days, is a matter of hundreds of W·m−2. The decline in evapotranspiration of 1 km2 due to drainage or degradation of the forest for an equivalent to 100 mg·m−2·s−1 water vapour represents 250 megawatts of solar en- ergy released from the area 1 km−2 into the atmosphere in the form of warm air (sensible heat). However, the real value of the sensible heat may be higher. From a thousand hectares (10 km2) of dry forest, 2000 megawatts of energy are released into the atmosphere in the form of sensible heat on sunny days, resulting in changes in airﬂow and gradual drying. From 1990 to 2012 7500 ha of evergreen forest was lost (approx. 30%) in the model area. A minimum of 15,000 megawatts of energy is released in the form of sensible heat in hot summer days from this area. numerous calamities since 1970 in various parts of Central and Eastern Europe, and have been culminating in recent years, which is linked to worse forest management (due to lack of people), water scarcity, drought and climate change. Similarly, bark beetle calamities occurred in Sweden, Norway, France, the Balkan peninsula, Ukraine, the Baltic republics. Extensive and less mentioned are calamities in the spruce forests of Russia from 55° to 63° N, in Ural region and in the western and eastern parts of Siberia. The individual calamities represented up to two tens of millions of m3 of dry wood. Many original studies of spruce calamities have not been written in the world languages, and the papers in which they were published were, and are, hard to reach. Skuhravý (2002) quotes 400 citations from Europe, including less accessible works in the original language of the former Soviet Union, Poland and the Balkan countries. Many authors interest mainly in the relationship of climate and climate change on bark beetle outbreaks (Fauria and Johnson 2009; Bentz et al. 4. Discussion 2010; Berg et al. 2006). There is not much attention paid to direct eﬀect of forest decay caused by bark beetle calamity on local climate in terms of active role of healthy forest in local precipitation and water cycle as described by Sheil and Murdiyarso (2009), Sheil (2018). Sagar and Waterhouse (2015) studied the changes in micro- climate (air and soil temperature, frost events and snow-free days) in the Chilcotian Plateau in British Columbia in relation to partial cuts and clear-cuts, as well as with outbreak of pine beetle (Dendroctonus pon- derosae). The eﬀect of forest structure changes on microclimate due to human activity and natural disturbances (such as insect’s outbreaks) reviewed by Chen et al. (1999). Classen et al. (2005) investigated the inﬂuence of herbivory insects on changes in vegetation (canopy change) and consequently on microclimate (soil moisture and tem- perature) and nutrient cycling in Northern Arizona. Deforestation caused by bark beetle outbreaks in Rocky Mountains (North America) was also responsible for water budget changes which caused decrease of transpiration with subsequent increase in groundwater contributions to streams (Bearup et al. 2014). The similar results provide Redding et al. (2008): “The current mountain pine beetle infestation and associated salvage harvesting have the potential to aﬀect the amount, timing, and quality of water originating from British Columbia’s forested water- sheds”. The authors elaborated the general recommendations for avoiding hydrological risks in management and planning for both the forested watersheds and the valley-bottom infrastructure. It should be pointed out that air heated by warm surfaces that as- cends into atmosphere contains water vapour. Landscapes lose water with this rising warm air driven by sensible heat. The amount of water in the air transported by sensible heat into atmosphere can be sub- stantially higher than that released by evapotranspiration. For example, air at 100% relative humidity at a temperature of 40 °C contains 50 g of water vapour in 1 m3 whereas air at 20% relative humidity contains 10 g of water vapour in 1 m3. Air driven by sensible heat from 1 m2 at a speed of 0.1 m·s−1 would transport 3.6 kg water into the atmosphere over the course of an hour, i.e. over 30 L per day. The eﬀect is known as advection of energy (warming of relative dry air) from overheated dry surfaces. These are water losses that are not measured as ﬂow rate in rivers. 4. Discussion In the summer, “air rivers” can drain invisibly more water than water courses. Discharge in rivers decreases and water gets high in the atmosphere. y The impact of bark beetle calamity on the hydrological balance of the Šumava national park territory is a widely discussed topic. There is the assumption that deforestation and decay of the tree ﬂoor will result in decreased evapotranspiration, which compensates for the loss of water caused by an eventual increase in runoﬀ. In addition, dead trees have a smaller surface area (leaf area index) than live trees, so more rainfall will reach the upper soil layer, which supports groundwater supply. Beudert et al. (2007) studied hydrological balance in connec- tion with bark beetle calamity in the Bavarian Forest. The forest decay caused signiﬁcant changes in the soil water balance and runoﬀ. Fast forest dieback, where 80% of trees died, resulted in a 39% decrease in evapotranspiration (ET) and a direct runoﬀincrease of 162%. Ad- ditionally, fast and slow groundwater runoﬀreached 125%, and 132%, respectively, when compared to reference data. After alternative ve- getation development, there was a signiﬁcant increase of ET, up to 78% and direct runoﬀreached 70%. Despite the fact that both groundwater levels reached the normal level, the fast component decreased to 74%, whereas the slow increased to 136%, which conﬁrms signiﬁcant eﬀects of deforestation on groundwater level. Bečka and Beudert (2016) dis- cuss several examples of hydrological response to disturbance (bark beetle calamity) and climate change. In the Forellenbach catchment area in the Roklan-Lusen area, annual transpiration from 1996 to 1997 decreased from approximately 700 l·m−2 to 450 l·m−2 because of the decline in mountain spruce forest. Absence of evapotranspiration leads to an increase in the amount of inﬁltrating water and faster recovery of groundwater supplies and a consequent increase in runoﬀ. Another Common values of ET are several mm (several litres per m2 per day). Very high values of ET are about 10 mm. From this point of view, ET can be considered as a process that slows down evaporation water losses from the landscape on regional level. ET binds surplus solar en- ergy into latent heat of water vapour and reduces release of sensible heat; water vapour is not driven up and stays close to the canopy. Tesař et al. 4. Discussion The current outbreak of bark beetle in the part of the southwest territory of the Czech Republic with overlapping to Austria and Bavaria was preceded by three calamities the largest in 1868–1878 and then the end of the First and the Second World Wars. Afterwards, the bark beetle spread in Germany and throughout the central Europe. There have been also heterogeneous; i.e. it is the average temperature of diﬀerent types of surfaces. The pixel heterogeneity and topography eﬀects on surface temperature are discussed in Hais and Kučera (2009). Despite this fact, changes in temperatures due to extensive changes in land cover are clearly visible on Landsat satellite imagery. Fig. 8. Snow cover height (in cm) in winter seasons 2014/2015 to 2017/2018 at the peaks of Poledník (1 310 m a.s.l.) and Smrčina (1330 m a.s.l.). Average daily temperature at the meteorological station Plechý (1344 m a.s.l.). (Data source: CHMI and I. Rolčík, DiS. – operator of Plechý meteorological station). Fig. 8. Snow cover height (in cm) in winter seasons 2014/2015 to 2017/2018 at the peaks of Poledník (1 310 m a.s.l.) and Smrčina (1330 m a.s.l.). Average daily temperature at the meteorological station Plechý (1344 m a.s.l.). (Data source: CHMI and I. Rolčík, DiS. – operator of Plechý meteorological station). 351 Ecological Engineering 120 (2018) 345–354 P. Hesslerová et al. study conducted from 1992 to 2013 examined changes in the hydro- logical balance of two streams lying in a non-intervention zone in the Roklan-Lusen area. After approximately 30% of spruce forests died in 1999, annual runoﬀincreased in both catchments by 146 l·m−2 (Fore- llenbach) and 127 l·m−2 (Große Ohe). The amount of precipitation remained almost the same and evapotranspiration decreased by 120 l·m−2 and 90 l·m−2, respectively; at the same time, the ground- water reserves increased. The third example presented by the authors compared the water conditions of large watercourses around the Ba- varian Forest National Park between 1978 and 2013. In areas with less than 36% of the forest vegetation, there was a decline in annual runoﬀ, but the total rainfall remained unchanged. Unchanged annual runoﬀis also typical for river basins where more than 50% of the trees have died. The authors associate this change with increased evapo- transpiration, which is caused by warming from May to August of ∼2 °C (measurements at 4 meteorological stations), even in April by 4 °C. 4. Discussion (2017). The sur- veys coincide with faster melting of the snow cover and increased spring runoﬀfrom model river basins and areas aﬀected by deadwood trees. therefore cannot describe real eﬀect of climate change. Even equal/ balanced temperature is the end of motion. For example, air tempera- ture in the forest is 18 °C in the morning and 22 °C in the afternoon. When we cut the forest down, the temperature is 15 °C in the morning and 25 °C in the afternoon. The climate has changed, however, the average temperature (20 °C) is still the same. Climate change caused by deforestation and, drainage is more signiﬁcant than it can be seen from an increase in average global temperature. References Andréassian, V., 2004. Waters and forests: from historical controversy to scientiﬁc debate. J. Hydrol. 291, 1–27. Bala, G., Caldeira, K., Wickett, M., Phillips, T.J., Lobell, D.B., Delire, C., Mirin, A., 2007. Combined climate and carbon-cycle eﬀects of large-scale deforestation. PNAS 104 (16), 6550-–6555. Bässler, C., 2008. Klimawandel—Trend der Lufttemperatur im Inneren Bayerischen Wald (Böhmerwald). Silva Gabreta 14, 1–18. Bearup, L.A., Maxwell, R.M., Clow, D.W., McCray, J.E., 2014. Hydrological eﬀects of forest transpiration loss in bark beetle-impacted watersheds. Nat. Clim. Change 4, 481–486. 5. Conclusions In: Puhlmann, H., Schwarze, R. (Eds.), IHP/HWRP Secretariat, Koblenz. IHP/HWRP‐ Report 6, 41–62. Schwarze, R. (Eds.), IHP/HWRP Secretariat, Koblenz. IHP/HWRP‐ Report 6, 41–62. Brom J Nedbal V Procházka J Pecharová E 2012 Changes in vegetation cover Brom, J., Nedbal, V., Procházka, J., Pecharová, E., 2012. Changes in vegetation cover, moisture properties and surface temperature of a brown coal dump from 1984 to 2009 using satellite data analysis. Ecol. Eng. 43, 45–52. Buchtele, J., Buchtelová, M., Tesař, M., 2006. Role of vegetation in the variability of water regimes in the Šumava Mts forest. Biologia 61 (19), S246–S250. h d kh i d d di i lib i Chander, G., Markham, B., 2003. Revised Landsat-5 TM radiometric calibration proce- dures and postcalibration dynamic ranges. IEEE Trans. Geosci. Remote 41 (11), 2674–2677. Climate extremes are not moderated by water and vegetation. Water and vegetation form the climate, equalizing diﬀerences in temperatures and pressures that cause torrential rains and storms. Man has deforested and drained large areas. Warm air rises from dry areas, dissolving clouds, and the landscape becomes even more heated. Deforestation releases sensible heat (heated air from the overheated surface of the landscape) in the range of hundreds of watts per m2. This has been known since the mid-20th century and can be easily measured. Increased concentrations of greenhouse gases caused an increase of radiation forcing by 1–3 W·m−2, which cannot be measured. Life is essentially immanent to balance diﬀerences in energy. In order to compensate for diﬀerences in pressure and temperature, life is main- tained and developed from these diﬀerences. Average temperature may not always be an opposite variable for assessing climate change, as it not reﬂects daily and seasonal temperature dynamics and extremes and Chen, J., Saunders, S.C., Crow, T.R., Naiman, R.J., Brosofske, K.D., Mroz, G.D., Brookshire, B.L., Franklin, J.F., 1999. Microclimate in forest ecosystem and landscape ecology: variations in local climate can be used to monitor and compare the eﬀects of diﬀerent management regimes. BioScience 49 (4), 288–297. Classen, A.T., Hart, S.C., Whitman, T.G., Cobb, N.S., Koch, G.W., 2005. Insect infestations linked to shifts in microclimate: important climate change implications. Soil Sci. Soc. Am. J. 69, 2049–2057. Geiger, R., 1957. The Climate Near the Ground, second ed. Harvard University Press, Cambridge, MA. Geiger, R., Aron, R.H., Todhunter, P., 2003. The Climate Near the Ground, sixth ed. Rowman and Littleﬁeld Publishers, Lanham, MD, USA. 4. Discussion (2006) assessed temperature regimes of three mountain forest basins in the Šumava at various stages of development (dead spruce forest with herbaceous undergrowth, clearings covered by herbaceous vegetation, and mature spruce forest). Results showed that both soil and air temperature are inﬂuenced by plant cover and the extremes in day and night-time air temperatures are a function of transpiring ve- getation height in hot and dry days, with higher daily maximums and lower night-time minimums for areas with smaller vegetation. For the period of the entire growing season, mean air temperature was 352 Ecological Engineering 120 (2018) 345–354 P. Hesslerová et al. independent of plant cover, but the magnitude of the dispersion var- iance followed the sequence in ascending order: mature forest – clearing – dead forest. Consequently, Bässler (2008), Bernsteinová et al. (2015), and Langhammer et al. (2015), observed increasing air tem- peratures in the Šumava for 80 years, most notably in the spring (+ ∼4 K in April). As a result of climatic changes and forest decay, the Vydra basin showed changes in the seasonality and variability of runoﬀ; a signiﬁcant amount of runoﬀthat historically occurred during the summer is now occurring in the spring period, when the melting of snow cover is accelerated by rising air temperatures. A number of studies have shown the positive inﬂuence of mature forest on slowing snowmelt and outﬂows from the catchment area (Troendle and Reuss, 1997; Kantor and Shach, 2002; Pomeroy et al., 2012). Even in extreme situations, as shown by Marks et al. (1998) with the Oregon ﬂood (USA, February 1996), the forest areas in the Cascade Mountains retained a signiﬁcant amount of rain in the snow compared to deforested ones. Hríbik et al. (2012) studied the inﬂuence of spruce (Picea abies (L.) Karst.) on the hydrophysical properties of the snow cover and results indicated that coniferous stands play an important role both in snow- melt and runoﬀformation. The results of a study conducted in Central Slovakia (in Polana Biosphere Reserve) showed a signiﬁcant eﬀect of spruce forests in slowing down snowmelt and spring runoﬀfrom the entire catchment area of the Hucava stream watershed. In the Šumava, the inﬂuence of spruce forests and the forest aﬀected by bark beetle (Ips typographus L.) on snow cover, melting speed, and the formation of runoﬀare discussed by Buchtele et al. (2006), Bernsteinová et al. (2015), Langhammer et al. (2015), and Jeníček et al. 5. Conclusions Bečka, P., Beudert, B., 2016. Kůrovec a voda. Jak bezzásahovost ovlivňuje vodní režim na Šumavě. (Barkbeetle and water. How non-intervention regime inﬂuences hydro- logical regime in Šumava). Šumava. Jaro 2016, 16–17 In Czech. The analyses of thermal satellite images show an increase in surface temperature in the area where the forest canopy layer died, due to bark beetle outbreaks, in the order of 2–4 °C. The meteorological station located in the center of the dieback forest area recorded a twice as fast increase in the average air temperature than the stations in the green forest areas. Similarly, it has been documented accelerated melting of the snow cover (by 11 days). Bentz, B.J., Régnière, J., Fettig, C.J., Hansen, E.M., Hayes, J.L., Hicke, J.A., Kelsey, R.G., Negrón, J.F., Seybold, S.J., 2010. Climate change and bark beetles of the Western United States and Canada: direct and indirect eﬀects. BioScience 60 (8), 602–613. Berg, E.E., Henry, J.D., Fastie, C.L., De Volder, A.D., Matsuoka, S.M., 2006. Spruce beetle outbreaks on the Kenai Peninsula, Alaska, and Kluane National Park and Reserve, Yukon Territory: Relationship to summer temperatures and regional diﬀerences in disturbance regimes. Forest Ecol. Manage. 227, 219–232. High surface temperature in the decayed forest indicates production of sensible heat instead of evapotranspiration, which cools adult live forests. Sensible heat drives the turbulent movement of air into the atmosphere. The ascending warm air transports water vapour into the atmosphere and dries the surrounding landscape by the so-called ad- vection eﬀect. Production of sensible heat reaches several hundred Watts per m2 on sunny days during the growing season. The area of c. 7500 ha, which lost high adult forest, and which shows an increase of surface temperature, produces at least 17,000 megawatts of sensible heat. This is transformed solar energy, which dries the landscape and aﬀects movement of air on at least the regional scale. Bernsteinová, J., Bässler, C., Zimmermann, L., Langhammer, J., Beudert, B., 2015. Changes in runoﬀin two neighbouring catchments in the Bohemian Forest related to climate and land cover changes. J. Hydrol. Hydromech. 63 (4), 342–352. g y y Beudert, B., Klöcking, B., Schwarze, R. 2007. Große Ohe: impact of bark beetle infestation on the water 24 and matter budget of a forested catchment. In: Puhlmann, H., Beudert, B., Klöcking, B., Schwarze, R. 2007. Große Ohe: impact of bark beetle infestation on the water 24 and matter budget of a forested catchment. Acknowledgements The paper was supported by the projects: Ministry of Education Youth and Sports of the Czech Republic – Institutional support for the long-term strategic development of research organization and Smart Regions – Buildings and Settlements Information Modelling, Technology and Infrastructure for Sustainable Development TE02000077 and HORIZON 2020 Grant Agreement Number: 689150 SIM4Nexus project supported by European Commission. Special ac- knowledgement to Mr. Antonín Vojvodík, owner of the amateur me- teorological stations Horská Kvilda and Březník and Mr. Ivo Rolčík, DiS., owner of the meteorological station Plechý, for providing air temperature data. 6, 302–307. of radiation surface temperature of diﬀerent landcover types in a temperate cultural landscape: consequences for the local climate. Ecol. Eng. 54, 145–154. Marks, D., Kimball, J., Tingey, D., Link, T., 1998. The sensitivity of snowmelt processes to climate conditions and forest cover during rain-on-snow: a case study of the 1996 Paciﬁc Northwest ﬂood. Hydrol. Process. 12, 1569–1587. Hojdová, M., Hais, M., Pokorný, J., 2005. In: Microclimate of Peat Bog and of the Forest in Diﬀerent States of Damage in the Šumava National Park. Silva Gabreta, Vimperk, pp. 13–24. Marsh, G.P., 1864. Man and Nature or, Physical Geography as Modiﬁed by Human Action. Scribner, Oxford University, New York. Hríbik, M., Vida, T., Škvarenina, J., Škvareninová, J., Ivan, L., 2012. Hydrological eﬀects of Norway spruce and European beech on snow cover in a mid-mountain region of the Polana Mts., Slovakia. J. Hydrol. Hydromech. 60, 319–332. Pomeroy, J., Fang, X., Ellis, C., 2012. Sensitivity of snowmelt hydrology in Marmot Creek, Alberta, to forest cover disturbance. Hydrol. Process. 26, 1891–1904. Huryna, H., Brom, J., Pokorný, J., 2014. The importance of wetlands in the energy bal- ance of an agricultural landscape. Wetlands Ecol. Manage. 22 (4), 363–381. Procházka, J., Rolčík, I., Vojvodík, A., Matoušek, M., 2017. Activities of amateur en- thusiasts for extending knowledge about the climate of the Šumava Mountains. Meteorol. Zprávy (Meteorol. Bull.) 70 (5), 143–148 in Czech. Ellison, D., Morris, C.E., Locatelli, B., Sheil, D., Cohen, J., Murdiyarso, D., Gutierrez, V., van Noordwijk, M., Creed, I.F., Pokorny, J., Gaveau, D., Spracklen, D.V., Tobella, A.B., Ilstedt, U., Teuling, A.J., Gebrehiwot, S.G., Sands, D.C., Muys, B., Verbist, B., Springgay, E., Sugandi, Y., Sullivan, S.A., 2017. Trees, forests and water: cool insights for a hot world. Global Environ. Change 43, 51–61. Redding, T., Winkler, R., Teti, P., Spittlehouse, D., Boon, S., Rex, J., Dubé, S., Moore, R.D., Wei, A., Carver, M., Schnorbus, M., Reese-Hansen, L., Chatwin, S., 2008. Mountain pine beetle and watershed hydrology. In Mountain Pine Beetle: from Lessons Learned to Community-based Solutions Conference Proceedings, June 10–11, BC. J. Ecosyst. Manage. 9 (3), 33–50. A.B., Ilstedt, U., Teuling, A.J., Gebrehiwot, S.G., Sands, D.C., Muys, B., Verbist, B., Springgay, E., Sugandi, Y., Sullivan, S.A., 2017. Trees, forests and water: cool insights for a hot world. Global Environ. Change 43, 51–61. g Fauria, M.M., Johnson, E.A., 2009. Large-scale climatic patterns and area aﬀected by mountain pine beetle in British Columbia, Canada. J. Geophys. Res. 114, G01012. 6, 302–307. IPCC, 2013. Climate Change 2013: The Physical science basis. In: Stocker, T.F., Qin, D., Sagar, R.M., Waterhouse, M.J. 2015. Microclimate Studies in Mountain Pine Beetle–Damaged Silvicultural Systems on the Chilcotin Plateau: The Itcha-Ilgachuz Project (1997–2013). Prov. B.C., Victoria, B.C. Tech. Rep. 089. Available from: http://www.for.gov.bc.ca/hfd/pubs/Docs/Tr/Tr089.htm. IPCC, 2013. Climate Change 2013: The Physical science basis. In: Stocker, T.F., Qin, D., Plattner, G.-K., Tignor, M., Allen, S.K., Boschung, J., Nauels, A., Xia, Y., Bex, V., Midgley, P.M. (Eds.), Contribution of working group I to the ﬁfth assessment report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA, pp. 1535. the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA, pp. 1535. Sheil, D.F., 2018. Forests, atmospheric water and an uncertain future: the new biology of the global water cycle. For. Ecosyst. 5, 19. http://dx.doi.org/10.1186/s40663-018- 0138-y. Jeníček, M., Hotový, O., Matějka, O., 2017. Snow accumulation and ablation in diﬀerent canopy structures at a plot scale: using degree-day approach and measured shortwave radiation. AUC Geogr. 52 (1), 61–72. Skuhravý, V., 2002. Lýkožrout smrkový (Ips typographus L.) a jeho kalamity. (Bark beetle (Ips typographus L.) and its outbreaks), Agrostroj, s.r.o. Praha. Jiménez-Muñoz, J.C., Sobrino, J.A., Skoković, D., Mattar, C., Cristóbal, J., 2014. Land surface temperature retrieval methods from Landsat-8 thermal infrared sensor data. IEEE Geosci. Remote Sens. Lett. 11 (10), 1840–1843. Š Sheil, D., Murdiyarso, D., 2009. How forest attract rain: an examination of a new hy- pothesis. Bioscience 59 (4), 341–347. Sobrino, J.A., Jiménez-Muñoza, J.C., Paolini, L., 2005. Land surface temperature retrieval from LANDSAT TM 5. Remote Sens. Environ. 90, 434–440. Š Kantor, P., Šach, F., 2002. Snow accumulation and melt in a spruce stand and on a clearcut in the Orlicke hory Mts. (Czech Republic). Ekologia–Bratislava 21 (1), 122–135. Tesař, M., Šír, M., Lichner, L., Zelenková, E., 2006. Inﬂuence of vegetation cover on thermal regime of mountainous catchments. Biologia (Bratisl) 61, 311–314. thermal regime of mountainous catchments. Biologia (Bratisl) 61, 311–314. Troendle, C.A., Reuss, J.O., 1997. Eﬀect of clearcutting on snow accumulation and water outﬂow at Fraser, Colorado. Hydrol. Earth Syst. Sci. 1, 325–332. Ú Langhammer, J., Su, Y., Bernsteinová, J., 2015. Runoﬀresponse to climate warming and forest disturbance in a mid-mountain Basin. Water 7, 3320–3342. Makarieva, A.M., Gorshkov, V.G., 2007. Biotic pump of atmospheric moisture as driver of the hydrological cycle on land. Hydrol. Earth Syst. 5. Conclusions Geiger, R., Aron, R.H., Todhunter, P., 2009. The Climate Near the Ground, seventh ed. Rowman and Littleﬁeld Publishers, Lanham, MD, USA. Hais, M., Kučera, T., 2008. Surface temperature change of spruce forest as a result of bark beetle attack: remote sensing and GIS approach. Eur. J. For. Res. 127, 327–336. Hais M Kučera T 2009 The inﬂuence of topography on the forest surface temperature Hais, M., Kučera, T., 2009. The inﬂuence of topography on the forest surface temperature retrieved from Landsat TM, ETM+ and ASTER thermal channels. ISPRS J. Photogram 64, 585–591. Hesslerová, P., Pokorný, J., Brom, J., Rejšková – Procházková, A., 2013. Daily dynamics 353 P. Hesslerová et al. Ecological Engineering 120 (2018) 345–354 Ecological Engineering 120 (2018) 345–354 6, 302–307. Sci. 11 (2), 1013–1033. Úlehla, V., 1947. Napojme prameny o utrpení našich lesů: sedm rozhlasových přednášek. Život a práce, Praha in Czech. WeForest Managing forests for water and for climate cooling, 2015. http://www bioticregulation.ru/oﬀprint/weforest.pdf (accessed 31 March 2018). Makarieva, A.M., Gorshkov, V.G., Li, B.-L., 2009. Precipitation on land versus distance from the ocean: evidence for a forest pump of atmospheric moisture. Ecol. Complex. 354
https://openalex.org/W2461498912	https://www.nature.com/articles/srep28908.pdf	English	null	(-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model	Scientific reports	2,016	cc-by	9,265	(-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model Xiaoyin Zhu1,2, Jingwei Tian2, Songmei Sun2, Qiuju Dong2, Fangxi Zhang2 & Xiumei Zhang1 received: 02 July 2015 accepted: 13 June 2016 Published: 28 June 2016 Chronic cerebral hypoperfusion (CCH) is one of the most common causes of vascular dementia (VaD) and is recognised as an etiological factor in the development of Alzheimer’s disease (AD). CCH can induce severe cognitive deficits, as assessed by the water maze task, along with neuronal loss in the hippocampus. However, there are currently no effective, approved pharmacological treatments available for VaD. In the present study, we created a rat model of CCH using bilateral common carotid artery occlusion and found that (-)-SCR1693, a novel compound, prevented rats from developing memory deficits and neuronal damage in the hippocampus by rectifying cholinergic dysfunction and decreasing the accumulation of the phospho-tau protein. These results strongly suggest that (-)- SCR1693 has therapeutic potential for the treatment of CCH-induced VaD. Vascular dementia (VaD) is the most common cause of dementia after Alzheimer’s disease (AD)1. VaD is defined as a loss of cognitive function resulting from ischaemic, ischaemic-hypoxic or haemorrhagic brain tissue lesions due to cardiovascular disease and cardiovascular pathological changes2. Chronic cerebral hypoperfusion (CCH) is a major cause of VaD and can result from disorders that affect the cerebral vascular system, including hyperten- sion, diabetes, generalised atherosclerosis, and cigarette smoking3. A study focusing on the pathogenetic mech- anism of VaD has revealed that, similar to AD, cholinergic abnormalities are associated with a disturbance in cognitive function in patients with VaD4. Cholinergic neurons that project into the hippocampus play a critical role in learning and memory function, and the cholinergic terminals in the presynaptic membrane are sensitive to ischaemic insults5. These findings suggest the possibility of using cholinergic substances as therapeutic interven- tions in patients with VaD. The inhibition of brain acetylcholinesterase (AChE) can increase synaptic concentra- tions of acetylcholine, which may improve cognitive dysfunction and neuropathology in patients suffering from cerebral ischaemic dementia6. However, many clinical studies have revealed that memantine and the AChE inhib- itors donepezil, galantamine and rivastigmine only have modest beneficial effects on the cognitive symptoms of VaD and provide no concomitant global or clinical benefits in most cases7. The use of AChE inhibitors is limited because of adverse drug reactions, which include increased patient mortality7. This limitation indicates that the inhibition of cholinergic abnormalities may not completely prevent the development of VaD. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results ( ) SCR1 (-)-SCR1693 prevents CCH-induced learning and memory deficits. Clinical observations and experimental research have indicated that CCH could lead to learning/memory deficits in subjects who are mid- dle-aged and older18. As shown in Fig. 1A, the ischaemic rats in the model group exhibited significantly longer escape latencies than the rats in the sham group from day 1 to day 5 (day 1, p = 0.012; day 2, p = 0.011; day 3, p = 0.019; day 4, p = 0.024; day 5, p = 0.016; n = 6) in the water maze task, which was initiated 4 weeks after BCCAo. On days 2–5, there were no significant differences in escape latencies between the rats in the donepezil and (-)-SCR1693 groups vs. the sham group, except in the 0.3 mg/kg (-)-SCR1693 group on day 3 (p = 0.036). We found that on days 3–5, 3 mg/kg of (-)-SCR1693 significantly shortened the escape latencies prolonged by permanent BCCAo (day 3, p = 0.036; day 4, p = 0.006; day 5, p = 0.026) compared with the model group. On day 4, the escape latencies of the rats in the group that received 1 mg/kg of (-)-SCR1693 were significantly shorter compared with the model group (p = 0.005). Although the mean escape latency of the rats that received 1 mg/ kg of donepezil was lower on days 4 and 5, no statistically significant difference was observed compared with the model group during the trial. g p g In the probe trial, Fig. 1B shows that the number of times the rats crossed the platform area for the experimen- tal and sham groups was 4.83 ± 1.07 and 2.67 ± 1.37 (p = 0.015, n = 6), respectively. Administering 1 mg/kg and 3 mg/kg of (-)-SCR1693 significantly increased the number of times the rats crossed compared with the model group (5.33 ± 1.25 and 5 ± 0.82, p = 0.038 and p = 0.028, respectively), which was similar to the sham group. The number of times the rats crossed the platform area in the group that received 1 mg/kg of donepezil was not different from that in the model group. In Fig. 1C, representative swim paths during the probe test are shown, and they reveal shorter swimming distances and less time spent in the target quadrant for the ischaemic rats, while (-)-SCR1693 ameliorated these changes. (-)-SCR1693 alleviates neuronal damage caused by CCH. (-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model Xiaoyin Zhu1,2, Jingwei Tian2, Songmei Sun2, Qiuju Dong2, Fangxi Zhang2 & Xiumei Zhang1 g y y The bilateral common carotid artery occlusion (BCCAo) rat model is a commonly used model of VaD. Surgical ligation of both the common carotid arteries in rats produces a CCH condition8. Previous studies have revealed that CCH induces severe cognitive deficits, as assessed by the water maze task, along with neuronal loss in the hippocampus9. In addition to cholinergic abnormalities, CCH treatment induced learning/memory alterations, increased microtubule-associated protein tau hyperphosphorylation, and caused imbalances in the phosphorylation system by activating glycogen synthase kinase 3β (GSK-3β) and Akt10. In humans, tau plays a key role in regulating microtubule dynamics, axonal transport and neurite outgrowth, and all of these func- tions of tau are modulated by site-specific phosphorylation11. Hyperphosphorylation of tau can result in the self-assembly of tangles of paired helical filaments and straight filaments, which are involved in the pathogenesis of AD and other tauopathies, such as VaD12. After phosphorylation at additional sites, including Ser396/404, 1Department of Pharmacology, Shandong Univeristy School of Medicine 44#, Wenhua Xi Road, Jinan, Shandong, 250012 P.R. China. 2School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, P.R. China. Correspondence and requests for materials should be addressed to X.Z. (email: zxy_sptenic@163.com) Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 1 www.nature.com/scientificreports/ tau can be cleaved, which increases the propensity of tau to oligomerise and eventually form filamentous aggre- gates13. The exact function that tau oligomers and filaments serve in the cell dysfunction/death process has not yet been clearly defined. The enzyme GSK-3β is one of a group of proline-directed kinases that can phosphorylate tau. Gene-knockout studies indicate that both tau and GSK-3β bind to the same region of presenilin 1 (PS1), residues 250–298, whereas the binding domain on tau is the microtubule-binding repeat region14. The ability of PS1 to bring tau and GSK-3β into close proximity suggests that PS1 may regulate the interaction between tau and GSK-3β14. Protein kinase B, also known as Akt, is activated by phosphorylation at serine 473 (Ser473) and threonine 308 (Thr308). Activated Akt phosphorylates a wide range of substrates, resulting in the activation of anti-apoptotic (survival) factors and the inactivation of pro-apoptotic factors15. Akt down-regulates the activities of GSK-3β by phosphorylating serine residue 9 (Ser9)16. (-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model Xiaoyin Zhu1,2, Jingwei Tian2, Songmei Sun2, Qiuju Dong2, Fangxi Zhang2 & Xiumei Zhang1 β y p p y g The novel compound (-)-SCR1693, whose name is gem-dimethyl-tacripyrine hydrochloride and whose chemical name is (-)-ethyl 5-amino-4-(2-chlorophenyl) -2,7,7-trimethyl-1,4,6,7,8,9-hexahydrobenzo[b][1,8] naphthyridine-3-carboxylate hydrochloride, shows multiple activities at the enzymatic and cellular levels, includ- ing the inhibition of both tau hyperphosphorylation and AChE activity. (-)-SCR1693 is being developed as a new drug for the treatment of dementia associated with AD, and its efficacy in AD has been demonstrated in animal models. Two major pathological pathways leading to the development of AD have been hypothesised: the amyloid cascade and vascular injury. The vascular hypothesis suggests that ischaemic changes and hypoperfusion asso- ciated with aging, as well as other vascular risk factors, disturb the blood supply and metabolism. This distur- bance leads to neuronal and/or neuroglial energy failure that not only causes injury but also accelerates amyloid over-production and reduces clearance17, eventually leading to AD pathology. This vascular hypothesis has also been suggested to contribute to VaD pathology. Thus, medications administered for AD are probably effective in the treatment of VaD. (-)-SCR1693 has been proposed to improve cognitive dysfunction and prevent neuro- degeneration in VaD patients by inhibiting AChE and increasing synaptic concentrations of acetylcholine, thus inhibiting tau hyperphosphorylation. Based on these properties, a new trial was initiated using BCCAo rats to test the effects of long-term (-)-SCR1693 administration on behaviour and on neuronal survival and tau pathology. Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 Results ( ) SCR1 Global ischaemia is known to often lead to delayed neuronal death in the CA1 region of the hippocampus. The cognitive deficits caused by ischaemia have been shown to be closely correlated with neuronal cell damage in the hippocampal CA1 region19. In addition, granule neurons of the dentate gyrus receive inputs from the entorhinal cortex through the perforant pathway and send signals to the CA3 pyramidal neurons through the mossy fibre pathway, which are critical pathways for memory processing and recording. Therefore, neuronal degeneration in these areas, together with synaptic deficits, may underlie the basis of the mild memory impairments observed in CCH mice20. Previous studies have shown neuronal damage, such as pyknosis of nuclei and vacuolation of neuropil, in pyramidal neurons of the CA1–CA4 region of the hippocampus and granule neurons of the dentate gyrus (DG) in post-ischaemic rats19.hf The neuroprotective effects of donepezil and (-)-SCR1693 were evaluated by measuring neuronal cell density in the hippocampus after 34 days of global cerebral ischaemia. Representative photomicrographs of the Nissl staining results for each group are shown in Fig. 2. In the sham group, the neurons in the DG, CA1, CA3, and CA4 regions were normal and did not show any cell damage. In ischaemic rats, the numbers of cells in these regions were markedly decreased and the neurons exhibited a shrunken morphology (solid arrows) and vacuolation (hollow arrows). These results demonstrate that ischaemia leads to obvious neuron loss, disordered arrangement, vacuolation of the cell body of neurons, pyknosis of nuclei, and coagulation necrosis in the DG, CA1, CA3, and CA4 regions of the model group. Similar changes were observed in the donepezil group, but these changes were ameliorated in the group that received 3 mg/kg of (-)-SCR1693. In 2 out of 6 rats in the 0.3 mg/kg group and 3 out of 6 rats in the 1 mg/kg group, the histological lesions in the hippocampus were attenuated, but there were no Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 2 www.nature.com/scientificreports/ p / Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, mean daily escape latencies during the training phase are shown. Results ( ) SCR1 (B) The number of times the rats crossed the place where the platform had been hidden during the training phase of the probe trial (swimming 120 s without platform) is shown. (C) Representative swim paths during the probe test are shown. Statistical analysis was performed using one-way ANOVA. When the ANOVA indicated significant treatment effects, the means were separated using Tukey’s multiple comparison test. Bars represent mean ± SEM for replicate samples (n = 6). p < 0.05, p < 0.01 vs. Sham group; #p < 0.05, ##p < 0.01 vs. Model group. Representative swim paths during the probe test showed that spatial memory deficits improved in ischaemic rats treated with (-)-SCR1693. statistically significant differences in the number of surviving neurons in the groups that received 0.3 or 1 mg/kg of (-)-SCR1693 compared with the model group. The histograms show that the number of surviving neurons in the model group was markedly decreased compared with that in the sham rats. Obvious recovery of neuron loss was observed in rats treated with 3 mg/kg of (-)-SCR1693, while the neuron loss could not be suppressed follow- ing treatment with 1 mg/kg of donepezil. (-)-SCR1693 inhibits AChE activity in the hippocampus and blood plasma. Inhibition of AChE activity could decrease the hydrolysation of Ach and increase the concentration of ACh. In Fig. 3a, we show that CCH significantly increased the activity of AChE in the rat hippocampus compared with the sham group (p = 0.002, n = 6). Hippocampal AChE activity in the donepezil group was significantly lower than in the model group (p = 0.018). All three doses of (-)-SCR1693 significantly decreased the activity of AChE compared with the model group (0.3 mg/kg, p = 0.025; 1 mg/kg, p = 0.001; 3 mg/kg, p = 0.000). g g g g g g g As shown in Fig. 3b, blood plasma AChE activity was slightly lower in the model group than in the sham group, but the difference was not statistically significant (p = 0.636). Donepezil (p = 0.001 vs. sham, p = 0.003 vs. model), 1 mg/kg of (-)-SCR1693 (p = 0.013 vs. sham, p = 0.038 vs. model), and 3 mg/kg of (-)-SCR1693 (p = 0.000 vs. sham, p = 0.001 vs. model) significantly decreased blood plasma AChE activity compared with the sham and model groups. Results ( ) SCR1 (B) The number of times the rats crossed the place where the platform had been hidden during the training phase of the probe trial (swimming 120 s without platform) is shown. (C) Representative swim paths during the probe test are shown. Statistical analysis was performed using one-way ANOVA. When the ANOVA indicated significant treatment effects, the means were separated using Tukey’s multiple comparison test. Bars represent mean ± SEM for replicate samples (n = 6). p < 0.05, *p < 0.01 vs. Sham group; #p < 0.05, ##p < 0.01 vs. Model group. Representative swim paths during the probe test showed that spatial memory deficits improved in ischaemic rats treated with (-)-SCR1693. Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, mean daily escape latencies during the training phase are shown. (B) The number of times the rats crossed the place where the platform had been hidden during the training phase of the probe trial (swimming 120 s without platform) is shown. (C) Representative swim paths during the probe test are shown. Statistical analysis was performed using one-way ANOVA. When the ANOVA indicated significant treatment effects, the means were separated using Tukey’s multiple comparison test. Bars represent mean ± SEM for replicate samples (n = 6). p < 0.05, *p < 0.01 vs. Sham group; #p < 0.05, ##p < 0.01 vs. Model group. Representative swim paths during the probe test showed that spatial memory deficits improved in ischaemic rats treated with (-)-SCR1693. Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral li ti f th tid t i i t ( ±SEM 6) (A) I th l i ti t t Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, h Figure 1. Effects of (-)-SCR1693 on water maze performance deficits caused by permanent bilateral ligation of the common carotid arteries in rats (mean ± SEM, n = 6) . (A) In the place navigation test, mean daily escape latencies during the training phase are shown. Results ( ) SCR1 (-)-SCR1693 (1 mg/kg) led to a similar decrease in hippocampal AChE activity and higher plasma AChE activity compared with donepezil, while 3 mg/kg of (-)-SCR1693 led to a similar change in plasma AChE activity and lower hippocampal AChE activity compared with donepezil. According to the data, (-)-SCR1693 may produce fewer side effects and may be more efficient in inhibiting AChE activity than donepezil. (-)-SCR1693 up-regulates the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. The enzyme-linked immunosorbent assay (ELISA) (Fig. 4) revealed that hippocampal BDNF levels in the model group were significantly higher than in the sham group (p = 0.049, n = 6). These results indi- cate that ischaemia leads to an increase in BDNF gene expression, which is in accordance with a previous study21. Exposure to 3 mg/kg of (-)-SCR1693 significantly elevated hippocampal BDNF expression (p = 0.013, vs. sham group; p = 0.045, vs. model group). Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bi of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollo neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the mode Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-ind damage to different degrees (scale bars=50μm) Histograms show the neuron number per field Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 4 www.nature.com/scientificreports/ Figure 3. The effects of (-)-SCR1693 on acetylcholinesterase (AChE) activity in the hippocampus and blood plasma (mean ± SEM, n = 6). (A) Hippocampal AChE activity. (B) Blood plasma AChE activity. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 3. The effects of (-)-SCR1693 on acetylcholinesterase (AChE) activity in the hippocampus and blood plasma (mean ± SEM, n = 6). (A) Hippocampal AChE activity. (B) Blood plasma AChE activity. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 4. The effects of (-)-SCR1693 on hippocampal brain-derived neurotrophic factor (BDNF) expression (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 4. Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. The effects of (-)-SCR1693 on hippocampal brain-derived neurotrophic factor (BDNF) expression (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. (-)-SCR1693 modulates the phosphorylation of AKT, GSK-3β and tau in the rat hippocamp T l th h i d l i lt d t h h l ti i d th lt ti f t t l (-)-SCR1693 modulates the phosphorylation of AKT, GSK-3β and tau in the rat hippocampus. To explore the mechanisms underlying altered tau phosphorylation, we examined the alterations of total Akt, p-Akt, total GSK-3β, and p-GSK-3β. Phosphorylation of Akt at Ser473 reflects an increased activity of Akt22. Akt phosphorylation could lead to a decreased activity of GSK-3β, thus reducing tau phosphorylation and activating the cell survival-supporting functions of the Akt pathway22. Western blotting (Fig. 5) showed that the levels of total Akt did not change dramatically, while p-Akt immunoreactivity in the hippocampus after CCH treatment decreased by 16%. (-)-SCR1693 (3 mg/kg) reversed this change. The ratio of p-GSK-3β to GSK-3β decreased by 12% following CCH and increased by approximately 10% in rats that were treated with (-)-SCR1693 com- pared with control rats. Total GSK-3β and p-GSK-3β levels did not change after CCH or after treatment with (-)-SCR1693 and donepezil. (-)-SCR1693 modulates the phosphorylation of AKT, GSK-3β and tau in the rat hippocampus. To explore the mechanisms underlying altered tau phosphorylation, we examined the alterations of total Akt, p-Akt, total GSK-3β, and p-GSK-3β. Phosphorylation of Akt at Ser473 reflects an increased activity of Akt22. Akt phosphorylation could lead to a decreased activity of GSK-3β, thus reducing tau phosphorylation and activating the cell survival-supporting functions of the Akt pathway22. Western blotting (Fig. 5) showed that the levels of total Akt did not change dramatically, while p-Akt immunoreactivity in the hippocampus after CCH treatment decreased by 16%. (-)-SCR1693 (3 mg/kg) reversed this change. The ratio of p-GSK-3β to GSK-3β decreased by 12% following CCH and increased by approximately 10% in rats that were treated with (-)-SCR1693 com- pared with control rats. Total GSK-3β and p-GSK-3β levels did not change after CCH or after treatment with (-)-SCR1693 and donepezil. Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 5 www.nature.com/scientificreports/ Figure 5. The effects of (-)-SCR1693 on the ratios of p-Akt/Akt, p-GSK-3β/GSK-3β and p-tau/tau (mean ± SEM, n = 6). Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. The full-length blots are shown in the Supplementary Materials. The gels were run under the same experimental conditions. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 5. The effects of (-)-SCR1693 on the ratios of p-Akt/Akt, p-GSK-3β/GSK-3β and p-tau/tau (mean ± SEM, n = 6). The full-length blots are shown in the Supplementary Materials. The gels were run under the same experimental conditions. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Assay Test Concentration (M) % Inhibition of Control Values % of Control Values Reference Compound IC50 Ref (M) nH Ref 1st 2nd Mean CDK5 /p35 3.0E-05 −5 105.2 104.1 104.6 staurosporine 2.1E-08 1.2 GSK3beta 3.0E-05 −3 106.4 98.8 102.6 staurosporine 9.9E-08 1.6 MARK1 3.0E-05 −4 100.7 107.8 104.2 staurosporine 1.5E-08 2.9 MARK2 3.0E-05 7 93.5 91.5 92.5 H-89 8.3E-06 2.9 MARK3 3.0E-05 4 95.8 96.9 96.3 staurosporine 1.7E-08 1.6 MARK4 3.0E-05 2 98.5 97.1 97.8 hymenialdisine 3.6E-08 1.4 T bl 1 E d ll b d fil f ( ) SCR1693 Table 1. Enzyme and cell-based assays profile of (-)-SCR1693. Tau can be phosphorylated at multiple sites, such as Ser396/404, which increases the propensity of tau to oli- gomerise and eventually form filamentous aggregates13. While the protein levels of total tau in all of the groups were similar, a dramatic increase in the levels of p-tau (phosphorylated at Ser396) was observed in the hip- pocampus of the BCCAo rats. (-)-SCR1693 rectified this CCH-induced change in p-tau, and this effect was dose dependent (Fig. 5). However, there were no obvious changes in the levels of p-Akt and p-GSK-3β between the ischaemic group and either the control group or the (-)-SCR1693 group. We consider the small variations in p-Akt and p-GSK-3β to be unrelated to the dramatic (-)-SCR1693-induced decrease in the levels of p-tau. The enzymatic activity of GSK-3β in vitro. To determine the effects of (-)-SCR1693 on the activity of the upstream enzymes of tau, an additional cellular functional assay was conducted by Cerep (Le Bois l’Eveque, France). The effects of the compounds on the activity of human CDK5/p35, GSK-3β, MARK1, MARK2, MARK3 and MARK4 were evaluated by measuring the phosphorylation of the corresponding substrates using human recombinant enzymes and the LANCE® detection method. Discussion h In the present study, we investigated the mechanisms underlying CCH-induced spatial learning and memory deficits, and we examined whether the novel compound (-)-SCR1693 could ameliorate these deficits in a CCH rat model. We also compared the effects of (-)-SCR1693 with the effects of donepezil, which has been shown to improve hippocampal neuronal damage and reduce learning and memory deficits in the hippocampus of a global ischaemic gerbil model23. Our findings suggest that (-)-SCR1693 exhibits beneficial effects on CCH-induced cognitive and memory deficits and that its effects on these cognitive deficits are mediated by attenuating CCH-induced dysfunction of the central cholinergic system and the tau protein in the brain. y g y Surgical ligation of both of the common carotid arteries in rats produces a chronic, global hypoperfusion state, which is less severe than the 4-vessel occlusion (4-VO) animal model24. Impaired learning and memory have been confirmed using the Morris water maze task at ~7 days post-surgery25. In our present behavioural experiment, learning performance in the water maze task was severely impaired in the BCCAo rats, as indicated by an increase in the time required to find the hidden platform and a decrease in the number of times the rats crossed the place where the hidden platform was previously located. These results are in agreement with previous studies showing that chronic cerebral hypoperfusion results in impaired learning performance26. Our behavioural experiment shows that (-)-SCR1693 prevents learning and memory deficits and is more effective than donepezil.h p g yif p Neurons have a high demand for oxygen and a limited endogenous reserve of glycogen. Thus, adult neurons are vulnerable to ischaemic conditions27. Hippocampal neurons are particularly sensitive and undergo selective and delayed degeneration in response to global ischaemia28. The pyramidal neurons in the hippocampus are crit- ically involved in spatial learning and memory, and degeneration of these neurons results in cognitive deficits29. We found that CCH caused hippocampal neural damage, including neuronal loss, disordered arrangement, pyk- nosis of nuclei, vacuolation of the neuronal cell body, and coagulation necrosis in the model group. (-)-SCR1693 significantly attenuated the bilateral ligation-induced histological lesions in the hippocampus, while vacuola- tion and pyknosis of neurons were still observed in the donepezil group. Figure 2. Typical neuropathological changes observed in the hippocampus 4 weeks after bilateral ligation of the common carotid arteries. Neuronal loss, shrinkage (solid arrows) and vacuolation (hollow arrows) of neurons were observed in the CA1, CA3, CA4 and DG regions of the hippocampus in the model group rats. Long-term administration of (-)-SCR1693 and donepezil attenuated chronic hypoperfusion-induced neuronal damage to different degrees (scale bars = 50 μm). Histograms show the neuron number per field of view (×400) in the CA1, CA3, CA4 and DG regions (mean ± SEM, n = 6). p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. The results shown in Table 1 demonstrate that (-)-SCR1693 did not affect the activities of human CDK5/p35, GSK-3β, MARK1, MARK2, MARK3 and MARK4 in vitro. The inhibitory effect of (-)-SCR1693 on tau hyperphosphorylation depends on the interaction of PS1 with tau. The ability of PS1 to bring tau and GSK-3β into close proximity suggests that PS1 may play an important role in regulating the phosphorylation of tau via GSK-3β14. In the present study, (-)-SCR1693 did not alter either the phosphorylation or enzymatic activity of GSK-3β. Thus, we speculated that the interaction between PS1 and tau may explain the effect of (-)-SCR1693 on tau. The anti-tau antibody was incubated with pro- tein extracts from the hippocampus. The immunocomplexes were then precipitated by incubation with Protein A+G Agarose and detected using the anti-PS1 antibody. As shown in Fig. 6, more PS1 was precipitated in the Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 6 www.nature.com/scientificreports/ Figure 6. Binding of PS1 to tau using co-immunoprecipitation. (A) Protein bands represent the levels of PS1 binding to tau. (B) The effects of (-)-SCR1693 on the interaction between tau and PS1 (mean ± SEM, n = 6). The full-length blots are shown in the Supplementary Materials. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. *p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. Figure 6. Binding of PS1 to tau using co-immunoprecipitation. (A) Protein bands represent the levels of PS1 binding to tau. (B) The effects of (-)-SCR1693 on the interaction between tau and PS1 (mean ± SEM, n = 6). The full-length blots are shown in the Supplementary Materials. p < 0.05 vs. Sham group; #p < 0.05 vs. Model group. **p < 0.01 vs. Sham group; ##p < 0.01 vs. Model group. ischaemia model group than in the control group, which indicates that CCH results in an increase in PS1 binding to tau. (-)-SCR1693 at doses of 1 mg/kg and 3 mg/kg prevented the binding of tau to PS1, but donepezil did not have this effect. Discussion h Thus, the neuroprotective effects of (-)-SCR1693 contribute to cognitive rehabilitation, and its efficacy is greater than that of donepezil.i gfi y g p Cholinergic deficits in VaD have been observed in preclinical and clinical studies30,31, and evidence of cho- linergic changes in animal models of VaD has recently been reviewed32. The VaD-induced increase in AChE activity in the hippocampal areas could lead to reductions in the efficiency of cholinergic neurotransmission, such as a decrease in ACh levels in the synaptic cleft. This effect would contribute to the progression of cognitive impairment and other types of neurological dysfunction seen in patients and rats with VaD33. Our data showed that long-term administration of (-)-SCR1693, a potent AChE inhibitor, significantly inhibited AChE activity in a dose-dependent manner. The inhibition of serum AChE activity by donepezil has been linked to its adverse side effects, which include nausea, vomiting, diarrhoea, abdominal pain, weight loss, anorexia34 and hepatotox- icity associated with serum alanine aminotransferase elevation in up to 50% of patients35. We could reasonably conclude that (-)-SCR1693 might have similar AChE-inhibiting effects in the hippocampus, but with less adverse side effects, as higher doses of (-)-SCR1693 did not cause more adverse side effects than donepezil. A previous study reported that BDNF is involved in adult hippocampal neurogenesis and memory recovery under ischaemic conditions36 and that BDNF expression may be involved in the CREB-dependent neuroprotective mechanisms of donepezil in ischaemic injuries23. Neurodegenerative disorders may indeed affect neurotrophic factor function by reducing the adaptation of neurons to disease-related alterations37. Our results indicate that CCH induces an increase in BDNF expression, which was previously considered to be induced by neurogenesis after ischae- mia38. (-)-SCR1693 also increased the levels of BDNF, which may contribute to the protection of neurons in the hippocampus. pp p To investigate the possible molecular mechanisms underlying CCH-induced cognitive impairment and the effects of (-)-SCR1693, we further investigated tau-related molecules and pathways in the CCH rat brain. Under pathological conditions in which there is an imbalance in the phosphorylation/dephosphorylation of tau, aber- rant tau phosphorylation at Ser396 can increase the propensity of tau to oligomerise and eventually form filamen- tous aggregates13. The present study found that the levels of p-tau increased significantly in the hippocampi of the BCCAo rats, while (-)-SCR1693 inhibited the CCH-induced phosphorylation of tau at Ser396. Treatment and control materials. Treatment and control materials. (-)-SCR1693 (light yellow solid, purity = 98%, provided by Jiangsu Simovay Pharmaceutical Co., Ltd) was suspended in 1% (w/v) carboxymethyl cellulose sodium (SCMC) solution. Donepezil (Donepezil Hydrochloride Tablets, Eisai, China), which was used as a positive control, was pulverised and suspended in 1% (w/v) SCMC solution. Rats in the sham group were given 1% (w/v) SCMC solution. Rat surgery and treatment. Male Sprague–Dawley (SD) rats weighing between 200 and 250 g were pur- chased from Shandong Luye Pharmaceutical Co. Ltd, China. Animals were housed at 50–70% humidity, a temper- ature of 22–24 °C, and under a 12:12 h light/dark cycle. All experiments were conducted in accordance with the guidelines of the Ministry of Health of PR China and the Animal Care Committee of China Medical University. The study protocol was approved by the Experimental Animal Research Committee of Yantai University. Food and water were freely available during all phases of the experiment. Rats were acclimated to the facility for 5 days prior to surgery. The rats were anaesthetised with 3 mL/kg of 10% (w/v) chloral hydrate administered by intra- peritoneal injection. Through a midline cervical incision, the bilateral common carotid arteries were exposed and gently separated from the carotid sheath and vagus nerve. Each artery of the rats assigned to the ischaemic group was ligated with a 5–0 silk suture, while rats in the sham group underwent the same operation, including the neck incision and isolation of arteries, but without ligation. During recovery, the rats were kept in the animal quarters with free access to food and water. After the operation, the ischaemic animals were randomly divided into 6 groups (n = 12): a sham group, a model group, a 1 mg/kg/day donepezil group, and (-)-SCR1693 groups consisting of 0.3, 1, or 3 mg/kg/day. The SCMC or drugs were administered once daily for 4 weeks by gavage at a dosing volume of 1 mL/kg. Four weeks later, the spatial memory retention of the rats was tested, after which the rats were sacrificed to conduct histopathologic and biochemical examinations. During the behavioural test, the drug was administered 60 min before the trials. Morris water maze. Spatial memory performance was evaluated 4 weeks after BCCAo or sham surgery using the Morris water maze29. The Morris water maze device is composed of a circular pool placed in a room with conspicuous symbols on the pool wall. Discussion h Active GSK-3β can Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 7 www.nature.com/scientificreports/ cause hyperphosphorylation of the tau protein at Ser39611, and its upstream enzyme, activated Akt, increases Aβ protease levels. This increase inhibits GSK-3β and activates multiple survival signals to promote neuron growth and survival39,40. The present study found that CCH led to a minor decrease in p-Akt (active form) and p-GSK-3β (inactive form), while (-)-SCR1693 increased p-Akt and p-GSK-3β expression to almost normal levels. The effects of (-)-SCR1693 on the phosphorylation of Akt and GSK-3β in the rat hippocampus are clearly not the main cause of the inhibition of tau hyperphosphorylation. In addition, cellular functional assays conducted by CEREP confirmed that (-)-SCR1693 did not affect the enzymatic activity of GSK-3β. Therefore, (-)-SCR1693 inhibited the hyperphosphorylation of tau at Ser396 without affecting the enzymatic activity of GSK-3β. PS1 has been proven to bring tau and GSK-3β into close proximity to regulate the phosphorylation of tau by GSK-3β14. In our co-immunoprecipitation study, CCH resulted in more interactions between tau and PS1, which may be the reason why the levels of p-tau increased without changes in p-GSK-3β levels. (-)-SCR1693 decreased the precipitation of PS1, which suggests that (-)-SCR1693 may inhibit the interactions between tau and PS1. Therefore, the phos- phorylation of tau by GSK-3β was inhibited. Donepezil did not show similar effects on PS1 and the tau protein. However, further research is needed to investigate the precise mechanism of action of (-)-SCR1693. g In summary, the present study shows that (-)-SCR1693 attenuates the learning/memory impairment that is due to CCH-induced neuron damage. The proposed mechanisms of the neuroprotective effects of (-)-SCR1693 include the rectification of cholinergic deficits, an increase in survival signals such as BDNF, and a decrease in the accumulation of the phospho-tau protein. Our results strongly suggest that (-)-SCR1693 has therapeutic potential for the treatment of VaD-induced cholinergic dysfunction and CCH and shows more therapeutic potential than donepezil. The pathological changes that occur after permanent bilateral ligation of the common carotid arteries are quite similar to those observed in patients suffering from multi-infarct dementia, Binswanger’s and AD41. Therefore, the present findings suggest that (-)-SCR1693 may be a promising therapeutic agent for the treatment of other diseases involving vascular dementia. However, further studies are needed to explore its precise mecha- nism of action. Materials and Methods Treatment and control materials. Treatment and control materials. The pool was 150 cm in diameter, 50 cm deep, and filled to a height of 30 cm with water to cover a platform (10 cm in diameter). The platform and the interior of the device were painted black to prevent visibility of the platform, which was submerged approximately 1.5 cm below the surface of the water and remained in the middle of quadrant II. The swimming activity of each rat was monitored by a video camera linked to a computer with an image analyser. On days 1–5, the rats were placed in the water (24 ± 1 °C) with their backs to the platform and allowed to swim until they found the platform (maximum swim time 60 s). The latency of each rat climbing onto the hidden platform was recorded. If the animal failed to locate the platform in 60 s, it was placed on the platform and left there for 20 s and the result was recorded as 60 s. Each rat was placed into the water at the same point in one quadrant and then the three other quadrants. On day 6, the platform was removed from the pool and the number of times rats crossed the place where the hidden platform had been pre- viously hidden in 120 s was recorded. Histopathological examination. Histopathological examination. After behavioural examination, six rats from each group were anaesthe- tised with 3 mL/kg of 10% (w/v) chloral hydrate administered by intraperitoneal injection. After myocardial per- fusion with 4% (w/v) paraformaldehyde solution, the brains of the 6 rats in each group were separated and fixed in paraformaldehyde solution overnight. The brains were then embedded in paraffin and cut into sections (5 μm) using a microtome (Leica, Nussloch, Germany; S100, TPI). The tissue slides were stained with Nissl staining 8 Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 www.nature.com/scientificreports/ solution. Neuronal damage was examined and evaluated by counting the number of surviving neurons per field of view (×400) in the DG, CA1, CA3, and CA4 regions using light microscopy (Leica, DMIRB). solution. Neuronal damage was examined and evaluated by counting the number of surviving neurons per field of view (×400) in the DG, CA1, CA3, and CA4 regions using light microscopy (Leica, DMIRB). solution. Neuronal damage was examined and evaluated by counting the number of surviving neurons per field of view (×400) in the DG, CA1, CA3, and CA4 regions using light microscopy (Leica, DMIRB). Measurement of AChE activity. The remaining rats were anaesthetised with 3 mL/kg of 10% (w/v) chloral hydrate administered by intraperitoneal injection, and blood samples were obtained from the abdominal aorta. After myocardial perfusion with 0.9% (w/v) sodium chloride solution, the skull was dissected and the hippocam- pus was separated on ice. The tissue was homogenised in ice-cold saline to determine the relative biochemical index. AChE activity in blood plasma and the hippocampus was assayed using an AChE assay kit (#A024, Nanjing Jiancheng Bioengineering Institute, China) via a spectrometric method42. Measurement of BDNF. BDNF expression in the hippocampus was measured using the BDNF Emax® ImmunoAssay System (#G7610, Promega Corporation, USA) as per the manufacturer’s instructions. Measurement of BDNF. BDNF expression in the hippocampus was measured using the BDNF Emax® mmunoAssay System (#G7610, Promega Corporation, USA) as per the manufacturer’s instructions. Western blot analysis. Protein samples were homogenised in lysis buffer (20 mM Tris, 150 mM NaCl, 1% (v/v) Triton X-100) containing 1 mM PMSF, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, and leupeptin. The samples were then incubated for 30 min at 4 °C and centrifuged for 10 min at 13,000 × g. The supernatant (lysate) was collected, and 15 μg protein was loaded into each lane. Histopathological examination. Samples were separated on 8–12% SDS-polyacrylamide gels and electro-blotted onto nitrocellulose membranes (Millipore). After blocking with 5% (w/v) non-fat milk, the blots were incubated with the following primary antibodies: rabbit anti-phospho-Akt (p-Akt-Ser473, 1:400, Santa Cruz), rabbit anti-Akt (1:400, Santa Cruz), rabbit anti-phospho-GSK-3β (p-GSK-3β-Ser9, 1:1000, Cell Signaling Technology), rabbit anti-GSK-3β (1:1000, Cell Signaling Technology), mouse anti-phospho-tau (p-tau-Ser396, 1:1000, Cell Signaling Technology), mouse anti-tau (1:1000, Cell Signaling Technology), rabbit anti-presenilin 1 (1:1000, Cell Signaling Technology), and β-actin (1:1000, Santa Cruz). Conjugated goat anti-rabbit or goat anti-mouse IgG was detected with enhanced chemilumines- cence (ECL) (Pierce® ECL Western Blotting Substrate). β-actin was used as an internal reference for relative quantification. Cellular functional assays in vivo. The effects of (-)-SCR1693 on the activities of human CDK5/p35, GSK3β, MARK1, MARK2, MARK3 and MARK4 were evaluated by measuring the phosphorylation of the respec- tive corresponding substrates using a human recombinant enzyme and the LANCE® detection method (Le Bois l’Evêque - B.P. 1 - 86600 Celle-L’Evescault - FRANCE). Co-immunoprecipitation. Protein extracts from the rat hippocampi of each group were preincubated with 1 μg of anti-tau antibody at 4 °C overnight and then incubated with 40 μL Protein A+G Agarose at 4 °C for 3 h. The mixtures were centrifuged at 2500 rpm for 5 min, and then the supernatant was discarded. After being washed 5 times in phosphate buffered saline, the immunoprecipitates were eluted by incubation with loading buffer at 100 °C for 5 min. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis for detection of tau and PS1. Statistical analysis. Statistical analysis of the data was conducted according to the Curran-Everett and Benos methods. All data were expressed as the mean ± SEM, n = 6. The behavioural data were analysed using one-way repeated-measures ANOVA or two-way repeated-measures ANOVA followed by the Tukey test for mul- tiple comparisons among different groups. Neurochemical data were compared using one-way ANOVA followed by the Tukey test for multiple comparisons. P < 0.05 was considered statistically significant. References 1. Prencipe, M. et al. Stroke, disability, and dementia: results of a population survey. Stroke 28, 531 536 (1997). 2. Roman, G. C. Vascular dementia revisited: diagnosis, pathogenesis, treatment, and prevention. Med Clin North Am 86, 477 (2002). ( ) 3. Valerio Romanini, C. et al. Neurohistological and behavioral changes following the four-vessel occlusion/internal carotid artery model of chronic cerebral hypoperfusion: comparison between normotensive and spontaneously hypertensive rats. Behav Brain Re 252, 214–221, doi: 10.1016/j.bbr.2013.05.043 (2013).h j ( ) 4. Gottfries, C. G., Blennow, K., Karlsson, I. & Wallin, A. The neurochemistry of vascular dementia. Dementia 5, 163–167 (1994) j ( ) 4. Gottfries, C. G., Blennow, K., Karlsson, I. & Wallin, A. The neurochemistry of vascular dementia. Dementia 5, 163–167 (1994).l h y 5. Tohgi, H., Abe, T., Kimura, M., Saheki, M. & Takahashi, S. Cerebrospinal fluid acetylcholine and choline in vascular dementia of Binswanger and multiple small infarct types as compared with Alzheimer-type dementia. J Neural Transm (Vienna) 103, 1211–1220 (1996). h 5. Tohgi, H., Abe, T., Kimura, M., Saheki, M. & Takahashi, S. Cerebrospinal fluid acetylcholine and choline in vascular dementia of Binswanger and multiple small infarct types as compared with Alzheimer-type dementia. J Neural Transm (Vienna) 103, 1211–1220 (1996).i ( ) 6. Wang, L. M., Han, Y. F. & Tang, X. C. Huperzine A improves cognitive deficits caused by chronic cerebral hypoperfusion in rats. Eur J Pharmacol 398, 65–72 (2000). J ( ) 7. Baskys, A. & Cheng, J. X. Pharmacological prevention and treatment of vascular dementia: approaches and perspectives. Exp Gerontol 47, 887–891, doi: 10.1016/j.exger.2012.07.002 (2012).f ( ) 7. Baskys, A. & Cheng, J. X. Pharmacological prevention and treatment of vascular dementia: approaches and perspectives. Exp Gerontol 47, 887–891, doi: 10.1016/j.exger.2012.07.002 (2012). , j g ( ) 8. Vicente, E. et al. Astroglial and cognitive effects of chronic cerebral hypoperfusion in the rat. Brain Res 1251, 204–212, doi: 10.1016/j. brainres.2008.11.032 (2009).fii j g 8. Vicente, E. et al. Astroglial and cognitive effects of chronic cerebral hypoperfusion in the rat. Brain Res 1251, 204–212, doi: 10.1016/j. brainres.2008.11.032 (2009).fii 9. de la Torre, J. C. et al. Chronic cerebrovascular insufficiency induces dementia-like deficits in aged rats. Brain Res 582, 186–195 (1992).t 10. Yao, Z. H., Zhang, J. J. & Xie, X. F. Enriched environment prevents cognitive impairment and tau hyperphosphorylation after chronic cerebral hypoperfusion. Curr Neurovasc Res 9, 176–184 (2012).f 10. Yao, Z. H., Zhang, J. J. Author Contributions X.Z. and X.Z. designed the research and wrote the manuscript; Xiaoyin Zhu performed the majority of the experiments; S.S., F.Z. and Q.D. supported several experiments. Xiumei Zhang and J.T. contributed materials and revised the manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 5. Brunet, A., Datta, S. R. & Greenberg, M. E. Transcription-dependent and -independent control of neuronal survival by the PI3K-Ak signaling pathway. Curr Opin Neurobiol 11, 297–305 (2001).h g g p y p 16. Kaytor, M. D. & Orr, H. T. The GSK3 beta signaling cascade and neurodegenerative disease. Curr Opin Neurobiol 12, 275 (2002). ( ) 7. Mawuenyega, K. G. et al. Decreased clearance of CNS beta-amyloid in Alzheimer’s disease. Science 330, 1774, doi: 10.1126 science.1197623 (2010). 8. Goshen, I. et al. Environmental enrichment restores memory functioning in mice with impaired IL-1 signaling via reinstatement o long-term potentiation and spine size enlargement. J Neurosci 29, 3395–3403, doi: 10.1523/jneurosci.5352-08.2009 (2009). 9 Block F Global ischemia and behavioural deficits Prog Neurobiol 58 279 295 (1999) 18. Goshen, I. et al. Environmental enrichment restores memory functioning in mice with impaired IL-1 signaling v long-term potentiation and spine size enlargement. J Neurosci 29, 3395–3403, doi: 10.1523/jneurosci.5352-08.20 long-term potentiation and spine size enlargement. J Neurosci 29, 3395–3403, doi: 10.1523/jneurosci.5352-08.2009 (2009). 19. Block, F. Global ischemia and behavioural deficits. Prog Neurobiol 58, 279–295 (1999). long term potentiation and spine size enlargement. J Neurosci 29, 3395 3403, doi: 10.1523/jneurosci.5352 08.2009 (2009). 19. Block, F. Global ischemia and behavioural deficits. Prog Neurobiol 58, 279–295 (1999). g p p g j 19. Block, F. Global ischemia and behavioural deficits. Prog Neurobiol 58, 279–295 (1999). i g ( ) 0. Zhao, Y. et al. Chronic cerebral hypoperfusion causes decrease of O-GlcNAcylation, hyperphosphorylation of tau and behaviora deficits in mice. Front Aging Neurosci 6, 10, doi: 10.3389/fnagi.2014.00010 (2014).f i 20. Zhao, Y. et al. Chronic cerebral hypoperfusion causes decrease of O-GlcNAcylation, hyperpho deficits in mice. Front Aging Neurosci 6, 10, doi: 10.3389/fnagi.2014.00010 (2014).f i g g g 1. Lindvall, O. et al. Differential regulation of mRNAs for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 h d l b f ll b l h d h l l A d S SA ( ) i g g g 21. Lindvall, O. et al. Differential regulation of mRNAs for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 in the adult rat brain following cerebral ischemia and hypoglycemic coma. Proc Natl Acad Sci USA 89, 648–652 (1992).f g yp g y , ( ) 22. Malm, T. M. et al. Pyrrolidine dithiocarbamate activates Akt and improves spatial learning in APP/PS1 mice without affecting beta- amyloid burden. www.nature.com/scientificreports/ J Neurosci 27, 3712–3721, doi: 10.1523/JNEUROSCI.0059-07.2007 (2007).it y 3. Min, D. et al. Donepezil attenuates hippocampal neuronal damage and cognitive deficits after global cerebral ischemia in gerbils Neurosci Lett 510, 29–33, doi: 10.1016/j.neulet.2011.12.064 (2012). Neurosci Lett 510, 29–33, doi: 10.1016/j.neulet.2011.12.064 (2012 j ( ) 4. Jiwa, N. S., Garrard, P. & Hainsworth, A. H. Experimental models of vascular dementia and vascular cognitive impairment: a systematic review. J Neurochem 115, 814–828, doi: 10.1111/j.1471-4159.2010.06958.x (2010).l y j 25. Pappas, B. A., de la Torre, J. C., Davidson, C. M., Keyes, M. T. & Fortin, T. Chronic reduction of cerebral blood flow in the adult rat: late-emerging CA1 cell loss and memory dysfunction. Brain Res 708, 50–58 (1996). 6. Ohta, H., Nishikawa, H., Kimura, H., Anayama, H. & Miyamoto, M. Chronic cerebral hypoperfusion by permanent internal carotid ligation produces learning impairment without brain damage in rats. Neuroscience 79, 1039–1050 (1997).t 27. Bendel, O. et al. Reappearance of hippocampal CA1 neurons after ischemia is associated with recovery of learning and mem Cereb Blood Flow Metab 25, 1586–1595, doi: 10.1038/sj.jcbfm.9600153 (2005). j j 28. Kirino, T. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239, 57–69 (1982). d l ’ f l d h h l l 29. Morris, R., Garrud, P., Rawlins, J. & O’Keefe, J. Place navigation impaired in rats with hippocampal lesions. Nature 297, 681 (1982). 30. Kimura, S. et al. Pathogenesis of vascular dementia in stroke-prone spontaneously hypertensive rats. Toxicology 153, 167–178 (2000). 1. Martin-Ruiz, C. et al. Nicotinic receptors in dementia of Alzheimer, Lewy body and vascular types. Acta Neurol Scand Suppl 176 34–41 (2000).i 32. Roman, G. C. & Kalaria, R. N. Vascular determinants of cholinergic deficits in Alzheimer disease and vascular dementia. Neurobiol Aging 27, 1769–1785, doi: 10.1016/j.neurobiolaging.2005.10.004 (2006). g g j g g 33. Silva, A. J., Stevens, C. F., Tonegawa, S. & Wang, Y. Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. Science 257, 201–206 (1992). 4. Imbimbo, B. P. Pharmacodynamic-tolerability relationships of cholinesterase inhibitors for Alzheimer’s disease. CNS drugs 15 375–390 (2001). ( ) 5. Wakita, H., Tomimoto, H., Akiguchi, I. & Kimura, J. Glial activation and white matter changes in the rat brain induced by chronic b l h f i i hi t h i l t d A t N th l 87 484 492 (1994) ( ) 35. Wakita, H., Tomimoto, H., Akiguchi, I. References & Xie, X. F. Enriched environment prevents cognitive impairment and tau hyperphosphorylation after chronic cerebral hypoperfusion. Curr Neurovasc Res 9, 176–184 (2012).f 11. Johnson, G. V. & Stoothoff, W. H. Tau phosphorylation in neuronal cell function and dysfunction. J Cell Sci 117, 5721–5729, doi: 10.1242/jcs.01558 (2004). 11. Johnson, G. V. & Stoothoff, W. H. Tau phosphorylation in neuronal cell function and dysfunction. J Cell Sci 117, 5721–5729, doi: 10.1242/jcs.01558 (2004). j ( ) 2. Alonso, A., Zaidi, T., Novak, M., Grundke-Iqbal, I. & Iqbal, K. Hyperphosphorylation induces self-assembly of tau into tangles o paired helical filaments/straight filaments. Proc Natl Acad Sci USA 98, 6923–6928, doi: 10.1073/pnas.121119298 (2001). pi gi p 13. Abraha, A. et al. C-terminal inhibition of tau assembly in vitro and in Alzheimer’s disease. J Cell Sci 113, 3737–3745 (2000). ii 13. Abraha, A. et al. C-terminal inhibition of tau assembly in vitro and in Alzheimer’s disease. J Cell Sci 113, 3737–3745 (2000). Scientific Reports \| 6:28908 \| DOI: 10.1038/srep28908 9 9 www.nature.com/scientificreports/ & Kimura, J. Glial activation and white matter changes in the rat brain induced by chronic cerebral hypoperfusion: an immunohistochemical study. Acta Neuropathol 87, 484–492 (1994).i 6. Shieh, P. B., Hu, S. C., Bobb, K., Timmusk, T. & Ghosh, A. Identification of a signaling pathway involved in calcium regulation o BDNF expression. Neuron 20, 727–740 (1998). 7. Bolognin, S. et al. An experimental rat model of sporadic Alzheimer’s disease and rescue of cognitive impairment with a neurotrophic peptide. Acta Neuropathol 123, 133–151, doi: 10.1007/s00401-011-0908-x (2012).t p p p p 38. Yamashima, T. et al. Vascular adventitia generates neuronal progenitors in the monkey hippocampus after ischemia. Hippocampus 14, 861–875 (2004). 39. Cole, G. M. & Frautschy, S. A. DHA may prevent age-related dementia. J Nutr 140, 869–874, doi: 10.3945/jn.109.113910 (2010). 40. Ma, Q. L. et al. Beta-amyloid oligomers induce phosphorylation of tau and inactivation of insulin receptor substrate via c-Jun N-terminal kinase signaling: suppression by omega-3 fatty acids and curcumin. J Neurosci 29, 9078–9089, doi: 10.1523/ jneurosci.1071-09.2009 (2009). j ( ) 41. Ni, J. W., Matsumoto, K., Li, H. B., Murakami, Y. & Watanabe, H. Neuronal damage and decrease of central acetylcholine level following permanent occlusion of bilateral common carotid arteries in rat. Brain Res 673, 290–296 (1995). 2. Ellman, G. L., Courtney, K. D., Andres, V. Jr. & Feather-Stone, R. M. A new and rapid colorimetric determination o acetylcholinesterase activity. Biochem Pharmacol 7, 88–95 (1961). 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 financial interests: The authors declare no competing financial interests. How to cite this article: Zhu, X. et al. (-)-SCR1693 Protects against Memory Impairment and Hippocampal Damage in a Chronic Cerebral Hypoperfusion Rat Model. Sci. Rep. 6, 28908; doi: 10.1038/srep28908 (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. 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W2900659783.txt	https://europepmc.org/articles/pmc6276438?pdf=render	en		Knowledge and Attitude of Health Professionals toward Telemedicine in Resource-Limited Settings: A Cross-Sectional Study in North West Ethiopia	Journal of healthcare engineering	2,018	cc-by	4,926	Hindawi Journal of Healthcare Engineering Volume 2018, Article ID 2389268, 7 pages https://doi.org/10.1155/2018/2389268 Research Article Knowledge and Attitude of Health Professionals toward Telemedicine in Resource-Limited Settings: A Cross-Sectional Study in North West Ethiopia Kirubel Biruk 1 2 1 and Eden Abetu 2 Debre Markos University, College of Medicine and Health Science, Department of Health Informatics, Debre Marqos, Ethiopia University of Gondar, College of Medicine and Health Science, Department of Clinical Pharmacy, Gondar, Ethiopia Correspondence should be addressed to Kirubel Biruk; birukkirubel@gmail.com Received 21 June 2018; Revised 3 August 2018; Accepted 30 August 2018; Published 18 November 2018 Academic Editor: Stefano Capolongo Copyright © 2018 Kirubel Biruk and Eden Abetu. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. In resource-limited environments, such as those categorized as underdeveloped countries, telemedicine becomes viewed as eﬀective channel for utilizing the scarce medical resources and infrastructures. The aim of this study was to assess knowledge and attitude toward telemedicine among cross section of health professionals’ working in three hospitals in North West Ethiopia. Methods. An institution-based cross-sectional study was conducted among 312 health professionals working in three diﬀerent hospitals of North Gondar Administrative Zone during November 13 to December 10, 2017. Data were collected using structured self-administered questionnaires. Data entry and analysis were done using SPSS version 20. The mean, percentage, and standard deviation were calculated to describe the characteristics of respondents. The chi-square test was used as appropriate, to evaluate the statistical signiﬁcance of the diﬀerences between the responses of the participants. A P value of < 0.05 was considered signiﬁcant. Result. A total of 312 study subjects were approached and included in the study from November 13 to December 10, and the response rate was 95.5%. The majority of respondents (195 (65.4%)) were male, and the majority of the respondents (66.1%) were in the age group of 21–29 years. A large number of respondents (224 (75%)) were bachelor’s degree holders. Only 37.6% of the respondents had demonstrated good knowledge of telemedicine, of which 74.1% were male, 65.2% of them were in the age group of 20–29 years, and 63.4% of them had >5 years of work experience. 191 (64.0%) respondents had good attitude toward telemedicine. Conclusion. The ﬁndings of the study suggest that although the respondents’ knowledge of telemedicine is limited, most of them have good attitude toward telemedicine. This study underlined the need of giving training on telemedicine in order to ﬁll the knowledge gap. 1. Introduction Innovative technological advancement has caused various changes in every sector. Medical science is not an exception, and new technologies have aﬀected both the advancement of the medicine and the way to deliver medical services [1]. This has caused a new area of health care in which medical practitioners, hospitals, health centers, and ﬁnancial and medical insurance experts cooperate together in a digital environment in order to improve fairness in distribution of medical services and the quality of these services as well as reducing the costs of services. The use of information technologies in the medical and health care ﬁelds shows great potential for improving the quality and eﬀectiveness of work done by medical organizations [2–4]. Allowing the ﬂow of expert medical knowledge from medical, research, and teaching institutions to distant remote locations where knowledge is needed but lacking medical experts, cost, and accessibility issues are the reasons why telemedicine has been identiﬁed as possible solution to medical problems evident in underdeveloped countries [5, 6]. Despite the great promise of telemedicine, to date, its implementation in Ethiopia and other developing countries has achieved little success with low utilization [5, 7, 8]. There are possible reasons discussed in literatures that why implementation of e-health systems continues to be challenging [9–11]. As the 2 Journal of Healthcare Engineering expansion and scale-up of the telemedicine system is still ongoing in sub-Saharan Africa, a key concern of the implementation team’s eﬀorts was that physicians’ and health professionals’ reactions to the telemedicine system would impact implementation success. [12] The success of any new technology depends on many factors including the knowledge and understanding of the concept, skills, acquired attitude, and working environment by the concerned professionals. This is applicable for any technologysupported medical service providing methods such as telemedicine where it is important to train the new concept and assess how far they are professionally ready to accept and provide telemedicine services. Since telemedicine is an emerging technology in the health sector of Ethiopia, to facilitate the adoption, it prominently requires information about the knowledge and attitude toward telemedicine among health professionals [13–15]. This study is, therefore, aimed at assessing knowledge and attitude of health professionals regarding telemedicine. 2. Methods 2.1. Study Design and Setting. An institution-based crosssectional study was conducted to assess the knowledge and attitude of health professionals among hospitals of North Gondar Administrative Zone, North West Ethiopia, from November 13 to December 10. North Gondar Zone has two district hospitals and one referral hospital serving a population of more than six million. During the study period, Debark hospital, University of Gondar specialized referral hospital, and Metema Hospital has 102, 833, and 136 health professionals, respectively. All health professionals who were on internship, unwilling health professionals to take part in this study, and those who were absent in the study place at the time of study were excluded. 2.2. Sample Size and Procedure. The sample for this study was calculated by using a single population proportion formula, with ﬁnite population correction, [16] with 95% conﬁdence interval (CI) and a proportion of telemedicine knowledge and attitude of 50%, since there is no previous study done in the same population with a relative precision to be 5%, and 10% nonresponse rate. Accordingly, the total sample size was 312. n′ � NZ2 P(1 − P) , d2 (N − 1) + Z2 (P)(1 − P) (1) where n′ � sample size with ﬁnite population correction, N � population size, Z � statistic for a level of conﬁdence (Z � 1.96), P � expected proportion (in proportion of one) (P � 50%), and d � precision (in proportion of one) (d � 0.05). n′ � (1071)1.962 (0.5)(0.5) � 283, + 1.962 (0.5)(0.5) 0.052 (1071 − 1) (2) anticipated nonrespondent rate 10% � 29, and total sample size � 283 + 29 � 312. Proportionate stratiﬁed simple random sampling technique was performed to select study participants from those public hospitals in North Gondar Administrative Zone. The Human Resource list of health professionals in each hospital was used as a sampling frame to identify potential study participants. We assumed that all health care providers working in the same hospital were homogenous regarding knowledge and attitude toward telemedicine. Study participants were then selected randomly using record identiﬁcation numbers retrieved from the sampling frame. Lottery method was used to randomly select a set of health care providers as respondents of this study. 2.3. Data Acquisition Instrument and Analysis. Data were collected by using structured self-administered questionnaire designed for the study, pretested on 35 health professionals of Felege Hiwot Hospital before it was distributed to research participants. The questionnaire was prepared by reviewing previous related studies [12, 17–21] and was translated to local language (Amharic) and then back to English in order to ensure that the translated version gives the proper meaning. The questionnaire consists of three main parts. Part 1 includes sociodemographic information of the participants (six items), part 2 is related to the health professionals’ knowledge of telemedicine (ten items), and part 3 investigates health professionals’ attitude of the relative advantages of telemedicine (seven items), compatibility of telemedicine (four items), complexity of deploying telemedicine (ﬁve items), trial ability of telemedicine application’s ease of use (four items), and observability of telemedicine (three items) (see supplementary materials section for detail). Respondents’ level of knowledge of telemedicine was assessed by questions to be answered in either “Yes” or “No.” A score of “1” will be given for “Yes” and “0” for “No.” One can score a minimum of 0 and a maximum of 10 in this section. In this study, the average score of 5 (50%) from the ten questions was used as a cutoﬀ point to determine the level of knowledge of telemedicine. The mean knowledge score less than 5 (50%) was labeled as poor knowledge of telemedicine, and more than average score of 5 (50%) was labeled as good knowledge of telemedicine. The perceived telemedicine attributes of relative advantage, compatibility, complexity, trial ability, and observability were rated on a 5-point Likert scale that ranged from “1 � strongly disagree” to “5 � strongly agree,” except for complexity attribute questions which were reversely scored (1 � strongly agree and 5 � strongly disagree). Scores for all statements for each perceived telemedicine attribute were be averaged to create the speciﬁc mean score. In this study, mean score of less than 2.5 (50%) is labeled as poor attitude, 2.6 (51%)–3.0 (60%) as moderate, and greater than 3.0 (60%) is labeled as good attitude. One-day training on the objective and relevance of the study, conﬁdentiality of data, respondents’ rights, informed consent, and data collection techniques was given to three individuals who were recruited to collect the data from study participants and one supervisor who conducted supportive supervision of the data collection process by the study investigators. Journal of Healthcare Engineering After collection was done, the data were checked, cleaned, edited, and analyzed by using SPSS version 20. The means and percentages were calculated to describe the proﬁle of the respondents. The chi-square test was used as appropriate, to evaluate the statistical signiﬁcance of the diﬀerences between the responses of the participants. A P value of <0.05 was considered signiﬁcant and presented. 2.4. Operational Deﬁnitions. In this study, telemedicine was deﬁned as “the delivery of healthcare services, where distance is a critical factor, by all healthcare professionals using information and communication technologies for the exchange of valid information for diagnosis, treatment and prevention of disease and injuries, research and evaluation and for the continuing education of healthcare providers, all in the interests of advancing the health of individuals and their communities” [4]. Health professionals were deﬁned as those employees with at least a diploma certiﬁcate in the health professions who are practicing clinical service in the study settings. Good knowledge of telemedicine was deﬁned as a score of the second part of the questionnaire more than 50%. It involves knowing the medical applications of telemedicine, knowing telemedicine infrastructure, knowing telemedicine tools (such as telesurgery and teleconsultation), and knowing the eﬀect of telemedicine on quality of care, cost, and time. Good health professionals’ attitude of telemedicine was deﬁned as more than 50% score of the perceived relative advantage, compatibility, complexity, trial ability, and observability of telemedicine. 2.5. Ethical Consideration. In conducting the study, ethical clearance was secured from Department of Health Informatics, College of Medicine and Health Sciences, Debre Markos University Ethical Review Committee. Additional permissions to access participants were obtained from the oﬃces of Hospital Directors, and verbal informed consent from the respondents was also attained. 3 Table 1: Responses by hospital. Hospitals Debark Metema Gondar university Total response Actual no. 30 39 243 312 Received no. 27 35 236 298 Received (%) 90 89.7 97.1 95.5 Table 2: Sociodemographic characteristics of study participants. Sociodemographic status Gender Male Female Age 20–29 30–39 >39 Educational status Diploma Degree Masters Others∗ Type of profession Physician Nurse Health oﬃcer Medical laboratory technician Pharmacist Midwifery Others∗∗ Year of experience <5 5–10 >10 Salary <1500 1500–3500 3500–5500 >5500 Number Percent 195 103 65.4 34.6 197 62 39 66.1 20.8 13.1 44 224 22 8 14.8 75.1 7.4 2.7 11 158 13 38 29 32 17 3.7 53.0 4.4 12.8 9.7 10.7 5.7 221 53 24 74.2 17.8 8.0 22 127 84 65 7.4 42.6 28.2 21.8 ∗ 3. Result A total of 312 study subjects were approached and included in the study from November 13 to December 10, and the response rate was 95.5%. The majority of respondents (195 (65.4%)) were male, and the majority age group was between 20 and 29 years (66.1%). A large number of respondents were bachelor’s degree holders (224 (75%)). Most of the study participants were nurses (158 (53.0%)), and regarding work experience, employees having less than 5 years’ work experience (221 (74.2%)) were the majorities. 127 (42.6%) respondents were employees earning monthly salary of 1500–3500 Ethiopian Birr. Responses by each hospitals and respondents’ sociodemographic characteristics are presented in Tables 1 and 2, respectively. Health professionals’ knowledge about telemedicine was observed from the study that 37.6% of the respondents had good knowledge of telemedicine, of which 74.1% were men, 65.2% of them were in the age group of 20–29, and 63.4% of them had >5 years of work experience. Only 22.1% (66) of Certiﬁcate and PhD. ∗∗ Physiotherapist, psychiatrist, anesthetists, radiologist, and optometrist. the respondents have seen a telemedicine system. Most of the information sources (57.4%) about telemedicine were from colleagues of the respondents. Among physicians, 54.5% of them have good knowledge of telemedicine. Among the health professionals who were considered having good knowledge of telemedicine, 82.1% were bachelor’s degree holders (Table 3 for details). This ﬁnding indicated that health care professionals had good attitude toward telemedicine with a “relative advantage” mean of 4.1, “compatibility” mean of 3.4, and “trialability” mean of 3.8 and had poor attitude regarding the complexity and observability of telemedicine (2.4 and 2.3, respectively) as summarized in Table 4. Among the total of 298 health professionals, 191 (64.0%) respondents had good attitude toward telemedicine. 111 (54.0%) respondents strongly agreed that telemedicine improves the quality of clinical decision, whereas 197 (66.0%) respondents agreed and strongly agreed that 4 Table 3: Health professionals with good knowledge of telemedicine. Sociodemographic Total count Percent P value Gender Male 83 74.1 0.000 Female 29 25.9 X2 � 23.181 Age 20–29 73 65.2 0.069 (ns) 30–39 26 23.2 X2 � 5.351 >39 13 11.6 Educational status Diploma 9 8.0 Degree 92 82.1 0.000 Masters 8 7.1 X2 � 18.140 3 2.7 Others∗ Type of profession Physician 6 5.4 Nurse 65 58.0 Health oﬃcer 4 3.6 0.000 Medical laboratory technician 6 5.4 X2 � 29.411 Pharmacist 12 10.7 Midwifery 8 7.1 11 9.8 Others∗∗ Year of experience <5 71 63.4 0.008 5–10 34 30.3 X2 � 9.576 >10 7 6.3 Salary <1500 4 3.6 0.008 1500–3500 42 37.5 3500–5500 39 34.8 X2 � 11.731 >5500 27 24.1 ∗ Certiﬁcate and PhD. ∗∗ Physiotherapist, psychiatrist, anesthetists, radiologist, and optometrist. telemedicine threatens information conﬁdentiality and patient privacy. 61% of health care professionals in this study appeared to know the beneﬁt of telemedicine in saving time, and 56.4% of them knew that it reduced unnecessary transportation cost. According to the ﬁnding of this study, most health professionals (155 (52%)) thought that telemedicine is complex with 229 (76.2%) respondents opinion that telemedicine requires lots of mental eﬀort. Sociodemographic status and health professionals’ attitude toward telemedicine are shown in Table 5. 4. Discussion The use of information technology in health care system is inﬂuenced by many issues. Among others, human-related components such as users’ knowledge and attitude toward technology are of high importance. A survey in Michigan State University, USA, and other similar studies show that attitude and perception is an important and key research question to explain how telemedicine is viewed and conceived by health professionals [17, 18, 22–25]. To deal with these issues, targeted strategies need to be taken into consideration to facilitate the deployment of the technology. The purpose of this study was to measure the level of knowledge and attitude of health professionals among three Journal of Healthcare Engineering hospitals in North West Ethiopia. The results suggest that most of respondents had poor knowledge of various aspects of telemedicine, and even fewer had ever heard of telemedicine (36.2%), which is by far less than the number of health professionals from the study of European regions, which is 84% [26]. From those who have heard about telemedicine, the proportion of health professionals’ source of information about the technology was mostly from their colleagues with 57.4%, which is as same as the study in Iran with 51.4% information source being colleagues [19]. The participants of this study had generally positive attitude toward telemedicine, but they also had signiﬁcant concerns on its complexity and observability that 65.8% of them agreed and strongly agreed that telemedicine increases staﬀ work load and 68.2% of them thought that telemedicine creates new responsibilities for the staﬀ. These ﬁndings indicate that much work is needed to be done to educate health care professionals about telemedicine in order to lay the groundwork for successful and sustainable adoption of the technology in the country. The proportion of respondents in this survey who have good knowledge of telemedicine (37.6%) was similar to the proportion of respondents of a cross-sectional study in India, which is about 41% [18] and slightly lower than the proportion of medical students in Sir Lanka (43%) from a survey of ﬁnalyear medical students studying at the Faculty of Medicine, Sri Jayewardenepura University (SJU), Sri Lanka [27]. A study in Mashhad, Iran, in selected teaching hospitals also showed that health professionals have low knowledge about telemedicine with total awareness of health professionals with 13 ± 5.5 out of 35, which signiﬁed as low, and from those health professionals participated in the study, most of them have positive attitude toward telemedicine with total score of 63.42 ± 9.5 out of 95 (65%), which indicates a total positive attitude [19] that is almost similar with the result of this study. Another ﬁnding in Isfahan, Iran, that 48.4% of the participants have good awareness and 63.3% of the participants have favorable attitude regarding telemedicine somehow conforms to this study [20]. Finally, this ﬁnding revealed that the majority of health professional’s knowledge was low, and their attitude toward telemedicine was found to be good, which is in line with the previous results of similar studies. 5. Conclusion As telemedicine is a new concept in health care service in Ethiopia, its implementation has not been as much satisfactory and successful. However, the respondents of this study conﬁrmed positive attitude toward telemedicine. According to these ﬁndings, there is an opportunity for telemedicine to be fully integrated in the health care system in Northern Ethiopia if appropriate training and education is provided for the health care professionals. Therefore, before the deployment of this technology, it is very important to consider improving users’ knowledge of the technology and demonstrate its competencies and beneﬁts because ample knowledge and aﬃrmative opinions of the technology are key factors to reassure users to use the technology in the future. Journal of Healthcare Engineering 5 Table 4: Attitude of health professionals toward telemedicine. Attributes of telemedicine attitude Relative advantage Reduce medical errors Facilitate diagnosis and treatment Increase communication among health care providers Telemedicine can reduce the number of visits to health care centers Enables me accomplish my task more quickly Improve clinical decisions Provide more comprehensive health care services Total mean score Compatibility In my opinion, telemedicine is compatible with all aspects of my work Telemedicine is completely compatible with my current situation I think telemedicine ﬁts well with the way I like to work Using telemedicine ﬁts well into my work style Total mean score Complexity I believe using telemedicine requires a lots of mental eﬀort∗ Learning to operate telemedicine is hard for me∗ I think telemedicine increases staﬀ work load∗ I think telemedicine creates new responsibilities for staﬀ∗ In my opinion, telemedicine threatens information conﬁdentiality and patient privacy∗ Total mean score Trial ability I believe to try telemedicine applications is a great opportunity I do not have to take very much eﬀort to try out telemedicine I believe, using telemedicine on a trial basis is enough to see what it could do I would like to try out telemedicine applications before using it Total mean score Observability I have seen what other hospital staﬀs do with telemedicine technologies Telemedicine technology is very visible in the hospital where I work In the hospital, I see telemedicine technology being used for many tasks Total mean score Overall attitude mean score ∗ Strongly disagree (%) Disagree (%) Neutral (%) Agree (%) Strongly agree (%) Mean score 7.0 2.0 9.4 3.7 14.1 2.7 36.9 60.1 32.6 31.5 3.8 4.2 1.3 2.7 4.7 56.4 34.9 4.2 6.0 7.4 10.4 42.3 33.9 3.9 3.4 1.0 7.4 3.0 16.3 9.4 37.1 32.6 35.8 54.0 3.9 4.4 0.7 1.3 7.4 37.9 52.7 4.4 4.1 8.7 14.4 10.7 41 25.2 3.6 10.4 11.1 11.8 38.9 27.8 3.6 12.1 18.1 13.4 29.6 26.8 3.4 18.5 22.5 26.2 20.8 12.0 2.9 3.4 10.7 9.4 3.7 46.3 29.9 2.2 12.8 18.7 7.9 42.1 18.5 2.7 11.1 13.0 10.1 34.9 30.9 2.4 9.4 12.7 9.7 39.3 28.9 2.3 9.1 11.1 13.8 28.7 37.3 2.3 2.4 2.0 3.0 1.7 52.0 41.3 4.3 11.4 16.1 22.1 27.9 22.5 3.3 16.1 15.8 13.1 34.5 20.5 3.3 6.1 7.0 5.0 36.6 45.3 4.1 3.8 32.9 28.2 5.7 17.1 16.1 2.6 36.2 40.0 5.0 16.1 11.7 2.4 38.0 42.6 3.7 9.7 6 2.0 2.3 3.2 Reverse scored. 6. Limitation of the Study The major limitation of this study was the small sample size, which was due to limited resource issue, and it was conducted among health professionals working in North Gondar Administrative Zone hospitals which were only three. Due to this reason, results may not be attributed to the whole health professional population. It would be more useful and generalizable if this study was conducted in the whole country to determine the knowledge and attitude of a larger sample of health professionals in more facilities and regions than we were able to cover. 6 Table 5: Health professionals with good attitude toward telemedicine. Characteristics Number Percent P value Gender Male 109 57.1 0.000 X2 � 17.921 Female 82 42.9 Age 20–29 106 55.5 0.000 30–39 48 25.1 X2 � 29.826 >39 37 19.4 Educational status Diploma 16 8.4 Degree 149 78.0 0.000 Masters 18 9.4 X2 � 77.512 8 4.2 Others∗ Type of profession Physician 11 5.8 Nurse 87 45.5 Health oﬃcer 9 4.7 0.000 Medical laboratory technician 25 13.1 X2 � 60.931 Pharmacist 24 12.6 Midwifery 21 11.0 14 7.3 Others∗∗ Year of experience <5 134 70.1 0.000 5–10 42 22.0 X2 � 44.959 >10 15 7.9 Salary <1500 9 4.7 0.000 1500–3500 81 42.4 X2 � 34.214 3500–5500 67 35.1 >5500 34 17.8 ∗ Certiﬁcate and PhD. ∗∗ Physiotherapist, psychiatrist, anesthetists, radiologist, and optometrist. Data Availability All data generated or analyzed during this study are included in this article and its supplementary information ﬁles. Ethical Approval Ethical clearance was secured from Department of Health Informatics, College of Medicine and Health Sciences, Debre Markos University Ethical Review Committee. Additional permissions to access participants were obtained from the oﬃces of Hospital Directors, and verbal informed consent from the respondents was also attained. Conflicts of Interest The authors declare that they have no conﬂicts of interest. Authors’ Contributions KB made substantial contributions to conception and design and acquisition of data, data collection supervision, data analysis, interpretation of data, and preparation of the manuscript. EA agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately Journal of Healthcare Engineering investigated and resolved. Both were involved in drafting and revising the manuscript and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Both authors read and approved the ﬁnal manuscript. Acknowledgments The authors would like to thank all health professionals for their time and eﬀort spent during the data collection period. Supplementary Materials Supplementary materials include the questioner used for data collection with 6 items related to sociodemographic characteristics of participants, 10 items related to telemedicine knowledge, and 22 items related to attitude toward telemedicine. (Supplementary Materials) References [1] R. L. Bashshur, G. W. Shannon, E. A. Krupinski et al., “National telemedicine initiatives: essential to healthcare reform,” Telemedicine and e-Health, vol. 15, no. 6, pp. 600– 610, 2009. [2] J. H. Thrall and G. Boland, “Telemedicine in practice,” Seminars in Nuclear Medicine, vol. 28, no. 2, pp. 145–157, 1998. [3] A. Wiborg and B. Widder, “Teleneurology to improve stroke care in rural areas. The telemedicine in stroke in swabia,” Stroke, vol. 34, no. 12, pp. 2951–2956, 2003. [4] S. Ryu, “Telemedicine: opportunities and developments in member states: report on the second global survey on eHealth 2009 (global observatory for eHealth series, volume 2),” Healthcare Informatics Research, vol. 18, no. 2, pp. 153–155, 2012. [5] D. Wamala and K. Augustine, “A meta-analysis of telemedicine success in Africa,” Journal of Pathology Informatics, vol. 4, no. 1, p. 6, 2013. [6] A. Dasgupta and S. Deb, “Telemedicine: a new horizon in public health in India,” Indian Journal of Community Medicine Oﬃcial Publication of Indian Association of Preventive and Social Medicine, vol. 33, no. 1, p. 3, 2008. [7] M. Kiﬂe, F. C. Payton, V. Mbarika, and P. Meso, “Transfer and adoption of advanced information technology solutions in resource-poor environments: the case of telemedicine systems adoption in Ethiopia,” Telemedicine and e-Health, vol. 16, no. 3, pp. 327–343, 2010. [8] K. Mengistu, V. W. A. Mbarika, and V. R. Bradley, “Global diﬀusion of the internet X: the diﬀusion of telemedicine in Ethiopia: potential beneﬁts, present challenges, and potential factors,” Communications of the Association for Information Systems, vol. 18, no. 30, pp. 612–640, 2006. [9] T. H. Broens, R. M. Huis in’t Veld, M. M. VollenbroekHutten, H. J. Hermens, A. T. van Halteren, and L. J. Nieuwenhuis, “Determinants of successful telemedicine implementations: a literature study,” Journal of Telemedicine and Telecare, vol. 13, no. 6, pp. 303–309, 2007. [10] T. R. F. Postema, J. M. Peeters, and R. D. Friele, “Key factors inﬂuencing the implementation success of a home telecare application,” International Journal of Medical Informatics, vol. 81, no. 6, pp. 415–423, 2012. Journal of Healthcare Engineering [11] M. Nazviya and S. Kodukula, “Evaluation of critical success factors for telemedicine implementation,” International Journal of Computer Applications, vol. 12, no. 10, pp. 29–36, 2011. [12] G. T. Olok, W. O. Yagos, and E. Ovuga, “Knowledge and attitudes of doctors towards e-health use in healthcare delivery in government and private hospitals in Northern Uganda: a cross-sectional study,” BMC Medical Informatics and Decision Making, vol. 15, no. 1, p. 87, 2015. [13] D. S. Edirippulige, A. C. Smith, J. Young, and R. Wootton, “Knowledge, perceptions and expectations of nurses in e-health: results of a survey in a children’s hospital,” Journal of Telemedicine and Telecare, vol. 12, no. 3, pp. 35–38, 2006. [14] D. Jayasinghe, R. M. Crowder, and G. Wills, “Model for the adoption of telemedicine in Sri Lanka,” SAGE Open, vol. 6, no. 3, article 2158244016668565, 2016. [15] N. Demartines, O. Freiermuth, D. Mutter, M. Heberer, and F. Harder, “Knowledge and acceptance of telemedicine in surgery: a survey,” Journal of Telemedicine and Telecare, vol. 6, no. 3, pp. 125–131, 2000. [16] L. Naing and B. N. Rusli, “Practical issues in calculating the sample size for prevalence studies,” Archives of Orofacial Sciences, vol. 1, pp. 9–14, 2006. [17] H. Ayatollahi, S. F. Z. P. Sabari, and M. Langarizadeh, “Clinicians’ knowledge and perception of telemedicine technology,” Perspectives in Health Information Management, vol. 12, p. 1, 2015. [18] Z. Zayapragassarazan and S. Kumar, “Awareness, knowledge, attitude and skills of telemedicine among health professional faculty working in teaching hospitals,” Journal of Clinical and Diagnostic Research: JCDR, vol. 10, no. 3, pp. 1–4, 2016. [19] A. Sheikhtaheri, M. Sarbaz, K. Kimiafar, M. Ghayour, and S. Rahmani, “Awareness, attitude and readiness of clinical staﬀ towards telemedicine: a study in Mashhad, Iran,” Studies in Health Technology and Informatics, vol. 228, pp. 142–146, 2016. [20] H. Keshvari, A. Haddadpoor, B. Taheri, and M. Nasri, “Determining the awareness and attitude of employees in Deputy of Health of Isfahan University of Medical Science toward telemedicine and its advantages,” Acta Informatica Medica, vol. 23, no. 2, pp. 97–101, 2015. [21] R. Parvin and M. Shahjahan, “Knowledge, attitude and practice on ehealth among doctors working at selected private hospitals in Dhaka, Bangladesh,” Journal of International Society for Telemedicine and eHealth, vol. 4, 2016. [22] P. Whitten, B. Holtz, and L. Nguyen, “Keys to a successful and sustainable telemedicine program,” International Journal of Technology Assessment in Health Care, vol. 26, no. 2, pp. 211–216, 2010. [23] R. Ward, C. Stevens, P. Brentnall, and J. Briddon, “The attitudes of health care staﬀ to information technology: a comprehensive review of the research literature,” Health Information and Libraries Journal, vol. 25, no. 2, pp. 81–97, 2008. [24] C. Dockweiler and C. Hornberg, “Knowledge and attitudes as inﬂuencing factors for adopting health care technology among medical students in Germany,” Journal of International Society for Telemedicine and eHealth, vol. 2, no. 1, p. 7, 2014. [25] F. Shiferaw and M. Zolfo, “The role of information communication technology (ICT) towards universal health coverage: the ﬁrst steps of a telemedicine project in Ethiopia,” Global Health Action, vol. 5, no. 1, article 15638, 2012. 7 [26] T. Mairinger, C. Gabl, P. Derwan, G. Mikuz, and O. FerrerRoca, “What do physicians think of telemedicine? A survey in diﬀerent European regions,” Journal of Telemedicine and Telecare, vol. 2, no. 1, pp. 50–56, 1996. [27] S. Edirippulige, R. B. Marasinghe, A. C. Smith et al., “Medical students’ knowledge and perceptions of e-health: results of a study in Sri Lanka,” Studies in Health Technology and Informatics, vol. 129, no. 2, pp. 1406–1409, 2007.
https://openalex.org/W2121200774	https://jeccr.biomedcentral.com/counter/pdf/10.1186/1756-9966-29-170	English	null	Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice	Journal of experimental & clinical cancer research	2,010	cc-by	5,220	RESEARCH Open Access Open Access © 2010 Zhou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Objective: The purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK) suicide gene system on mice hepatoma. Methods: Forty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1) PBS; (2) HSV-TK (3) HSV-TK+ ultrasound (HSV-TK+US); (4) HSV-TK+ultrasound +microbubbles (HSV-TK+US+MB). The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups. Results: The TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p < 0.05), and can significantly improve the survival time of tumor-bearing mice. Conclusion: Ultrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer. Recently a large number of studies have shown that ultrasound-targeted microbubble destruction is a safe, effective, non-invasive, and physical gene transfection technology, which brings a new hope for gene therapy in liver cancer [6-8]. Ultrasound microbubbles mostly contain gas [9]. The composition of its shell may include albumin, lipids, saccharide, non-ionic surfactants, poly- mers and other materials [10]. At present the size has been developed to nano-scale and it has the ability to penetrate the vascular endothelium [11]. Microbubbles containing gas will be compressed and expansed under the action of ultrasound with a certain intensity and fre- quency. When the sound energy reaches certain inten- sity, the microbubbles are immediately crushed. This will produce cavitation effect and mechanical effect to increase the permeability of cell membrane structure in target region, make the microvessels with the diameter ≤7 μm break down, widen the intercellular gap of vascu- lar endothelial cells. The exogenous genes can easily penetrate into the tissues and cells through capillary vessels to improve the gene transfection rate and * Correspondence: gongjianping11@126.com; liuchanganys@yeah.net 1Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, Chongqing 400010, PR China Full list of author information is available at the end of the article Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice iji Zhou1, Shengwei Li1, Zuojin Liu1, Yong Tang1, Zhigang Wang2, Jianping Gong1, Changan L Introduction Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence around the world [1,2]. More than one million new cases appeared each year, particularly in the Asia-Pacific region. This disease has rapid progress, high recurrence rate and traditional treatments have limited. With the continuous develop- ment of molecular biology, gene therapy for liver cancer has become a research hotspot and direction [3]. However, the safety of viral vector, ineffectiveness of non-viral gene vectors and other problems limit its further development [4,5]. Therefore, the search for an efficient, well targeting and safe gene transfection system for cancer gene therapy has become a focus of resea- chers inteset. Page 2 of 6 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 ATGGCCTCGTACCCCGGCCATCAACAC) and down- stream primer TKR (CGCGGATCCTCAGTTAGCCTC CCCCATCTCCCGGG) to obtain about 1.2 kb target HSV-TK fragment. Then directionally connect HSV-TK target gene fragment and pIRES2-EGFP (Invitrogen, USA) vect with the help of DNA ligase to obtain recom- binant plasmid pIRES2-EGFP-TK. The recombinant plasmid was transformed into DH5a Escherichia coli competent cells and spread on onkanamycin resistant LA plate for culture of 12-16 h. When the colonies grew out, we selected positive clones to extract plasmid, followed by Sal I and BamH I enzymes cut identification and sequencing by TaKaRa Company. ATGGCCTCGTACCCCGGCCATCAACAC) and down- stream primer TKR (CGCGGATCCTCAGTTAGCCTC CCCCATCTCCCGGG) to obtain about 1.2 kb target HSV-TK fragment. Then directionally connect HSV-TK target gene fragment and pIRES2-EGFP (Invitrogen, USA) vect with the help of DNA ligase to obtain recom- binant plasmid pIRES2-EGFP-TK. The recombinant plasmid was transformed into DH5a Escherichia coli competent cells and spread on onkanamycin resistant LA plate for culture of 12-16 h. When the colonies grew out, we selected positive clones to extract plasmid, followed by Sal I and BamH I enzymes cut identification and sequencing by TaKaRa Company. expression [12,13]. Cavitation effect can also damage cells, inhibit cell proliferation, and promote tumor cell apoptosis. When ultrasound-targeted microbubble generates strong cavitation effects, it can also damage blood vessel wall, active endogenous or exogenous coa- gulation, induce large-scale capillary embolism and block nutrient supply to cancerous cells, leading to dis- appearance of tumor tissues [14,15]. Suicide gene therapy has been widely used in liver can- cer treatment and showed a good application prospect. Especially the herpes simplex virus thymus kinase/ganci- clovir (HSV-TK/GCV) therapy system is most widely applied. Preparation of lipid microbubbles Preparation of lipid microbubbles Dipalmitoyl phosphatidylcholine (DPPC) (sigma, USA), distearoyl phosphatidyl ethanolamine (DSPE) (sigma, USA), diphenyl phosphoryl azide (DPPA) (sigma, USA), glycerol, PBS were mixed according to a certain propor- tion and were placed in a 1.5 ml vial, The vials were incubated at 40°C for 30 minutes. Each vial was filled with the perfluoropropane gas (C3F8), then the vials were mechanically shaken for 45 seconds in a dental amalgamator (YJT, Shanghai Medical Instrument Co., Ltd.) and quiescence for 5 min. This solution was diluted by phosphate-bufferedsaline, sterilized by Co60 and stored at 4°C;. Then the self-made lipid microbub- bles were made. The average diameter was 1.82 ± 0.45 μm; the average concentration was 1.2 × 1010/ml; the average potential was -24.7 ± 0.56 mV (n = 4). Introduction HSV-TK is a prodrug enzyme gene which can express and produce TK in the tumor cells, catalyze nucleoside analogue to form mono- phosphate products, and further form a triphosphoric acid product under the effect of phosphokinase in the cell. As a chain terminator, it will interfere with DNA synthesis during cell division, leading to tumor cell death [16,17]. A large number of stu- dies have shown that suicide gene system also has a “bystander effect”. The effect will kill non-transfected cells with the transfected cells, which overcomes the shortcom- ings of the low gene transtection rate and greatly enhances the anti-tumor effect of suicide gene therapy [18]. Connection of microbubbles with plasmid According to the method of preparation of gene-loaded lipid microbubbles from the reference of Zhaoxia Wang [19]. We mixed the prepared blank lipid microbubbles and poly-L- lysine (1 mg/ml) (Sigma Corporation, USA), and cultured at 37°C; for 30 min. Subnatant was soaked and deserted and washed twice by PBS. Naked plasmid (1 mg/ml) was added and incubated at 37°C; for 30 min, and washed by PBS twice. The manipulation was repeated three times. then gene-loaded lipid microbubbles were made. It was measured the average diameter of the HSV-TK wrapped microbubbles was between 2 μm to 4 μm and the concentration was 6.9 × 109/ml. The potential was -3.7 ± 0.56 mv (n = 4) and the plasmid concentration was 0.1 μg/μl. In this study, ultrasound microbubbles wrapped HSV- TK suicide gene had targeted release in mice liver tis- sues, and improved gene transfection efficiency with the features of ultrasound and microbubbles. In addition, the bystander effect of suicide gene fully played the anti- tumor role. The study provided an efficient, relatively targeted, non-invasive, and physical gene transfection method for HSV-TK/GCV system. Animal model Th t d The study protocol was approved by the Animal Research Committee of our institution.40 Kunming mice, cleaning grade, body weight (20 ± 2 g), male, 6 to 8 weeks old, were purchased from the Laboratory Animal Center of Third Military Medical University. H22 tumor cells (from Institute of ultrasonography, the second affiliated Hospital of Chongqing Medical University as a gift) were cultured in the RPMI 1640 medium (Hyclone, China) containing 10% betal bovine serum (FBS) at 37°C; with 5% CO2. We used serum-free RPMI1640 medium to adjust cell concentration to about 1 × 107/ml, followed by placenta blue exclusion dye test. The detected cell activity was >90%. Each mouse was inoculated 0.2 ml cell suspension subcutaneously in the right flank of Kunming mice. The tumor diameter was 0.5-1.0 cm after one week with the tumor rate of 100%. Experimental animals were randomly divided into four groups (10/per group) :(1) PBS group; (2) HSV-TK group; (3) HSV-TK+ US group; (4) HSV-TK+MB+US group. Assessment of therapeutic effect Measure the tumor size regularly to calculate the inhibi- tion rate: during treatment use calipers to measure the maximum diameter a (cm) and the shortest diameter b (cm) of tumors every 3 d, and apply the formula V = ab2/2 to calculate the tumor volume with the unit of cm3. The tumor inhibition rate = (the average size of tumors in control group- mean tumor volume in treat- ment group)/mean tumor volume in control group × 100%. According to the size of the measured tumor volume, draw the growth curves. Take five mice in each group for the observations of survival time. The obser- vation lasts for 80 days and survival curves were drawn. Plasmid The pORF-HSVTK plasmid was carried out PCR ampli- fication with upstream primer TKF(ACGCGTCGAC The microbubbles containing HSV-TK plasmid were injected through the tail vein of mice, 200 μl for each Page 3 of 6 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 time. The mice were injected once every 3d and conse- cutively injected for 3 times. Group A: PBS (200 μl); Group B: HSV-TK (200 μl, 0.1 μg/μl); group C: US +HSV-TK (200 μl, 0.1 μg/μl); Group D: US+HSV-TK +MBs (200 μl, 0.1 μg/μl). Self-made ultrasonic gene transfection instrument (UTG 1025, Institute of Ultra- sound Imaging of Chongqing Medical Sciences, Chongqing, China) was applied on C and D groups for irradiation after the target gene injection, with the radia- tion frequency of 1 MHz, sound intensity of 2 W/cm2, and used pulse irradiation method for 5 min, with the interval time of 10 s. Each mouse was intraperitoneally injected 0.2 ml (100 mg·kg-1·d-1) GCV (Roche, Switzer- land) 48 h after irradiation, which last for 14 days. acid and ethanol, washing, and sealing with conventional resin. Then under the optical microscope with 400 times magnification, five tumor cell areas were randomly selected. Count the number of total cells and apoptotic cells to calculate the percentage of TUNEL staining positive cells, i.e., apoptotic index (AI). AI = (number of apoptotic cells/the total number of tumor cells) × 100%. Western-blot Proteins were extracted using protein extraction reagent,48 hours after transfection and save at -20°C;, following a protocol provided by the manufacture. TK protein expression was detected with western-blot. 40 ml/L concentrated gel, 100 ml/L separation gel, pre- stained protein Marker 3.0 μL, 20 μg/hole sample total protein. Add sample into 100 mL/L SDS-PAGE followed by electrophoresis at 60 V. Change voltage to 100 V after 30 min. Get the gel when bromophenol blue ran to the bottom after 90 min. Synchronously transfer the protein to PVDF membrane at 20 V for 50 min. Seal for 4 h with 50 mL/L skim milk TBST at room temperature after trarsmembrane; add primary antibody (TK1 Poly- clonal antibody, 1:500) (Abcam, United Kingdom) fol- lowed by incubation for 2 h at room temperature and staying overnight at 4°C;. Use TBST to wash membrane three times with 15 min/time. Add appropriate concen- tration of secondary antibody combined with HRP (1:5000) for incubation followed by jiggle at room tem- perature for 2 h, washing membrane, imaging and expo- sure. The protein bands were normalized with b-actin, and all blots were quantified with Software Quantity One (Bio Rad). Statistic analysis The SPSS17.0 statistic software was used to make a sta- tistic analysis. The measurement data was expressed as mean ± SD. The analysis of variance was used to assess the inhibition rate. LSD-t test was used for pairwise comparison. Kaplan-Meier method was applied for sur- vival analysis. A P value less than .05 was considered indicative of a statistically significant difference. HSV-TK in vivo transfection effect 48 h after the transfection of ultrasound microbubble mediated HSV-TK in mice, the TK protein expression was detected in tissues by western-blot. It was observed that a single band appeared in each group at 25 kd. The band in HSV-TK+US+MBs group was the most obvious (Figure 1). Treatment effect As the tumor increases, the mice show obviously ema- ciated body, appetite loss, dull furs, activity reduction, body weight loss and so on. However, after treatment the mice growth in the GCV treatment group is significantly better than the control group. It can be seen from the tumor growth curve (Figure 3) that the tumor growth in group D (HSV-TK+US+MB) slows down significantly. Compared with the tumor size of control group A (PBS), the tumor sizes of group D were smaller than group A at all time points with statistical significance (P < 0.01). The tumor inhibition rates of group A, B, C and D were: 0%, 3.90% ± 1.80%, 22.70% ± 2.86% and 41.25% ± 3.20%. Take five mice tumor-bearing in each group as an 80-day con- tinuous observation of their survival time. It can be seen from the survival curves (Figure 4) that group D has a sig- nificant difference (P < 0.05) with other groups in improv- ing the survival time of tumor-bearing mice. In this study, microbubble wrapped HSV-TK plasmid was intravenously injected into mice, followed by ultra- sound irradiation to tumors in order to smash the microbubbles for the targeted release of HSV-TK gene. 48 h after transfection, TK protein expression in HSV- TK+ US+MB+GCV (group D) was significantly higher. The valid expression of TK protein in the target area is the premise for tumor treatment HSV-TK/GCV. From the final treatment effect, the anti-tumor effect of HSV- TK+US+MB group was the highest amony other groups, and the survival time of tumor-bearing mice could be prolonged. Detection of tumor cell apoptosis with TUNEL staining Detection of tumor cell apoptosis with TUNEL staining After the treatment, the tumor tissues were routinely paraffin-embedded and made into 5 μm slices. The sections were dewaxed with xylene followed by gradient alcohol hydration. Add 20 μg/ml free-DNase protease K and keep at 37°C; for 15 minutes. Then wash three times with PBS followed by incubation in 3% hydrogen peroxide (H2O2) at room temperature for 10 minutes. Then wash three times with PBS. Add 10 μl b-11-DUTP and 10 μL TDT to 1 ml Tunel buffer followed by reac- tion at 37°C; for 1 h and at room temperature for 1 h; Streptavidin-HRP (1:400) reaction for 30 min; 0.04% DAB+0.03% H2O2 color development for 10 min; hema- toxylin contrast dye, differentiation with hydrochloric Figure 1 The expression of TK protein was detected by Western-blot 48 h after transfection. Each group has a single band at 25 kDa and the TK protein expression was the highest in the HSV-TK+ US+MB group (A. PBS group; B. HSV-TK; C. HSV-TK+US; D. HSV-TK+US+MB). Figure 1 The expression of TK protein was detected by Western-blot 48 h after transfection. Each group has a single band at 25 kDa and the TK protein expression was the highest in the HSV-TK+ US+MB group (A. PBS group; B. HSV-TK; C. HSV-TK+US; D. HSV-TK+US+MB). Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Page 4 of 6 Table 1 The apoptotic index of tumor tissues in each group (x– s) Group PBS group HSV1-TK group HSV1-TK +US HSV1-TK+US +MB AI(%) 12.1 ± 2.0 16.8 ± 2.3 23.5 ± 3.1# 38 ± 3.6# Compared with control group, #p < 0.05; compared with other groups, p < 0.001. Table 1 The apoptotic index of tumor tissues in each group (x– s) Apoptosis In order to further confirm that microbubble mediated HSV-TK/GCV treatment system can induce apoptosis of tumor cells. We applied TUNEL staining to detect tumor cell apoptosis in each group. When cells under- went apoptosis, DNA double-strand broke and dUTP could be marked at the DNA breakage. As can be seen from each group, the tumor cells in each group appeared apoptosis in different degrees. The tumor cell apoptosis in HSV-TK+US+MBs+ GCV group was the most obvious (Figure 2). Apoptotic index comparison: group D vs group C, P < 0.05; group D vs group A, P < 0.001; group A vs group B, P > 0.05 (Table 1). However, ultrasound-targeted microbubble destruction technology provides a good physical gene transfection method. The ultrasound can be applied to monitor and crush the microbubbles in target tissues at the specific time and space to achieve the accuracy and targeting for gene therapy. The cavitation and mechanical effects gen- erated by ultrasound-targeted microbubble destruction can increase membrane permeability in target areas and widen the gap of vascular endothelial cells, making it easier for foreign gene into the target tissue. Most stu- dies have indicated that under certain ultrasonic irradia- tion conditions, ultrasound did not destroy the transfection gene, but enhanced the transfection efficiency of target genes [20,21]. Discussion Liver cancer gene therapy requires a non-invasive, effi- cient, targeting and safe gene transfection technology. At present, most of the studies in which microbubbles were chosen as gene carriers applied the method of Figure 2 Apoptosis expression in four groups of mice liver cancer tissues (original magification × 400). Terminal deoxyuridine nick end- labeling results showed that cells stained brown in nuclei were apoptotic cells. The tumor cells in two groups appear apoptosis in varying degree. (a. HSV-TK+US group, b. HSV-TK+US+MB). Figure 2 Apoptosis expression in four groups of mice liver cancer tissues (original magification × 400). Terminal deoxyuridine nick end- labeling results showed that cells stained brown in nuclei were apoptotic cells. The tumor cells in two groups appear apoptosis in varying degree. (a. HSV-TK+US group, b. HSV-TK+US+MB). Page 5 of 6 Page 5 of 6 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 Figure 3 It can be seen from the tumor growth curve that the tumor growth in HSV-TK+US+MB group was significantly inhibited. Compared with control group, P < 0.01; compared with HSV-TK+US group, P < 0.05.A. PBS; B. HSV-TK; C. HSV-TK+US; D. HSV-TK+US+ MB). expressions for sustainable long time. The studies from Aoi A et al have shown that in this method target gene will obviously decreased 48 h after transfection, which may be related to the rapid degradation after plasmid DNA transfection [23]. In this study, the method of multiple dosing of HSV-TK gene was applied to over- come the shortcoming that exogenous genes can not constantly express in transient transfection. The method of multiple dosing of target gene also shows a great help for the treatment of tumor. At the same time a lot of studies have shown that microbubble is a safe, reusable carrier which will cause immune response rarely which provides an evidence for multiple dosing of gene in this study [24]. Figure 3 It can be seen from the tumor growth curve that the tumor growth in HSV-TK+US+MB group was significantly inhibited. Compared with control group, *P < 0.01; compared with HSV-TK+US group, P < 0.05.A. PBS; B. HSV-TK; C. HSV-TK+US; D. HSV-TK+US+ MB). HSV-TK suicide gene in this study is a pro-drug enzyme gene. Author details 1Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, Chongqing 400010, PR China. 2Institutional of Ultrasound Imaging, the Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong District, Chongqing 400010, PR China. Acknowledgements Thi h This research was supported by National Natural Scientific Foundation of China (No.3087 2977) and Municipal Health Burean Science Foundation of Chongqing (2008-2-192). Discussion It can transform non-toxic pro-drugs GCV into cytotoxic drugs by phosphorylation to play an anti-tumor effect. The TK gene will cause tumor cell death ultimately with the process of apoptosis [25]. We used TUNEL staining to assess the tumor apoptosis in all groups. Compared with the control group, the tumor cell apoptosis in US+HSV-TK group and HSV-TK+US +MB group was more obvious. The apoptosis index of HSV-TK+US+MB group was the highest in the four groups. This phenomenon illustrates that the microbub- ble wrapped HSV-TK can significantly increase the TK gene transfection under the ultrasonic irradiation and enhance the anti-tumor effects of HSV-TK/GCV system. On the other hand, the bystander effect of HSV-TK/ GCV system is also strong. Those cells which have not been transfected can be supplemented by “bystander effect” to play a good anti-tumor effect [26]. mixture of microbubble and gene for transfection [22]. Using this approach for gene transfection may affect the foreign gene transfection efficiency in the target tissues, making the targeted expression of foreign gene decrease. In this study, the method of preparation of microbubble from Wang et al was selected [19]. Through the princi- ple of electrostatic adsorption, the target genes become a part of the microbubble shells. This will not only increase the amount of gene carried by microbubbles, but also make use of microbubble shells to prevent the foreign gene from being degraded by DNA enzymes in the blood. Thereby target gene expression in the target tissue was increased. Ultrasound-targeted microbubble destruction technol- ogy for gene transfection is a kind of transient transfec- tion. Gene expression time in organizations is relatively short, rather than other virus-mediated foreign gene In conclusion, we used an ultrasound contrast agent as a new type of gene delivery vector, and the anti- tumor efficacy of HSV-TK was markedly improved. Ultrasound- targeted microbubble destruction technol- ogy is expected to become a new gene delivery means and may provide a novel strategy for targeted cancer therapy. Figure 4 The survival time of five tumor-bearing mice in each group is observed for 80 days. It can be seen from the survival curves of tumor-bearing mice that the survival time of tumor- bearing mice in HSV-TK+US+MB group is significantly prolonged. References Proc Natl Acad Sci USA 2005, 102:279-84. 26. Gentry BG, Boucher PD, Shewach DS: Hydroxyurea induces bystander cytotoxicity in cocultures of herpes simplex virus thymidine kinase- expressing and nonexpressing HeLa cells incubated with ganciclovir. CancerRes 2006, 66:3845-51. 26. Gentry BG, Boucher PD, Shewach DS: Hydroxyurea induces bystander cytotoxicity in cocultures of herpes simplex virus thymidine kinase- expressing and nonexpressing HeLa cells incubated with ganciclovir. CancerRes 2006, 66:3845-51. 6. Daigeler A, Chromik AM, Haendschke K, Emmelmann S, Siepmann M, Hensel K, Schmitz G, Klein-Hitpass L, Steinau HU, Lehnhardt M, Hauser J: Synergistic effects of sonoporation and taurolidin/TRAIL on apoptosis in human fibrosarcoma. Ultrasound Med Biol 2010, 36(11):1893-906. 6. Daigeler A, Chromik AM, Haendschke K, Emmelmann S, Siepmann M, Hensel K, Schmitz G, Klein-Hitpass L, Steinau HU, Lehnhardt M, Hauser J: Synergistic effects of sonoporation and taurolidin/TRAIL on apoptosis in human fibrosarcoma. Ultrasound Med Biol 2010, 36(11):1893-906. doi:10.1186/1756-9966-29-170 Cite this article as: Zhou et al.: Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice. Journal of Experimental & Clinical Cancer Research 2010 29:170. doi:10.1186/1756-9966-29-170 Cite this article as: Zhou et al.: Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice. Journal of Experimental & Clinical Cancer Research 2010 29:170. 7. Luo J, Zhou X, Diao L, Wang Z: Experimental research on wild-type p53 plasmid transfected into retinoblastoma cells and tissues using an ultrasound microbubble intensifier. J Int Med Res 2010, 38(3):1005-15. 7. Luo J, Zhou X, Diao L, Wang Z: Experimental research on wild-type p53 plasmid transfected into retinoblastoma cells and tissues using an ultrasound microbubble intensifier. J Int Med Res 2010, 38(3):1005-15. 8. Suzuki J, Ogawa M, Takayama K, Taniyama Y, Morishita R, Hirata Y, Nagai R, Isobe M: ltrasound-microbubble-mediated intercellular adhesion molecule-1 small interfering ribonucleic acid transfection attenuates neointimal formation after arterial injury in mice. J Am Coll Cardiol 2010, 55(9):904-13. 9. Chomas JE, Dayton P, Allen J, Morgan K, Ferrara KW: Mechanisms of contrast agent destruction. IEEE Trans Ultrason Ferroelectr Freq Control 2001, 48:232-48. 10. Zhao YZ, Luo YK, Zhang Y, Mei XG, Tang J: Property and contrast- enhancement effects of lipid ultrasound contrast agent: a preliminary experimental study. Ultrasound Med Biol 2005, 31:537-43. 11. Lanza GM, Abendschein DR, Hall CS, Scott MJ, Scherrer DE, Houseman A, Miller JG, Wickline SA: In vivo molecular imaging of stretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles. J Am Soc Echocardiogr 2000, 13:608-614. Received: 4 November 2010 Accepted: 23 December 2010 Published: 23 December 2010 21. Kodama T, Tan PH, Offiah I, Partridge T, Cook T, George AJ, Blomley MJ: Delivery of oligodeoxynucleotides into human saphenous veins and the adjunct effect of ultrasound and microbubbles. Ultrasound Med Biol 2005, 31:1683-91. Authors’ contributions Figure 4 The survival time of five tumor-bearing mice in each group is observed for 80 days. It can be seen from the survival curves of tumor-bearing mice that the survival time of tumor- bearing mice in HSV-TK+US+MB group is significantly prolonged. SZ, JG, CL participated in the experiments design of the study and coordination. The plasmidpIRES2-EGFP -TK is constructed by SZ and YT. H22 cells and cultivation is finished by SZ. Experimental of mice model finished by SZ and SL. Apoptosis and Western-blot is finished by SZ and ZL. SZ and Page 6 of 6 Page 6 of 6 Zhou et al. Journal of Experimental & Clinical Cancer Research 2010, 29:170 http://www.jeccr.com/content/29/1/170 ZL participated in the performed the statistical analysis. SZ and ZW participated in the preparation of lipid microbubbles. All authors read and approved the final manuscript. 19. Wang ZX, Wang ZG, Ran HT, Ren JL, Zhang Y, Li Q, Zhu YF, Ao M: The treatment of liver fibrosis induced by hepatocyte growth factor-directed, ultrasound-targeted microbubble destruction in rats. Clin Imaging 2009, 33:454-61. References 1. Pisani P, Bray F, Parkin DM: Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int J Cancer 2002, 97(1):72-81. 1. Pisani P, Bray F, Parkin DM: Estimates of the world-wide prevalence of cancer for 25 sites in the adult population. Int J Cancer 2002, 97(1):72-81 22. Endoh M, Koibuchi N, Sato M, Morishita R, Kanzaki T, Murata Y, Kaneda Y: Fetal gene transfer by intrauterine injection with microbubble-enhanced ultrasound. Molecular Therapy 2002, 5(5):501-8. 2. Bosch FX, Ribes J, Díaz M, Cléries R: Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004, 127:S5-16. 2. Bosch FX, Ribes J, Díaz M, Cléries R: Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004, 127:S5-16. y 23. Aoi A, Watanabe Y, Mori S, Takahashi M, Vassaux G, Kodama T: Herpes simplex virus thymidine kinase- mediated suicide gene therapy using nano/microbubbles and ultrasound. Ultrasound Med Biol 2008, 34:425-34. 23. Aoi A, Watanabe Y, Mori S, Takahashi M, Vassaux G, Kodama T: Herpes simplex virus thymidine kinase- mediated suicide gene therapy using nano/microbubbles and ultrasound. Ultrasound Med Biol 2008, 34:425-34. 3. Touchefeu Y, Harrington KJ, Galmiche JP, Vassaux G: Review article: gene therapy, recent developments and future prospects in gastrointestinal oncology. Aliment Pharmacol Ther 2010, 32(8):953-68. 3. Touchefeu Y, Harrington KJ, Galmiche JP, Vassaux G: Review article: gene therapy, recent developments and future prospects in gastrointestinal oncology. Aliment Pharmacol Ther 2010, 32(8):953-68. 24. Pitt WG, Husseini GA, Staples BJ: Ultrasonic drug delivery-A general review. Expert Opin Drug Deliv 2004, 1:37-56. gy 4. Uren AG, Kool J, Berns A, van Lohuizen M: Retroviral insertional mutagenesis: past, present and future. Oncogene 2005, 24:7656-72. 4. Uren AG, Kool J, Berns A, van Lohuizen M: Retroviral insertional mutagenesis: past, present and future. Oncogene 2005, 24:7656-72. 25. Shibata MA, Horiguchi T, Morimoto J, Otsuki Y: Massive apoptotic cell death in chemically induced rat urinary bladder carcinomas following in situ HSVtk electrogene transfer. J Gene Med 2003, 5(3):219-31. 5. Roy I, Ohulchanskyy TY, Bharali DJ, Pudavar HE, Mistretta RA, Kaur N, Prasad PN: Optical tracking of organically modified silica nanoparticles as DNA carriers: a nonviral, nanomedicine approach for gene delivery. Proc Natl Acad Sci USA 2005, 102:279-84. 5. Roy I, Ohulchanskyy TY, Bharali DJ, Pudavar HE, Mistretta RA, Kaur N, Prasad PN: Optical tracking of organically modified silica nanoparticles as DNA carriers: a nonviral, nanomedicine approach for gene delivery. Competing interests 20. Suzuki R, Takizawa T, Negishi Y, Hagisawa K, Tanaka K, Sawamura K, Utoguchi N, Nishioka T, Maruyama K: Gene delivery by combination of novel liposomal bubbles with perfluoropropane and ultrasound. J Control Release 2007, 117:130-136. The authors declare that they have no competing interests. Received: 4 November 2010 Accepted: 23 December 2010 Published: 23 December 2010 References 12. Zhigang W, Zhiyu L, Haitao R, Hong R, Qunxia Z, Ailong H, Qi L, Chunjing Z, Hailin T, Lin G, Mingli P, Shiyu P: Ultrasoun-mediated microbubble destruction enhances VEGF gene delivery to the infarcted myocardium in rats. Clin Imaging 2004, 28:395-398. 13. Zhang Q, Wang Z, Ran H, Fu X, Li X, Zheng Y, Peng M, Chen M, Schutt CE: Enhanced gene delivery into skeletal muscles with ultrasound and microbubble techniques. Acad Radiol 2006, 13:363-7. 14. Furusawa Y, Zhao QL, Hassan MA, Tabuchi Y, Takasaki I, Wada S, Kondo T: Ultrasound -induced apoptosis in the presence of Sonazoid and associated alterations in gene expression levels: A possible therapeutic application. Cancer Lett 2010, 288(1):107-15. 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 15. Feril LB Jr, Kondo T, Zhao QL, Ogawa R, Tachibana K, Kudo N, Fujimoto S, Nakamura S: Enhancement of ultrasound-induced apoptosis and cell lysis by echo-contrast agents. Ultrasound Med Biol 2003, 29:331-7. Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 16. Mesnil M, Yamasaki H: Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gapjunctional intercellular communication. Cancer Res 2000, 60:3989-99. 17. Fillat C, Carrio M, Cascante A, Sangro B: Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclovir system: fifteen years of application. Curr Gene Ther 2003, 3:13-26. 18. Freeman SM, Abboud CN, Whartenby KA, Packman CH, Koeplin DS, Moolten FL, Abraham GN: The “bystander effect": Tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 1993, 53:5274-83.
https://openalex.org/W4388991034	https://tidsskrift.dk/FPPU/article/download/141834/185698	Danish	null	Mellem nærhed og distance i participatorisk praksisforskning	Forskning i pædagogers profession og uddannelse	2,023	cc-by	5,447	Artiklen kan ﬁndes på https://tidsskrift.dk/FPPU DOI: 10.7146/fppu.v7i2.141834 Resume Liesanth Yde Nirmalarajan Ph.d.-studerende ved Institut for Sociologi og Socialt Arbejde, Aalborg Universitet samt Adjunkt ved VIA University College lin@socsci.aau.dk Nøgleord: praksisforskning, reﬂeksivitet, nærhed, distance, autoetnograﬁ Mellem nærhed og distance i participatorisk praksisforskning – metodologiske reﬂeksioner over fælles vidensproduktion med brugere Resume Artiklen diskuterer nærhed og distance i forholdet mellem undersøgeren, og det der undersø- ges. Grundlaget er et empirisk eksempel fra en logbog udarbejdet i et participatorisk praksis- forskningsprojekt. Artiklens relevans udspringer af udviklinger inden for praksisforskning med øget fokus på involveringen af brugere i udsatte positioner. Der kan være store forskelle i sociale baggrunde mellem forskeren og brugerne i udsatte positioner, hvilket nødvendiggør reﬂeksio- ner over dette forhold. Autoetnograﬁ kan understøtte reﬂeksioner over forskellene, ved at vende blikket indad mod forskeren og udad mod det, der undersøges. Ved at eksplicitere klasse- og kul- turbaggrund og institutionelle forhold, som forskeren er indlejret i, opnås større mulighed for at reﬂektere over vidensproduktionens vægtninger, prioriteringer og tilsidesættelser. Indledning g I udviklingen af socialt arbejde og (social)pædagogiske indsatser er der aktuelt stort fokus på, at forskellige aktører bør samarbejde i praksis og i forskningen, hvilket har aktualiseret diskussioner af nærhed og distance i parcipartoriske forskningstilgange (Nielsen & Repstad 2013). Forskeren kan beﬁnde sig i positioner imellem insider eller outsider (Headland et al. 1990) og have indefra eller udefra perspektiver (Cohen 2000). Når betegnelsen ”forskere” anvendes i denne artikel, inkluderer dette også studerende, som foretager undersøgelser. Med inspiration fra Uggerhøj et al. (2018), har centrale elementer i dette participato- riske praksisforskningsprojekt været samarbejde, dialog og reﬂeksion. Faciliteret gennem fremtidsværksteder (Jungk & Müllert 1991) var for- målet om at involvere brugernes perspektiver i dialoger om projektets design, formål, metoder og analyse eks. gennem diskussion af formule- ringen af forskningsspørgsmål og vægtningen af dem. Gennem et uddrag fra min logbog demonstreres det, hvordan mine erfaringer indenfor socialt og pædagogisk arbejde påvirker min måde at betragte brugere i udsatte positioner. Potentialet består i, at autoetno- graﬁ kan hjælpe med at identiﬁcere mine egne nære og distancerede positioner samt perspektiver på det, jeg undersøger. Dette potentiale er ikke kun relevant for mig, men også for andre forsker, der ønsker at benytte autoetnograﬁen som tilgang. Relevansen af dette aspekt styrkes yderligere grundet udviklinger i praksisforskningen, der ifølge Uggerhøj & Wisti (2020) ønsker at involvere brugere mere centralt i forskningsprocessen. For det første er der behov for at reﬂektere over, om forskere ekskluderer disse grupper i forskningsplanlægningen (Rømer & Müller 2022). For det andet kræves reﬂeksioner over forsker- subjektiveringen, fordi der kan være store forskelle i egen baggrund og dem man ønsker at undersøge. Udfordringerne kan opstå i forhold Projektets placering i praksisforskning skyldes en dobbelt interesse for at styrke involvering af familier i udsatte positioner i såvel empirisk arbejde som i forskningsproces. Praksisforskning kan producere viden tæt på praksis og brugernes perspektiver. Hvis forskere ikke formår at reﬂektere tilstrækkeligt over egne positioner og perspektiver, er der risiko for at overtage andre personers klassiﬁceringer eller at klassiﬁcere tilfældigt baseret på subjektive relationer til feltet. Ifølge Bourdieu og Wacquant (1992: 44) kan det ubevidste projicere sig ind i feltet og for- vride resultaterne. Det kan derfor være nødvendigt at deklarere egne positioner og perspektiver. Kvalitative forskere har en afgørende rolle i vidensproduktionen og ansvarlig for denne. Abstract Between closeness and distance in Participatory Practice Research – Methodological reﬂections of shared knowledge production with service users This article discusses closeness and distance within relationships between the researcher and the investigated. Through an example from a logbook, the article highlights the evolving landscape of practice research with increased focus on the involvement of service users in vulnerable posi- tions. Autoethnography supports reﬂections on diﬀerences between the researcher and service users, by directing the gaze towards the researcher and outward towards the subject being investigated. By explicitly addressing how the researcher’s background is embedded in class and culture, and institutional contexts, a greater opportunity emerges to reﬂect on the weighting, prioritization, and omissions in the production of knowledge. Keywords: practice research, reﬂexivity, closeness, distance, autoethnography Artiklen kan ﬁndes på https://tidsskrift.dk/FPPU DOI: 10.7146/fppu.v7i2.141834 LIESANTH YDE NIRMALARAJAN MELLEM NÆRHED OG DISTANCE I PARTICIPATORISK PRAKSISFORSKNING 5 5 Hvis forskere ikke formår at reﬂektere tilstrækkeligt over egne positioner og perspektiver, er der risiko for at overtage andre personers klassiﬁceringer Formålet med denne artikel er at diskutere empiriske eksempler fra forskningens logbog gennem Bourdieus betragtninger om reﬂeksivitet af forholdet mellem undersøgeren og det, der undersøges. I praksis- forskningen diskuteres erkendelsesprocesser med udgangspunkt i behov for, at forskeren og forskningen udsættes for et dobbeltbrud gennem såvel deltagernes sunde fornuft og forskerens position. Bryd- ninger sker ved systematiske undersøgelser af de “utænkelige katego- rier af tanker, der sætter grænserne for det tænkelige og forudbestemte sind” (Bourdieu 1982: 10, i Andersen et al. 2020:65, egen oversættelse). Baseret på tidligere diskussioner af forskersubjektivering, argumenterer jeg for at anvende autoetnograﬁer (Ellis & Bochner 2000) til at reﬂek- tere over disse brydninger gennem systematisk undersøgelse. Auto- etnograﬁen kan understøtte ekspliciteringer af nogle af de tanker og positioner, som forskeren er formet af og samtidigt former. Indledning Øget reﬂeksion og ekspli- citering medfører dobbelte implikationer, der placerer undersøgeren i nære eller distancerede positioner i forhold til det, der undersøges. FORSKNING I PÆDAGOGERS PROFESSION OG UDDANNELSE VOL 7 \| NO 2 \| 2023 \| TEMA: PÆDAGOGIK & FORSKNINGSMETODER 6 tive konsekvenser for dialogen, som dokumenteret i studiet af Høgh et al. (2018). Med dialogerne som centrale for ﬂere valg i projektet blev hensynet til transparensen diskuteret. Supplerende til ovennævnte metoder, valgte jeg fra begyndelsen at udarbejde en logbog med egne noter fra disse dialoger. til, hvordan viden vægtes, prioriteres eller tilsidesættes grundet disse forskelle. Når jeg advokerer for autoetnograﬁens potentiale som et reﬂeksions- værktøj i kvalitativ, participatorisk forskning, skyldes det den aktive betragtning af den situerede viden afhængig af involverede perspekti- ver (Andersen et al. 2020). Efter en præsentation af projektet, redegør jeg empirisk og teoretisk på baggrund af mine nære og distancerede erfaringspositioner med en af mine egne autoetnograﬁer inspireret af Ellis og Bochner (2000). Herefter analyseres de metodiske udfordringer og efterreﬂeksioner over udvalgte implikationer af viden produceret i tæt samarbejde med familier i udsatte positioner og de professionelle omkring dem. Logbogen bidrog til at strukturere mine personlige og professionelle erfaringer og udfordringer med nærhed og distance på tværs af projek- tets empiriske arbejde. Gennem dialog med andre kvalitative forskere stod det klart, at logbogen er forenelig den autoetnograﬁske metode. Logbogen rummer mine reﬂeksioner og identiﬁceringer af egne posi- tioner og perspektiver – såvel i konsensus og konﬂikt med brugernes eller de øvrige deltageres perspektiver. Denne form for autoetnograﬁ kan betragtes som, det Bourdieu har deﬁneret som, en selvanalyse. Her skærpes blikket for, hvorfra man udtaler sig og de asymmetrier, der er en konsekvens af forskellige sociale positioner (Svensson & Brandt 2023:43). Projektets karakter Baggrunden for artiklen består af erfaringer fra mit ph.d.- projekt, der startede i 2022. Projektets fokus er, hvordan familier i udsatte posi- tioner oplever brugerinddragelse med udvalgte digitale teknologier i pædagogisk og socialt arbejde. Det kan fx være ”Digital Post”, chat- tjenester, digitale ansøgnings- eller selvbetjeningsplatforme og andre IT-systemer anvendt til pædagogiske indsatser (fx AULA eller Bosted). Øget digitalisering rejser diskussioner om inddragelsen i skriftlige materialer (fx underretninger, statusbeskrivelser og undersøgelser etc.) og kan udfordre både familier og socialarbejdere (Nirmalarajan & Høybye-Mortensen 2023). Nedenstående tabeller viser en oversigt over projektet og de foreløbige data. Autoetnograﬁ til reﬂeksion af nære og distancerede positioner Ved at integrere autoetnograﬁen i praksisforskning kan nære og distancerede positioner gøres eksplicitte og berige viden indenfor socialt arbejde (Witkin 2022). Nielsen & Repstad (1993:21) frem- hæver, at metodelitteraturen sjældent har fokus på betydning af at forske i eget felt, hvorfor insiderkundskaben kan (for-)blive implicit. Ellis et al. (2011:273) deﬁnerer autoetnograﬁ som en tilgang til forsk- ning og skrivning med sigte mod at beskrive og systematisk analysere (graphy) personlige erfaringer (auto) for at forstå kulturel erfaring (etno) med varierende strukturer. Ellis giver forskere større selvstæn- dighed til at deﬁnere, hvad der undersøges, og hvordan det beskrives, hvorved læsere får indsigt i det naturlige kaos, forfatteren beﬁnder Igennem dialoger blev fordele og ulemper afvejet i forhold til lydopta- gelse af møderne med advisory boardet. Optagelser blev fravalgt som følge af bekymring for at det kunne hæmme og have kontraproduk- Tabel 1: Oversigt over projektet Formål og forskningsspørgsmål At involvere familier i udsatte positioner og undersøge, hvordan de oplever brugerinddragelsen med udvalgte digitale teknologier i socialt arbejde. Deltagere Deltagerne har været unge, forældre, social- og borgerrådgivere, akademiske medarbejdere samt softwareudviklere. Rekruttering Rekrutteringen har primært fundet sted igennem civilsamfundsorganisationer, kommuner og private organisationer. Tabel 2: Oversigt over foreløbig empiri Metode Formål Dokumentstudie (N = 1031) Studie af krydsfeltet mellem oﬀentlige digitaliseringsstrategier og socialretlig lovgivning. Fremtidsværksteder (5, N = 22) At involvere familierne i forskningsprocessen og samtidigt interesse sig for, hvordan de oplever inddragelse i egen sag aktuelt. Interviews (18, N = 22) At involvere familiernes mere speciﬁkke erfaringer. Feltnoter fra dialogmøder med advisory board At strukturere de forskellige inputs fra de involverede stakeholders. Tabel 1: Oversigt over projektet Formål og forskningsspørgsmål At involvere familier i udsatte positioner og undersøge, hvordan de oplever brugerinddragelsen med udvalgte digitale teknologier i socialt arbejde. LIESANTH YDE NIRMALARAJAN MELLEM NÆRHED OG DISTANCE I PARTICIPATORISK PRAKSISFORSKNING Uddra- get sætter krop og ansigt på det oftest kontekstløse forskersubjekt gennem en efterreﬂeksion skrevet på en tankstation på hjemturen fra København til Aarhus – kort efter et fremtidsværksted med forældre blev afholdt hjemme hos en deltager. En af fædrene fra fremtidsværkstedet fortæller om at føle sig frem- medgjort. Deltagerne genkender følelsen og udtrykker en aversion imod min anvendelse af borgerbegrebet og kan bedre genkende sig som andenrangsborgere. Min tilbageholdenhed med at føle, relaterer sig ofte til denne fremmedgørelse som en minoritet i et majoritetssamfund. Jeg slår tankerne hen på et par feminister, der har været et stillads gennem mit liv. Den ene, Judith Butlerbeskriver at selve livet bliver udelukket, når den ’rigtige’ måde er bestemt på forhånd (2004:225). Den anden, Sarah Ahmed (2012), advokerer for nødvendigheden for fællesskabets bevidsthed, der kan overvinde uligheden i de institutionelle rum. Udfordringen er dog, at disse ’brick walls’ oftest er mest synlige for de marginaliserede og usynlige for majoriteten, der forstår institutionerne som demokratiske. Jeg har gennem en årrække arbejdet ved en kommune, en privat aktør og en interesseorganisation for børns rettigheder. I arbej- det har jeg mødt ﬂere borgere, som har været genstand for mine beslutninger og vurderinger. Jeg er bekendt med sprogbrugen i og på tværs af de oﬀentlige systemer, som typisk omkranser familier. I min undervisning og forskning interesserer jeg mig for brugernes perspektiv. Jeg prøver at systematisere mine tanker om netop denne interesse, men også de strukturelle uretfærdigheder, som relaterer sig til dette. Gennem min uddannelse og akademiske karriere har jeg opnået mulighed for at være forsker. Jeg er taknemmelig – men føler mig som en fragmenteret forsker. I vores kodeks for integritet i forskningen, er en central værdi at være ærlig. Men hvor meget skal og bør man dele med brugere, som har valgt at deltage i forsknings- projektet? Med Butler og Ahmed i tankerne, er det her strukturerne møder aktørerne: Familierne oplever at slå panden imod muren, fordi deres familieliv udfordrer den ’rigtige’ forståelse og konfronterer de for majoriteten usynlige magtmekanismer. At opleve modstand og marginalisering er måske en præmis for de involverede familier. Jeg frygter, at min egen følelse af at være en fragmenteret forsker knytter sig an til disse strukturelle forhold, som gør det vanskeligt at leve et familieliv på tværs af kontekster, klasser og kategorier, hvis man erfaringer fra en marginaliseret position (Logbog, december 2022). Projektets karakter Deltagere Deltagerne har været unge, forældre, social- og borgerrådgivere, akademiske medarbejdere samt softwareudviklere. Rekruttering Rekrutteringen har primært fundet sted igennem civilsamfundsorganisationer, kommuner og private organisationer. Tabel 2: Oversigt over foreløbig empiri Metode Formål Dokumentstudie (N = 1031) Studie af krydsfeltet mellem oﬀentlige digitaliseringsstrategier og socialretlig lovgivning. Fremtidsværksteder (5, N = 22) At involvere familierne i forskningsprocessen og samtidigt interesse sig for, hvordan de oplever inddragelse i egen sag aktuelt. Interviews (18, N = 22) At involvere familiernes mere speciﬁkke erfaringer. Feltnoter fra dialogmøder med advisory board At strukturere de forskellige inputs fra de involverede stakeholders. LIESANTH YDE NIRMALARAJAN MELLEM NÆRHED OG DISTANCE I PARTICIPATORISK PRAKSISFORSKNING 7 sig i. Autoetnograﬁer er ærlige, tankevækkende og følelsesmæssigt intense (implicitte) førstepersonsfortællinger (Bochner & Ellis 2022), hvor forholdet mellem nære og distancerede positioner ekspliciteres. Ifølge Dam et. al (2019:14) kan autoetnograﬁen indeholde potentialer til at reﬂektere over ubevidste erfaringer, tavs viden og kropsliggjorte erfaringer. Autoetnograﬁen kan (formentlig) ikke eksplicitere alt, men styrke forudsætningen for øget bevidsthed om forskellige positioner. ”Forskeren eller undersøgeren vil med andre ord altid være positioneret i et »somewhere«, aldrig i et »nowhere«” (Dam et. al. 2019:23). Herved er det vanskeligt eller umuligt at indtage en neutral position og sætte sig selv uden for ligningen (Cohen 2000: Andersen et al. 2020: Dam et al. 2019). svært ved at føle, hvad jeg føler og mistede næsten kontakten med deltagerne. Samtidig havde jeg netop bedt otte forældre om at for- tælle, hvilke følelser de havde i forbindelse med at møde et system, som har anbragt deres børn uden samtykke. Jeg mærker mest lyst til at undgå spørgsmålet, men synes samtidig diskussionen om ærlighed er vigtig. Jeg reﬂekterer over min historie med forældre, der har ﬂygtninge- baggrund, og mine vanskelige opvækstvilkår formentlig er for meget at fortælle. Jeg er selv lige ved at vænne mig til, at jeg i nogle af livets henseender har brug for det oﬀentliges hjælp. Mens jeg skriver i log- bogen på tankstationen, tikker en besked ind på min digitale post app. Brevet har fokus på en række pligter, men jeg skal selv kontakte kommunen, hvis jeg har brug råd og vejledning. Jeg skal registrere noget digitalt, da nogle af systemerne ikke kan tale sammen. Mine socialrådgivererfaringer kan faktisk bruges i forhold til mig selv. Jeg forskånes formentlig for at møde en tidligere kollega eller stude- rende denne gang med de digitale kommunikationsveje. I følgende uddrag af min logbog beskrives det, hvordan min posi- tion og erfaringer får betydning i min forskning. Uddraget omhandler udfordringer, der kan opstå i den kvalitative forsknings interesse for andres subjektivitet. Forskningsfeltet kan være præget af perspektiver om middel/-overklasse, hvidhed, maskulinitet eller ”able-bodied”, som modvirker bevidsthed om diskrimination og barrierer ved deltagelse i samfundet (Ellis et al. 2011:25). I lighed med Ellis’ fokus (2004), peger uddraget af logbogen på, hvordan magt på forskellige niveauer kon- struerer kategorier og får betydning for det personlige narrativ. LIESANTH YDE NIRMALARAJAN MELLEM NÆRHED OG DISTANCE I PARTICIPATORISK PRAKSISFORSKNING Jeg kommer til at tænke på, at ﬂere deltagere under fremtidsværk- stedets møde reagerede på min motivation og indignation med spørgsmål om, hvorvidt jeg selv har erfaringer med oﬀentlige syste- mer. Allerede da de spurgte, tænkte jeg kortvarigt, at jeg netop lider af en sygdom, hvor undgåelsesadfærd er et symptom. Jeg tænkte derfor, det kunne udgøre en undskyldning for undgåelsen. Jeg har I ovenstående adresseres nære og distancerede positioner, som inter- agerer med hinanden som konsekvens af forskellige sociale positioner. Autoetnograﬁen tjener til at demonstrere min ikke-neutrale position, mit ”somewhere”, som Dam et al. (2019) benævner det, netop er indlej- FORSKNING I PÆDAGOGERS PROFESSION OG UDDANNELSE VOL 7 \| NO 2 \| 2023 \| TEMA: PÆDAGOGIK & FORSKNINGSMETODER FORSKNING I PÆDAGOGERS PROFESSION OG UDDANNELSE VOL 7 \| NO 2 \| 2023 \| TEMA: PÆDAGOGIK & FORSKNINGSMETODER 8 Jeg tog udgangspunkt i en facilitering med de demokratiske værdier, der er gældende i praksisforskningen (Andersen et al. 2020), hvor deltagerne får mulighed for at være subjekter snarere end objekter i processen (Uggerhøj et al. 2018). Mine manglende reﬂeksioner af forskersubjektiveringen medvirkede måske i nogle tilfælde som en katalysator for andetgørelse af brugerne (Julkunen & Rauhala 2013), for diskrimination eller epistemisk uretfærdighed (Burke & Newman 2020; Gillard et al. 2012). ret i bestemte kulturer og verdensbilleder. Mine egne levede erfarin- ger indgår i relationelle dynamikker eller myriader af magt med viden produceret gennem forhandlinger i mødet med deltagerne. Uddraget viser også, hvordan forældrene positionerer mig som forsker. Sådanne reﬂeksioner har ifølge Keyan et al. (2013:582) potentiale til at undersøge systematisk hvordan, forskeren indgår i ligningen, når viden produceres. Dobbelte implikationer af nærhed og distance i forskningen Med afsæt i uddraget analyserer jeg tre udvalgte udfordringer i relation til forskerpositionen, når brugere i udsatte positioner involveres i et participatorisk praksisforskningsprojekt. Som eksempel kunne de involverede brugere erfare manglende tillid til velfærdsstaten eller kommune. Mine tidligere ansættelse i det oﬀent- lige gjorde mig om muligt blind på forholdet, at mange af familierne var genstand for ikke-frivillige undersøgelser og interventioner. Flere brugere fremhævede, at de oftest søgte hjælp andre steder end i kom- munen som en konsekvens af manglende tillid, manglende bureau- kratiske og digitale kompetencer (Nirmalarajan & Høybye-Mortensen 2023). Hjælpen blev søgt i fx civilsamfundsorganisationer, krisecentre eller private (facebook)grupper. 1. Brugerpositioner som ”nobodies” og ”somebodies” . B uge pos t o e so obod es og so ebod es Den første udfordring er den styrkede involvering af brugere i diskus- sionen af praksis og forskning i socialt og pædagogisk arbejde. Projektet har dobbelt fokus på deres involvering i at udvikle viden med brugerne i stedet for at udvikle til dem. At vægte brugerperspektiver med denne dagsorden udfordrer ifølge Beresford & McLaughlin (2020) den kon- ventionelle forskning. Intentionen var at få brugerne til at indtræde fra positioner fra ”nobodies” til ”somebodies” ved at give dem mulighed for at være konsulterende, medforskere og partnere. Jeg anvendte derfor en teoretisk model baseret på Arnsteins deltagelsesstige (1969:217) til at konceptualisere brugerinddragelsen. Igennem dialog og forhandlin- ger med brugerne blev det konkretiseret, hvordan og hvorvidt involve- ringen skulle ﬁnde sted. I en post-reﬂeksiv proces anvendte jeg Bourdieus begreber om socio- og autoanalyse i min logbog til systematisk at undersøge min egen baggrund i forhold til mit genstandsfelt. Dette viste mig, hvordan jeg selv konstruerede forskningsobjekter eller levede videre med antagel- ser allerede deﬁneret i forskning eller politikker som mit dokumentstu- die af krydsfeltet mellem socialt arbejde og digitalisering med udsatte familier viste (Nirmalarajan & Høybye-Mortensen 2023). Jeg stræbte efter øget kritisk reﬂeksion over, hvordan jeg konstruerede forsk- ningsobjekter gennem min egen indlejring i verden, og hvordan dette farvede og forstyrrede mit udsyn. Udfordringen var, at jeg i min tilgang som forsker, havde svært ved at indleve mig i hverdagslivet udenfor akademia. Forudsætningerne for at følge med i, hvordan brugerne tænkte blev derfor genstand for det Bourdieu (1977) kalder primitive eller overintellektualiseret tilgange fx i forbindelse med bidrag i nyheds- breve eller oplæg. Jeg valgte derfor at involvere mine egne personlige erfaringer fra minoritetspositioner for at forebygge sådanne tilgange. I praksisforskningsprojektet er brugernes og socialarbejdernes per- spektiver relevante og nødvendige at undersøge i relation til hinanden (Andersen et al. 2020; Uggerhøj et al. 2018). Facilitering af dialoger og forhandlinger kan udvikle eller påvirke socialt arbejde og pædagogiske interventioner, som brugere i udsatte positioner oplever (Andersen et al. 2020; Beresford & McLaughlin 2020). Når brugernes viden anven- des som ”expert by experience” (McLaughlin, 2009:1111, Beresford & McLaughlin 2020), har det med logbogen været gavnligt at reﬂek- tere over, hvordan samtidige epistemologier er på spil og undersøge, hvordan mine egne positioner er situerede. Tilhørsforhold til sociale strukturer og kategorier (race, klasse, køn etc.) kan synliggøres gennem autoetnograﬁ (Ellis & Bochner; Scott 2022). 2. Forskerpositionen fra ”nowhere” til ”somewhere” 2. Forskerpositionen fra ”nowhere” til ”somewhere” Den anden udfordring handler om valget af participatorisk prak- sisforskning som tilgang med et stort fokus på dialog og reﬂeksion (Andersen et al. 2020). De første fremtidsværksteder blev indledt med min ret formelle forklaring af formålet med forskningsprojektet, men i takt med logbogens reﬂeksioner udviklede sig, begyndte jeg i højere grad at formulere min egen motivation. Forskere med en praksisforsk- ningstilgang kommer ifølge Andersen et al. (2022:17) oftest selv fra socialt arbejde med “commitment” og nærhed til feltet, mens nærhe- den samtidig kritiseres. Min indignation knytter sig til sammenhæn- gen mellem mine personlige møder med ”brick walls” og den ulighed, der eksisterer i de institutionelle rum samt de professionelle erfaringer, Min baggrund som socialrådgiver medførte oftest en distanceret posi- tion i begyndelsen af fremtidsværkstederne. I nogle tilfælde, blev jeg mødt med fjendtlighed, idet jeg blev opfattet som en repræsentant, der i højere grad var på systemets side i modsætning til at være på til brugernes side. Samtidigt havde det en signiﬁkant betydning, at jeg har undervist og arbejdet indenfor socialt arbejde med fokus på bruger- inddragelse. Dette gav en mere nær forskerposition, som deltagerne udviste håb og tiltro til. LIESANTH YDE NIRMALARAJAN MELLEM NÆRHED OG DISTANCE I PARTICIPATORISK PRAKSISFORSKNING 9 Spørgsmålet er derfor ikke, om der er bias, men hvordan man bliver bevidst om dem og takler dette praksisforskningsprojekter vil typisk have ansvaret for at styrke samar- bejde, reﬂeksioner og læring, hvorfor viden om samvirkende positio- ner i forhold til ”somewhere” er vigtig. Disse reﬂeksioner er nødvendige for at modvirke at brugernes viden afvises eller diskrimineres som en konsekvens af deres marginaliserede position (Beresford 2013). der sigter mod at fremme større social retfærdighed. At være situeret med multiple positioner og mangfoldige erfaringer fra og med socialt arbejde kan være nødvendige at anerkende, særligt i tilfælde, hvor der kan være synergier imellem kulturmønstre (Svensson & Brandt 2023). Der kan være fordele med et forsker-, praktiker- og brugerperspektiv grundet adgangen til situeret viden (Andersen et al. 2020). Logbogen har primært handlet om ambivalenser i indbyrdes forhold mellem forskellige positioner samt afsløringen disse. I dansk forskning ﬁndes eksempler på, at autoetnograﬁ og erfaringer fra minoriserede positio- ner integreres i vidensproduktion, og anses som en styrke for forsk- ningsmiljøer. Mine egne erfaringer fra minioriserede positioner kan medvirke til at give privilegeret adgang til, det Haraway deﬁnerer som, situeret viden (Thorsen 2019). Mine egne erfaringer med at støde på “brick walls” (Ahmed 2012) kan derfor også give mig andre forudsæt- ninger for at stille spørgsmål, som jeg ikke nødvendigvis ville have gjort, hvis jeg kun havde betragtet feltet ud fra et majoritetsblik fri af ”brick walls” og forstået institutionerne som demokratiske. Når jeg advokerer for autoetnograﬁ, skyldes det den ekstra dimension i forhold til ”ærlighed”, som beskrives i ”Det danske kodeks for integri- tet i forskningen” (UFM). Dette indeholder imidlertid det ”potentielt selvudleverende element”, der ”fremhæver den sårbarhed [Ellis 2007], som man som forsker kan opleve ved at afsløre sig selv samt ved ikke at have kontrol over, hvordan læseren modtager resultaterne” (Svensson & Brandt 2023:89). 3. Marginaliseret og privilegeret viden Den tredje udfordring handler om involveringen af brugere i margi- naliserede positioner som en del af udviklingen i praksisforskningen, der tidligere primært har orienteret sig mod praktikere og institutio- ner (Uggerhøj & Wisti 2020). I mit projekt anvendes åbne tilgange, da de giver bedre mulighed for at fremkalde ”autentiske beretninger om subjektiv oplevelse” (Miller & Glassner 2004:127, egen oversættelse). Når ambitionen er øget brugerinvolvering, er det nødvendigt, at forsknin- gen ikke overser eller negligerer marginaliserede brugeres perspekti- ver. Forskere ”der ofte tilhører relativt privilegerede grupper, vil have en udfordring med at forstå, hvordan virkeligheden ser ud fra et mindre privilegeret perspektiv” (Hansen & Kusk 2020:28). Denne afstand kan i yderste konsekvens medføre bias eller blinde pletter. Disse forhold kan være forankret på et institutionelt niveau med diskrimination eller undertrykkelse af brugernes perspektiver (Burke & Newman 2020). Selvom nogle af mine personlige erfaringer kan have ligheder med at være i en marginaliseret (og nær) position, er konteksten i forsknings- projektet præget af distance. Dialogerne ved fremtidsværkstederne er mit professionelle arbejde, mens emnet for brugerne handler om deres liv. Jeg har inviteret dem, og har designet og forhandlet deres deltagelse. Dette er resultatet af privilegier forbundet med at være forsker. Min undgåelsesadfærd beskrevet i logbogen demonstrerer, hvordan positionerne integrerer eller fragmenterer sig i min kvalitative forskning – en erkendelse opstået gennem reﬂeksioner i min logbog. Forskere i FORSKNING I PÆDAGOGERS PROFESSION OG UDDANNELSE VOL 7 \| NO 2 \| 2023 \| TEMA: PÆDAGOGIK & FORSKNINGSMETODER FORSKNING I PÆDAGOGERS PROFESSION OG UDDANNELSE VOL 7 \| NO 2 \| 2023 \| TEMA: PÆDAGOGIK & FORSKNINGSMETODER 10 Spørgsmålet er derfor ikke, om der er bias, men hvordan man bliver bevidst om dem og takler dette. reﬂeksionerne over forskerens egen forskersubjektivering styrkes, forsvinder udfordringer med at involvere brugere i udsatte positio- ner ikke nødvendigvis. Myriader af magtforhold vil fortsætte med at påvirke studie- og forskningsprojekter, når viden udvælges og vægtes (Keyan et al. 2016:576: Ahmed 2012). Her er det relevant at fokusere på hvilke perspektiver, der inkluderes eller ekskluderes, da der er risiko for at overse eller negligere brugerperspektiver grundet deres udsatte position (Hansen & Kusk 2020). Her kan Butlers betragtninger (2004) hjælpe med at bryde traditionelle forståelser af det ”rigtige”, der typisk er formet gennem sociale, kulturelle og politiske forhold. 3. Marginaliseret og privilegeret viden Autoetno- graﬁen som metodisk tilgang kan potentielt styrke social retfærdighed ved at adressere og konfrontere ulige magtforhold, som en konse- kvens af marginalisering, normativ kulturel bevidsthed og repræsen- tation (Scott 2022). Samlet kan disse reﬂeksioner styrke forskningens forudsætninger til ligeværdig og retfærdig inklusion af brugernes mangfoldige oplevelsesverdener (Duﬀy & Beresford 2020) og integritet (McLaughin 2010). Mødet mellem forskere og deltagere i participatorisk praksisforskning bevæger sig mellem at være med nærhed og distance alt afhængig af samarbejdskonteksten. Hvor viden produceres, mødes magtpositio- ner og epistemologier. Autoetnograﬁ kan være nyttig til at undersøge og eksplicitere, hvordan forskere er situerede i disse forhold og i sociale rum (Bourdieu 1977; Svensson & Brandt 2023). Witkin (2022) peger på, autoetnograﬁ kan udgøre et alternativ til kvalitetssikring af den producerede viden. Dette forudsætter dog reﬂeksioner over indefra- og udefra perspektiver (Cohen 2000), så den viden, der genereres til og med brugere, (potentielt) kan kvaliﬁcere det pædagogiske og sociale felt. Afsluttende reﬂeksioner om nærhed og distance Udviklinger i praksisforskningen aktualiserer, hvordan forskere kan tilnærme sig brugeres perspektiver i socialt og pædagogiske arbejde. Autoetnograﬁ tjener til at reﬂektere over det dobbelte brud i forholdet mellem forskeren og det undersøgte, hvormed den kan afsløre mag- tasymmetrier i sociale positioner og tydeliggøre, hvorfra man udtaler sig (Svensson & Brandt 2023). Igennem reﬂeksion over forskersubjek- tivering åbnes for accept og anerkendelse af forskerens subjektivitet, personlige følelser og erfaringer (Ellis et al. 2011:274). Autoetnograﬁen er ikke blot et fokus på “os” som forskere, men en måde at reducere distancen mellem fx subjekter, brugere og andre partnere. Selvom LIESANTH YDE NIRMALARAJAN MELLEM NÆRHED OG DISTANCE I PARTICIPATORISK PRAKSISFORSKNING 11 Ellis, C. (2004): A Methodological Novel about Autoethnography. Alta- mira Press. Ellis, C. (2004): A Methodological Novel about Autoethnography. Alta- mira Press. Litteratur Ahmed, S. (2012). On being included. Racism and Diversity in Institu- tional Life. Duke University Press. Ellis, C. og Bochner, A. P. (2000): Autoethnography, Personal Narra- tive, Reﬂexivity: Researcher as subject.: I Denzin, N. K og Lincoln, Y.S. (Red.), The handbook of Qualitative Research, 2nd. ed. (pp. 733- 769). Thousand Oaks: Sage Publications. Andersen, M. L. L., Mejlvig, K., & Uggerhøj, L. (2022). Praksisforskning og delt ejerskab. I Jensen, C. K. M. & Nielsen, H.S. (red.), Praksisnær forskning i socialt arbejde: Nordiske perspektiver. (pp. 14-40). Fryden- lund Academic. Ellis, C., Adams, T. E., & Bochner, A. P. (2011). Autoethnography: an overview. Historical social research/Historische sozialforschung, s. 273-290. Andersen, M.L., Brandt, L I, Henriksen, K, Mejlvig, K., Nirmalarajan, L., Rømer, M., Uggerhøj, L. & Wisti, P. (2020). Underlying theoreti- cal positions, perceptions, and foundations in practice research. I Joubert, L. & Webber, M. (Eds.), The Routledge Handbook of Social Work Practice Research (pp. 57-68). Routledge Gillard, S., Simons, L., Turner, K., Lucock, M., & Edwards, C. (2012). Patient and public involvement in the coproduction of know- ledge. Qualitative Health Research, 22, s. 1126–1137. doi:10.1177/ 1049732312448541 Arnstein, S. R. (2019). A Ladder of Citizen Participation. Journal of the American Planning Association, 85(1), 24–34. https://doi.org/10.1080 /01944363.2018.1559388 Hansen, R. S., & Kusk, M. L (2020). En empirisk forankret metodologi for studiet af værdier i professionerne. Tidsskrift for professionsstudier, 16(30), 26-37. https://tidsskrift.dk/tipro/article/view/119281 Beresford, P. (2013). From ‘other’ to involved: user involvement in research: an emerging paradigm. Nordic Social Work Research, 3(2), 139–148. https://doi.org/10.1080/2156857x.2013.835138 Headland,T., Pike, K. L., & Harris, M. (1990). Emics and etics: the insider/ outsider debate. Sage. Beresford, P. and McLaughlin, H. (2020). Service user involvement in research: What diﬀerence does it make? I McLaughlin, H., Beresford, P., Cameron, C., Casey, H. & Duﬀy, J. (Eds), The Routledge Handbook of Service User Involvement in Human Services Research and Educa- tion (pp. 515-520). Routledge. Høgh, V., Cummings, E., Frederiksen, K., & Delmar, C. (2018). Balancing between human intimacy and analytical distance: a special task for a clinical nurse investigating their own clinical practice. Nordisk Sygeplejeforskning, 8(4), 276–287. https://doi.org/10.18261/issn.1892- 2686-04-03x Bochner, A. P., & Ellis, C. (2022). Why Autoethnography? Social Work and Social Sciences Review, 23(2), 8–18. https://doi.org/10.1921/ swssr.v23i2.2027 Julkunen, I., & Rauhala, P.-L. (2013). Otherness, social welfare and social work – a Nordic perspective. Nordic Social Work Research, 3(2), 105–119. https://doi.org/10.1080/2156857x.2013.834266 Bourdieu, Pierre (1977): Outline of a theory of practice. Note Jeg er taknemmelig for kommentarer fra Lektor Lene Ingemann Brandt, Lektor Rasmus Sommer Hasen og Ph.d.-studerende Pernille Wisti, der kontinuerligt har bidraget til at kvaliﬁcere og diskutere disse reﬂeksio- ner. LIESANTH YDE NIRMALARAJAN MELLEM NÆRHED OG DISTANCE I PARTICIPATORISK PRAKSISFORSKNING Litteratur Cambridge: Cambridge University Jungk, R., & Müllert, N. R. (1991). Håndbog i fremtidsværksteder. København: Politisk Revy. Bourdieu, P. & Wacquant, L. (1992): An Invitation to Reﬂexive Sociology. Cambridge: Polity Press. McLaughlin. (2009). What’s in a name: “client”, “patient”, “customer”, “consumer”, “expert by experience”, “service user” - what’s next? The British Journal of Social Work, 39(6), 1101–1117. https://doi. org/10.1093/bjsw/bcm155 Burke, B. & Newman, A. (2020). Ethical involvement of service users. I McLaughlin, H., Beresford, P., Cameron, C., Casey, H. & Duﬀy, J., The Routledge Handbook of Service User Involvement in Human Services Research and Education (pp. 54-63). Routledge. McLaughlin. (2010). Keeping service user involvement in research honest. The British Journal of Social Work, 40(5), 1591–1608. https:// doi.org/10.1093/bjsw/bcp064https://doi.org/10.1093/bjsw/bcp064 Butler, J. (2004). Undoing gender. Routledge. Butler, J. (2004). Undoing gender. Routledge. Miller, J., & Glassner, B. (2004). The ‘inside’and the ‘outside’: Finding real- ities in interviews. I Qualitative research. Theory, Method and Pratice (red. Silverman, D.), 2. edition, (pp. 125-139). Sage Publications Cohen, J.H. (2000). Problems in the ﬁeld - Participant observation and the assumption of neutrality. Field Methods, 12(4)/ 316-333 Dam, T., Kvols, A. M., & Brandt, L. I. (2019). Må jeg skrive mig selv ind? Autoetnograﬁ på socialrådgiver-, pædagog- og læreruddannelsen. Frydenlund. Nielsen, J. R., & Repstad, P. (1993). Fra nærhet til distance og tilbake igjen. I Nielsen, J. R. (red.). Anderledes tanker om livet i organisatio- ner. København: Nyt fra samfundsvidenskaberne. Duﬀy, J. & Beresford, P. (2020) ‘Critical issues in the development of service user involvement’, I McLaughlin, H., Beresford, P., Cameron, C., Casey, H. & Duﬀy, J. (eds), The Routledge Handbook of Service User Involvement in Human Services Research and Education, New York, (pp. 9-16) Routledge. Nirmalarajan, L. Y. & Høybye-Mortensen (2023). Hvordan inddrages udsatte familier i den digitale forvaltninger? Uden for nummer: Tids- skrift for forskning og praksis i socialt arbejde, 46. https://socialraad- giverne.dk/publikationer/ 12 FORSKNING I PÆDAGOGERS PROFESSION OG UDDANNELSE VOL 7 \| NO 2 \| 2023 \| TEMA: PÆDAGOGIK & FORSKNINGSMETODER FORSKNING I PÆDAGOGERS PROFESSION OG UDDANNELSE VOL 7 \| NO 2 \| 2023 \| TEMA: PÆDAGOGIK & FORSKNINGSMETODER 12 UFM. (2014). Den danske kodeks for integritet i forskning. https://ufm. dk/publikationer/2015/ﬁler/ﬁle Rømer, M., & Müller, M. (2022). Udsatte stemmer i institutionel forsk- ning på det sociale og socialpædagogiske område - metodiske og etiske reﬂeksioner. Forskning i Pædagogers Profession og Uddan- nelse, 6(1), 7-22. https://doi.org/10.7146/fppu.v6i1.132311 Uggerhøj, L. & Wisti, P. (2020). Social Work Practice Research devel- opments. Litteratur Four statements and ten years later. I L. Joubert & M. Webber (Eds.), The Routledge Handbook of Social Work Practice Research (pp. 32-42), Routledge. Uggerhøj, L. & Wisti, P. (2020). Social Work Practice Research devel- opments. Four statements and ten years later. I L. Joubert & M. Webber (Eds.), The Routledge Handbook of Social Work Practice Research (pp. 32-42), Routledge. Scott, J. B. (2022) Writing ourselves back into the story: Using autoeth- nography to advance social justice. In Johnson, C. W. and Parry, D. C. (Eds.) Fostering Social Justice Through Qualitative Inquiry: A meth- odological guide (pp. 144-159). Routledge. Uggerhøj, L., Henriksen, K. & Andersen, M. L. (2018) Participatory Prac- tice Research and Action Research: Birds of a feather?, China Journal of Social Work, 11:2, 186-201 Uggerhøj, L., Henriksen, K. & Andersen, M. L. (2018) Participatory Prac- tice Research and Action Research: Birds of a feather?, China Journal of Social Work, 11:2, 186-201 Svensson, C. F., & Brandt, L. I. (2023). Introduktion til autoetnograﬁ. Syddansk Universitetsforlag Witkin, S. (2022). Autoethnography and social work: Strange bedfel- lows or complementary partners? Social Work and Social Sciences Review, 23(2), 19–35. https://doi.org/10.1921/swssr.v23i2.2030 Witkin, S. (2022). Autoethnography and social work: Strange bedfel- lows or complementary partners? Social Work and Social Sciences Review, 23(2), 19–35. https://doi.org/10.1921/swssr.v23i2.2030 Thorsen, T. S. S. (2019). Minoritetsbeskatning - et værktøj til at forstå opretholdelse af strukturelle uligheder i dansk akademia. Kvinder, Køn & Forskning, 28(1-2), 31–43.
https://openalex.org/W2887576581	https://discovery.ucl.ac.uk/10055187/7/Beck%20VoR%20s41431-018-0219-y.pdf	English	null	Registered access: authorizing data access	European journal of human genetics	2,018	cc-by	8,645	ARTICLE ARTICLE Abstract The Global Alliance for Genomics and Health (GA4GH) proposes a data access policy model—“registered access”—to increase and improve access to data requiring an agreement to basic terms and conditions, such as the use of DNA sequence and health data in research. A registered access policy would enable a range of categories of users to gain access, starting with researchers and clinical care professionals. It would also facilitate general use and reuse of data but within the bounds of consent restrictions and other ethical obligations. In piloting registered access with the Scientiﬁc Demonstration data sharing projects of GA4GH, we provide additional ethics, policy and technical guidance to facilitate the implementation of this access model in an international setting. Introduction advantage of the richer access-control policies current technology is capable of enforcing. Access-control policies, and the technology that enforces them, must enable rapid and efﬁcient access to data that is shared only for speciﬁc purposes to a wide range of users while effectively mana- ging ethical and legal risks. As data sharing policies in genomics strive to keep pace with the state of data-intensive science [1, 2], current poli- cies offer little choice for sharing genomic research data beyond the two established mechanisms of open access, when data are freely published on the World Wide Web, and controlled access (also called managed or restricted access), whereby qualiﬁed researchers apply for access on a project-by-project basis and their research plans are reviewed, often by a committee [3–5]. Both open and controlled access policy models have historically served the research community’s needs, scientiﬁc progress and clinical care. However, plans for greater integration of datasets and informatics platforms [6], along with ever greater sharing of health-related datasets and growing interest by clinicians and patients in also accessing genomic data, call for new streamlined models of data access that take greater Registered access: authorizing data access Stephanie O. M. Dyke1,35 ●Mikael Linden 2,3 ●Ilkka Lappalainen2,3,4 ●Jordi Rambla De Argila5,6 ●Knox Carey ● David Lloyd4,7 ●J. Dylan Spalding4 ●Moran N. Cabili8 ●Giselle Kerry4 ●Julia Foreman9 ●Tim Cutts9 ● Mahsa Shabani10 ●Laura L. Rodriguez11 ●Maximilian Haeussler12 ●Brian Walsh13 ●Xiaoqian Jiang14 ● Shuang Wang14 ●Daniel Perrett9 ●Tiffany Boughtwood15 ●Andreas Matern16 ●Anthony J. Brookes 17 ● Miro Cupak18 ●Marc Fiume18 ●Ravi Pandya19 ●Ilia Tulchinsky20 ●Serena Scollen3 ●Juha Törnroos2 ●Samir Das21 ● Alan C. Evans21 ●Bradley A. Malin22 ●Stephan Beck23 ●Steven E. Brenner24 ●Tommi Nyrönen 2,25 ● Niklas Blomberg 3 ●Helen V. Firth9 ●Matthew Hurles9 ●Anthony A. Philippakis8 ●Gunnar Rätsch26 ● Michael Brudno27,28 ●Kym M. Boycott29 ●Heidi L. Rehm 8,30 ●Michael Baudis 31 ●Stephen T. Sherry32 ● Kazuto Kato33 ●Bartha M. Knoppers1 ●Dixie Baker 34 ●Paul Flicek 4 Received: 26 February 2018 / Revised: 8 May 2018 / Accepted: 20 June 2018 / Published online: 2 August 2018 © The Author(s) 2018. This article is published with open access Received: 26 February 2018 / Revised: 8 May 2018 / Accepted: 20 June 2018 / Published online: 2 August 2018 © The Author(s) 2018. This article is published with open access European Journal of Human Genetics (2018) 26:1721–1731 https://doi.org/10.1038/s41431-018-0219-y European Journal of Human Genetics (2018) 26:1721–1731 https://doi.org/10.1038/s41431-018-0219-y European Journal of Human Genetics (2018) 26:1721–1731 https://doi.org/10.1038/s41431-018-0219-y * Stephanie O. M. Dyke stephanie.dyke@mcgill.ca Extended author information available on the last page of the article * Stephanie O. M. Dyke stephanie.dyke@mcgill.ca The registered access policy model Our proposals arise from discussions with a range of stake- holders engaging in international data sharing initiatives as members of the Global Alliance for Genomics and Health (GA4GH) [7]. GA4GH is an international coalition dedicated to improving human health by maximizing the potential of genomic medicine through effective and responsible data sharing, as founded on the Framework for Responsible Sharing of Genomic and Health-Related Data [8]. Our work has led us to conclude that there are speciﬁc datasets where existing consent agreements and ethical approval are com- patible with a novel data access policy model called regis- tered access [9]. This model would capitalize on the well- established role-based access control (RBAC) model for Extended author information available on the last page of the article S. O. M. Dyke et al. 1722 information technology security enforcement [10–14] and is based on the notion that potential users could be granted online access to data according to their roles (e.g., bona ﬁde researcher or clinical care professional) and risk analysis, rather than on the basis of a speciﬁcally described project as is normally required in the controlled access models commonly implemented for research purposes. RBAC is widely imple- mented in government and industry throughout the world [15]. By capitalizing on RBAC-based access-control tech- nologies, registered access could, in theory, provide access to all data shared in this way, following a uniﬁed general registration process and without the need for individualized data access committee review. searches, and mention on institutional websites. Users without a scientiﬁc track record (e.g., trainees) can be validated by a more senior colleague. Data entered into PhenomeCentral can then be shared either with chosen researchers or with pre-deﬁned groups (consortia) who have leads responsible for approving membership. In addition to these intra-consortia, coordinator-approved registration policies, several other current projects are pro- viding, or plan to provide, registration-based access to the research community beyond their projects (see Box 1). These resources either grant an account following a review of an applicant’s credentials (based on submitted or public information) or following a simple registration of their identity. All involve online agreement to data use terms and conditions. The registered access policy model CAGI, the Critical Assessment of Genome Interpretation, active since 2010, has several tiers of access according to the sensitivity of datasets [22], which are available to registered users ranging from unafﬁliated researchers to trainees entering the ﬁeld and individuals at companies to well-identiﬁed accomplished researchers. Vouching (e.g., of a mentor for a student) can also allow appropriate escalation of access. Examples of registered access Registration as a means to limit access to data to approved users—albeit with different approval processes—has already been used in several genomics projects. For example, the Wellcome Trust Case-Control Consortium required regis- tration for access to summary allele frequency datasets once it was demonstrated that these data could potentially lead to the re-identiﬁcation of study participants [16, 17]. While this risk was considered to be low, limiting access to consortium researchers seemed to be a reasonable mitigation strategy at the time and was judged by the Consortium Data Access Committee to be consistent with the participant consent agreements. More recently, the “Bravo” project requires a simple form of registration via logging in to access data. Recent policy recommendations based on risk assessment for such data aim to discriminate between a lower and higher risk of potential resulting harm in the case of re-identiﬁca- tion, for example, limiting access to aggregate data accord- ing to whether data were associated with more sensitive health or demographic information (e.g., ethnicity informa- tion about small or vulnerable populations) [18, 19]. Implementation in GA4GH Registered access: authorizing data access 1723 Box 1 Examples of current projects enabling registration-based access Resource Access requirements Critical Assessment of Genome Interpretation (CAGI) https://genomeinterpretation.org • Review of users • Digital signing of data use agreement Simons Foundation Autism Research Initiative (SFARI) https://www.nextcode.com/ssc/ • Review of users • Online agreement to data use conditions mPower Public Researcher Portal [39] http://sagebase.org/research-projects/mpower-researcher-portal/ • Veriﬁcation of user identity and training • Online agreement to data use conditions AACR Project GENIE https://www.synapse.org/#!Synapse:syn7222066/wiki/410922 • Veriﬁcation of user identity and training • Online agreement to data use conditions Bravo (http://bravo.sph.umich.edu) • Login with ID provider (Google ID) linked to work email address • Online agreement to data use conditions Box 1 Examples of current projects enabling registration-based access For the Beacon Project, which enables the discovery of genetic variants across multiple world-wide datasets, registered access is envisaged as a means to share more details than simple existence of genetic variants (e.g., that they are present in individuals with a speciﬁc health con- dition). In conjunction with Beacon partners ELIXIR (Europe’s infrastructure for life science information) and NCBI (the US National Center for Biotechnology Infor- mation), registered access is being developed for access to appropriate metadata from controlled access datasets. Such metadata access is similar to current access protocols at dbGaP [25] and the European Genome-phenome Archive (EGA) [26]. Speciﬁcally, users with an eRA account (for the NIH Commons research grant system) are dbGaP registered users with access to some information about available controlled access datasets [24]. Similarly, EGA users who have obtained access to at least one EGA con- trolled access dataset have access to speciﬁc EGA infor- mation about available controlled access datasets after logging in with their EGA account. of a matched case, including variants in a speciﬁc gene and high-level phenotypic information for their purposes as a scientiﬁc investigator working to understand the causes of rare diseases. The goal of the BRCA Challenge is to translate the rapid expansion of sequencing capacity into useful knowledge and, in particular, learn how to rapidly interpret variant data to generate clinical utility. Its intent is to provide an umbrella under which many groups can collaborate and bring together data to improve the precision of asses- sing variants across both BRCA1 and BRCA2. Implementation in GA4GH While its main resource on BRCA variant interpretation is publicly available, overlaying registered access would allow enrichment of the dataset with data that cannot be shared openly: for example, patient data supporting clinical inter- pretations of variants may not be consented for open release but would be available to expert review teams, researchers, or clinicians. To support these pilot implementations of registered access in GA4GH, we expand on our initial ethical–legal feasibility study and review of projects that are pioneering registration-based access policy (see Box 1) to describe plans for an international, uniﬁed approach that could lead to a standardized registration process allowing for access to a wide range of data resources. All three stages of registered access (authentication, attestation, and authorization) pose signiﬁcant ethical–legal and technical challenges, which we attempt to address by providing pol- icy and technical guidance. The MME is a federated network connecting databases of genomic and phenotypic data using a common appli- cation programming interface to facilitate rare disease gene discovery, including from DECIPHER (open sub- set), the PhenomeCentral platform [21], GeneMatcher [27], MyGene2 [28], Patient Archive (patientarchive. org), and matchbox. In its current iteration, it requires two-sided inquiry (i.e., a search from two parties with a similar patient) and, in this way, connects two investiga- tors looking for a match for the same candidate gene and disease. Each user must be registered in one of the data- bases in order for data to be deposited and queries made. Future iterations of MME will expand functionality and facilitate a one-sided inquiry, with bona ﬁde investigators identiﬁed by a registered access process able to see details Implementation in GA4GH Our model of registered access in the GA4GH context comprises a three-stage “Triple-A registration” process (Authentication, Attestation, and Authorization [9]), which aims to ensure both user identiﬁcation and agreement to a standard set of general responsibilities while considerably simplifying the data access application process. Through the identiﬁcation and authentication process, the individual provides “proof” that an asserted identity is their own. The attestation process establishes that the potential data user meets the requirements expected by the consent agreements and ethical approval of datasets in question and includes agreement to comply with the terms of data use required of registered users. Finally, authorization is the overall process by which users are granted access to data and permission to perform speciﬁc actions. We provide concrete examples of, and guidance for, each stage in the process based on three GA4GH Demonstration Projects with which we ﬂeshed out standards that would be broadly applicable. Another current example of a registration-based data access policy is the DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources (DECI- PHER [20]). Users who have been approved by the project coordinator (a senior physician working at the center depositing the data) are granted registered access to that project data. DECIPHER projects can be linked to form a consortium, allowing intra-consortium sharing. Phenome- Central is another example of a registered access policy for the identiﬁcation of additional cases for ultra-rare disorders [21]. Along with DECIPHER, PhenomeCentral is part of the GA4GH Matchmaker Exchange (MME) initiative. PhenomeCentral users are required to be bona ﬁde researchers or clinicians. This is validated through institu- tional email addresses, as well as through user-provided and publicly available information such as prior publications, scientiﬁc activity at conferences identiﬁed through web The Beacon Project (manuscript in press), the Match- maker Exchange [23], and the BRCA Challenge (manu- script submitted) are among the initial demonstration projects that aimed to drive learning, identify requirements, assess value, and coordinate activity within the ﬁrst phase of GA4GH. For each of these, we explored options for using registered access to improve and streamline access to data that had previously been available either through a controlled access application process and/or bound by protocol-speciﬁc restrictions. Authentication A potential advantage of the registered access policy model is to efﬁciently provide data access to a relatively large 1724 S. O. M. Dyke et al. Box 2 The “layered” registration system. Shows the main routes to user authentication for the categories of bona ﬁde researcher and clinical care professional A person may receive bona ﬁde researcher status if: 1. Their home institution conﬁrms they are researchers, OR 2. A person who satisﬁes condition (1) corroborates (“vouches for”) their researcher status (as a reference) A person may receive clinical care professional status if: 1. Their home institution conﬁrms they are clinical care professionals, OR 2. They have a physician or other clinical care professional license (ID/permit number) Box 2 The “layered” registration system. Shows the main routes to user authentication for the categories of bona ﬁde researcher and clinical care professional A person may receive bona ﬁde researcher status if: 1. Their home institution conﬁrms they are researchers, OR 2. A person who satisﬁes condition (1) corroborates (“vouches for”) their researcher status (as a reference) A person may receive clinical care professional status if: 1. Their home institution conﬁrms they are clinical care professionals, OR 2. They have a physician or other clinical care professional license (ID/permit number) number of authorised individuals and alleviate the con- siderable administrative burden on data custodians of managing controlled access requests. This model is pre- mised on the trust that broad categories of registered users, such as researchers and clinical care professionals, will use the data accessed with the same appropriate care as they would manage controlled access data. Deﬁning categories of users as bona ﬁde researchers or clinical care professionals in this context rests largely on the information provided at the time of registration (user attri- butes) and the attestation they agree to. The attributes requested from users for the registration process, and particularly their veriﬁcation, will have important implica- tions for access to data protected by registered access authorization methods. organization, they are an important means of strengthening accountability and traceability of registered users and can be simply veriﬁed by web searches or calls to institutional switchboards. Authentication We considered additional information that could demonstrate a research user’s professional status such as: researcher identity systems (e.g., ORCID or ISNI); PubMed publication IDs; and researcher accounts such as those with funding agencies (e.g., NIH Commons’ eRA), universities (email addresses or user accounts), and the major public archives (e.g., MyNCBI and PubMed Commons). Evidence of academic publication (in the context of a research position) is typically relied upon in the controlled access application process as an indication of researchers’ ability to use data [29]. However, concern was expressed regarding the value of journal publications and some researcher IDs as an indicator of professional activity, especially current activity. There was also concern about the rise in so-called “predatory” academic journals, leading to publications of dubious quality [30]. Based on an ethical–legal analysis of research ethics and other legal and administrative frameworks applicable to data sharing and access, it was previously proposed that several elements of controlled access review should be retained in registered access, including for how users might be authorized based on their “competence.” We considered whether it might be necessary to set a few differing levels of stringency for the registered access model (e.g., Registered, Registered+) to cater to different projects’ views of the requisite access and data sensitivity. However, we agreed that a minimal standard (basic registration criteria) could be established, thereby enabling mutual recognition between registration systems established in different parts of the world (e.g., ELIXIR and NCBI). This does not preclude policies that provide different levels of access to data to different categories of users. Indeed, such policies are enforceable using a combination of RBAC and attribute-based access control. We eventually decided on a “layered” registration system whereby bona ﬁde researchers or clinical care professionals could either demonstrate their status directly (by providing evidence of professional status, such as license numbers for clinical care professionals) or alter- natively have their status “vouched for” by another registered user within their category (for researchers) or their employing institution (for researchers and clinical care professionals) (see Box 2). One use case for such a voucher approach would be for students or trainees who may have neither professional appointment nor publica- tions, where the expectation would be for an advisor to support the registration. Responsibilities of institutions To qualify as either a bona ﬁde researcher or clinical care professional, ﬁrst of all, individuals will need to provide the following details of their identity and research/clinical activity: name; title; position; afﬁliation; and institutional email address, phone number, website, and mailing address. As these details may also be provided by an individual’s Accountability of registered users is central to the registered access model. Within data access policy models, various approaches have been proposed to hold users accountable. One is co-signing of a data access agreement by the (home) 1725 Registered access: authorizing data access institution of the users and recognizing this institution as the ultimate responsible entity. Within this perspective, that institution can be legally held accountable if the researcher or clinician commits any wrongdoing. Although registered access does not require signing such an agreement between the home institution and the data custodians, one could argue that, if any wrongdoing happens, the users’ institution will in all likelihood be contacted and asked to enforce administrative disciplinary measures in an analogous way as is currently done in some cases of scientiﬁc misconduct, such as plagiarism or publishing falsiﬁed data. In turn, in addition to the attestation registered users will have agreed to in registering for access to data, home institutions may require researchers and clinicians—who plan to use internal or external health data—to sign up to procedures and gui- dance documents such as a “Code of Conduct”, in order to bind them with the institutional rules and sanctions in this respect. registered user’s license number. As examples, in the UK, users could provide their General Medical Council licence number; in Germany, their Lebenslange Arztnummer; in France, their numéro RPPS (répertoire partagé des pro- fessionnels de santé); in Australia, their Australian Health Practitioner Regulation Agency registration number; and in Canada, their Royal College of Physicians and Surgeons identiﬁcation number. Registration for clinical care profes- sionals will in most cases be linked to professional over- sight and disciplinary governance frameworks. In case these routes did not allow registration of atypical potential users, as an additional route to registration, any individual would also be able to apply to a standard Data Access Committee (DAC), the committees that oversee access to controlled access data, to be assessed on a case- by-case basis for registered access status. Vouching For the second route to registration for bona ﬁde researchers, a person who has already been registered via their institution could corroborate another researcher’s status, as a reference. The vouching researcher would need to conﬁrm that they know and have identiﬁed the researcher they are registering. To promote accountability and community control, regis- tered users would be able to see who has vouched for whom. This is akin to having a witness to one’s competence and professional activity. The issue here is one of validation based solely on a personal statement and of potential liability for the researcher registering this way as they may not have institutional backup. “Vouchers” could also potentially be held liable. It is worth noting that a large-scale, successful community, the Debian community, maintains operating system software using a vouching approach based on Pretty Good Privacy (PGP) key signing. A member of the com- munity must have their PGP public key signed by at least one existing member of the community before their key can be admitted into the Debian keyring (which then enables them to modify and upload software, participate in elections, etc.). There are strict guidelines on the level of proof required for signature—meeting in person, both parties show government photo ID, etc. There are also other similar prerequisites, such as accepting the social contract and advocation by another member, and violations result in removal of access by the community. Responsibilities of institutions DACs may also help register users whose organizations have yet to establish the organizational or technical protocols to facilitate regis- tration (see discussion under “Accessibility” below). clinical care professional. The seven statements shown in quotation marks form the attestation stage of the process Fig. 1 The registered access policy model. The ﬁgure shows the authentication and attestation requirements of the GA4GH registered access policy model for the user categories of bona ﬁde researcher and clinical care professional. The seven statements shown in quotation marks form the attestation stage of the process clinical care professional. The seven statements shown in quotation marks form the attestation stage of the process registered users’ commitments to using data for appropriate research or clinical care purposes and to further the aims of public transparency. Another interesting suggestion regarding transparency was to request and publish links to public researcher proﬁles for all registered researchers. would allow for additional attestation statements attaching extra conditions of use for some datasets or for data from some providers. For example, Australian Genomics is considering the model that researchers need to supply proof of Human Research Ethics Committee (HREC) approval (HREC number, title, etc.), which can then be easily veriﬁed by web search. Attestation The seven statements shown in quotation marks form the attestation stage of the process Attestation Integral to the deﬁnitions of bona ﬁde researcher and clin- ical care professional are the statements and agreements included in the attestation stage of the registration process (see Fig. 1). Indeed, controlling the purpose of data use is a key component of data protection principles and the European Union (EU) General Data Protection Regulation (GDPR) [31]. One of the attestation statements refers to respecting consent-based data use permissions and restrictions, which should ideally be expressed as Consent Codes [32]. The GA4GH Consent Codes are a structured way of recording consent permissions so they can be made clear to users and to enable maximum data aggregation (with the same or broader permissions). Another attestation statement prohi- bits any attempt to identify individuals based on combining shared data with other public or non-public data sources. It allows for exceptions to this condition in some circum- stances, with “prior written permission of the provider’s sponsoring institution.” This is to enable the recontact of participants if warranted (e.g., for the return of individual research results) or for permission to conduct research into privacy risks. We plan to provide general guidance for the attestation statement about keeping data secure, such as that the data should be kept encrypted at rest and in transit between systems, and that only authorized individuals have access to the keys (http://genomicsandhealth.org/work- products-demonstration-projects/security-infrastructure). By providing several routes to registration, we hope to enable access to as wide a group of potential data users as possible while maintaining a strong level of accountability. For clinical care professionals in the USA, the National Provider Identiﬁer issued by the Centers for Medicare and Medicaid Services could be requested in addition to the We also plan to include an educational module as part of the registration process. Ultimately, we aim to enable a single format for the registration process but support a model that S. O. M. Dyke et al. 1726 Fig. 1 The registered access policy model. The ﬁgure shows the authentication and attestation requirements of the GA4GH registered access policy model for the user categories of bona ﬁde researcher and clinical care professional. The seven statements shown in quotation marks form the attestation stage of the process Fig. 1 The registered access policy model. The ﬁgure shows the authentication and attestation requirements of the GA4GH registered access policy model for the user categories of bona ﬁde researcher and clinical care professional. Authorization In agreeing on these deﬁnitions of bona ﬁde researcher/ clinical care professional and level of security, cross- federation becomes possible (e.g., to enable European bona ﬁde researchers to present queries to US Beacons at the registered access level and vice versa). The current attri- butes chosen to deﬁne registered users in these categories are designed to cover most of the use cases. When excep- tions arise, there would have to be a very strong need for the new deﬁnition to make everyone deploy it (and populate values to the existing users retrospectively). Importantly, such exceptions would only be considered valid if driven by informed consent requirements or national laws. In agreeing on the proposed routes to registration, we have effectively delegated the authorization of registered users for the two categories described here to established pro- fessional employment, accreditation, or accomplishment. The data sharing environment is therefore assumed from individuals’ bona ﬁdes (including work practices and the security aspect) along with the basic set of requirements set out in the registration attestation. Along with efforts to automate registered access, this potentially limits the amount of manual authorization that will be required. Our pilot implementation of the ﬁrst registration route for academic researchers (their home institution conﬁrms they are researchers) is the simplest in terms of liability for the category of “bona ﬁde researchers,” and therefore the “safest” place to start. ELIXIR is piloting an approach Although our model does not include a review and approval of the user’s speciﬁc research or data use plans, we considered requesting abstracts of general planned data use in lay terms that would be published to enhance trans- parency. This may be reconsidered, especially to reinforce Registered access: authorizing data access 1727 NCBI in the US could deploy the technology needed to operate as an OpenID Provider for their constituencies. where an ELIXIR user authenticates their identity through their own research organization’s account, and the organi- zation conﬁrms researcher's status. Organizational valida- tion is assumed to improve the provenance of the researcher’s professional status because home organizations are vetted by funders, are expected to know their researchers, and can also provide the authentication credentials securely to their researchers. A challenge will be to deﬁne the requirements an organization needs to meet to become trusted in a global GA4GH registered access system (e.g., for federated identity in research, the UK has minimal checks https://www.ukfederation.org.uk/ content/Documents/EligibleOrganisations). Authorization Registered access relying parties are the entities that consume OpenID authorizations and enforce access rights and privileges based on the registered access status and attributes of the users. To be able to use registered access claims, a relying party needs to trust one or several OpenID Providers. In order to establish a federation of OpenID Providers and relying parties, they need to agree on the exact semantics of registered access status and attributes; how credentials are veriﬁed by the OpenID Provider and expressed to the relying party; what technical protocols are used to share between the registry and the relying party; and how to protect the conﬁdentiality, integrity, and availability of the communication. Our experience is that this is often a more controversial and difﬁcult challenge than the veriﬁcation of individuals’ identity and role. For instance, institutions that might oversee clinicians and researchers wanting access to genetic data could include a range of clinical genetics centers (publicly funded/charitable/private); primary care centers, which treat certain inherited conditions and other contexts in which genetic testing may be commissioned or communicated without genetics specialists; and research institutions (university/other public/charitable/ private). The challenge, therefore, may be to establish standards for those entities facilitating registration, including the institutions hosting registered users. A particularly crucial element of the institutional aspect of access control is the identiﬁcation of accounts that no longer meet the access criteria. There needs to be well- deﬁned, well-understood mechanisms for reviewing and revoking status, and registries of users will need to demonstrate that they successfully ensure sponsors do so in a timely manner. Examples of situations which access control workﬂows may need to account for include staff moving from one role to another (which may alter the user’s clinical care professional vs. researcher category) or leaving the profession. User attributes and attestations are provided to relying parties through the standardized OpenID Connect protocol, which is based on OAuth 2.0 [33]. These standards provide a mechanism through which OpenID Providers may authenticate users and provide “claims”—data structures that encode various user attributes—that can be crypto- graphically validated by relying parties and used in med- iating access to data. Once identity has been authenticated and registered access attributes shared, OAuth 2.0 will mediate the requested access based on the data holder’s access policy. Authorization The GA4GH is working to deﬁne a set of custom claims for registered access that all OpenID Providers and relying parties can adopt (Library Cards [34]) providing interoper- ability across the ecosystem of registered access adopters. Strong identity-prooﬁng will be required within a uniﬁed identity framework, especially in the future, for registration that is independent of institutional listing or peer vouching. We plan to use existing guidelines [35] for how to establish and maintain trust in digital identities. These frameworks rank a spectrum of assurance levels, and relying parties can report (in claims) which of these levels was used to perform identity prooﬁng. From a technical point of view, we split the registered access architecture into two components, which can be separated organizationally and geographically: a component that manages the individual’s identity and attributes, and the party that relies upon this component to conﬁrm identity and attributes. The OpenID Connect technical standard (http://openid.net/connect/) refers to these two components as the “OpenID Provider” and the “relying party” respec- tively. There may be several registries and relying parties managed by different organizations in different geo- graphical locations. Researcher attributes and registered access status count as personal, identiﬁable information, which is protected by privacy laws, including the new GDPR in the EU. To protect the privacy of researchers and respect data protec- tion laws, it is proposed that OpenID Providers limit the amount of personal data shared with relying parties. This would mean communicating only a pseudonymous identi- ﬁer of the researcher (i.e., an alphanumeric code, which is needed for thwarting re-identiﬁcation and other attacks on multiple relying parties simultaneously) and their registered access status (which is needed for verifying the requestor’s status), including its route and provenance, i.e., which registry delivered the status. Consent is one of the six lawful bases to process personal information in the GDPR [36]. Article 4(11) deﬁnes consent as: “any freely given, speciﬁc, The OpenID Provider is responsible for authenticating a registered user’s identity and for sharing attributes that the relying party may use to authorize access (see Box 2 and Fig. 1). Given OpenID Connect’s broad use worldwide, we suppose that organizations such as ELIXIR in Europe or S. O. M. Dyke et al. 1728 technology and that are bridged with a system called edu- GAIN (https://www.edugain.org; a sister service of eduroam, https://www.eduroam.org/). Authorization A beneﬁt of using an identity federation for registered access is that the researcher status is not self-asserted by the researcher but instead claimed by the research institution employing the researcher. The home institution is also able to provide more ﬁne-grained information on the person’s afﬁliation (http://software.internet2.edu/eduperson/internet2-mace-dir- eduperson-201602.html#eduPersonAfﬁliation) than a sim- ple institutional e-mail address check, which often does not differentiate between researchers, students, and administrative staff. Additional details that could support registered access through federated identity management would be the categorization of bio/health researchers or even “registered following GA4GH standards.” A challenge of identity federation is that currently there is no widely deployed framework for the level of assurance of the identity and authentication of users. Data protection laws also make some institutions hesitate to release researchers’ personal data to other jurisdictions. Collaborations such as the Federated Identity Management for Research Colla- boration (FIM4R) aim to establish common standards that meet the needs of various research communities [38, 39]. informed and unambiguous indication of the data subject’s wishes by which he or she, by a statement or by a clear afﬁrmative action, signiﬁes agreement to the processing of personal data relating to him or her”. For the registration process, this would entail providing users with a way to consent to the sharing of their personal data for the purposes of gaining registered status, which ELIXIR has integrated into its pilot system. Different datasets, even within an institution, may have different requirements, such as the Consent Codes asso- ciated with data. Such datasets may require additional Attestation statements, beyond those recommended by GA4GH, for access (see Fig. 1); a data steward [37] (or the data custodian or guardian as referred to in different locations) must specify and enable such Attestations, and they will usually be guided by research ethics committees and institutional review boards in these responsibilities. While access conditions must reﬂect the use permissions of the dataset, additional Attestations/restrictions may complicate or prevent the aggregation of data from many sources. Accessibility In the interests of efﬁciency and alleviating administrative burden on data custodians—particularly given the number of potential registered users—efforts should be made to automate the registered access process. Additionally, from an information security perspective, self-asserted attributes provide little accountability and raise the possibility of identity theft. We therefore sought to incorporate automated (or delegated, e.g., institutional) checks of user attributes. As our plans for the processing of registered access attri- butes for bona ﬁde researcher registration draw on pre- existing academic infrastructure, we envisage minimal investment from an institutional perspective, reducing bar- riers to adoption of this system. It will be important to install a comparable system for access by researchers in industry. Conclusion While there remain many challenges in implementing registered access, especially at scale and with respect to the legal and administrative tools to facilitate registration through the proposed range of routes, the GA4GH pilots have allowed us to ﬂesh out various aspects and better understand its practical utility. The main goal of registered access is to streamline access to datasets that require acceptance of terms and conditions due to consent agree- ments or because of a level of ethical and legal risk, and to enable access to multiple datasets at once as well as to facilitate data discovery and use. We also envisage that the simplicity, and clarity, of the standard conditions of data access and use in registered access (the attestation) will both encourage greater use of the data and respect for its ethical use, as seen with licensing terms, such as GNU General Public License and Creative Commons. Since 2005, research and education institutions have been operating technical frameworks called identity fed- erations that allow researchers to use their home institu- tion’s credentials (such as user accounts and passwords) to access services that are outside their home institutions. To register their bona ﬁde researcher status and make the related attestations within such federations, a researcher would ﬁrst need to log in at their home institution, which then delivers their fresh and validated role and afﬁliation information to the registration process. Currently, there is some form of national research and education identity federation in 72 countries (https://refeds.org/federations) using many different systems but usually the same The registered access model and services described above must correctly maintain protections that were agreed to by study participants as well as researchers and clinicians who wish to study their data in order to eventually advance biomedical knowledge and beneﬁt society. The registered access policy model will then need to be recognized and supported by many stakeholders, including research ethics boards, such that the language used in consent forms and research agreements are 1729 Registered access: authorizing data access compatible with this access model. This will make a big difference in how “silo-ed” data continue to be. Ultimately, the conﬁdence the research community will gain in the system will determine the extent of the resources it will ultimately provide. References 1. Bobrow M. Funders must encourage scientists to share. Nature. 2015;522:129. We expect registered access will inform and may even replace many controlled access mechanisms as the level of accountability that it can achieve is demonstrated over time. Data Access Committees may come to play new roles, such as deciding which data are suited to registered access, as well as reviewing applications of atypical potential users and handling other aspects of data govern- ance (e.g., data use breaches or retractions). 2. Auffray C, Balling R, Barroso I, Bencze L, Benson M, Bergeron J, et al. Making sense of big data in health research: towards an EU action plan. Genome Med. 2016;8:71. 3. Toronto International Data Release Workshop Authors, Birney E, Hudson TJ, Green ED, Gunter C, Eddy S, et al. Prepublication data sharing. Nature. 2009;461:168–70. 4. Ramos EM, Din-Lovinescu C, Bookman EB, McNeil LJ, Baker CC, Godynskiy G, et al. A mechanism for controlled access to GWAS data: experience of the GAIN Data Access Committee. Am J Human Genet. 2013;92:479–88. We believe that it is ethically desirable to use less restrictive access controls, wherever suitable, to increase the chances of having the best research from the most people using the data that has been contributed. To needlessly reduce appropriate access likely undermines the intentions and desires of research participants as well as hindering the course of research progress. 5. Brenner SE. Be prepared for the big genome leak. Nature. 2013;498:139. 6. Manolio TA, Fowler DM, Starita LM, Haendel MA, MacArthur DG, Biesecker LG, et al. Bedside back to bench: building bridges between basic and clinical genomic research. Cell. 2017;169:6–12. 7. Global Alliance for Genomics and Health. GENOMICS. A fed- erated ecosystem for sharing genomic, clinical data. Science. 2016;352:1278–80. Acknowledgements We thank Dr. David Kelsey (STFC Rutherford Appleton Laboratory) for helpful comments and discussion of regis- tered access. 8. Knoppers BM. Framework for responsible sharing of genomic and health-related data. Hugo J. 2014;8:3. 9. Dyke SO, Kirby E, Shabani M, Thorogood A, Kato K, Knoppers BM. Registered access: a ‘Triple-A’ approach. Eur J Human Genet. 2016;24:1676–80. Funding SOMD is supported by the Canadian Institutes of Health Research (EP1-120608; EP1-120609; CEE-151618), Genome Que- bec, Genome Canada, the Government of Canada, the Ministère de l’Économie, Innovation et Exportation du Québec (Can-SHARE grant 141210), and the Canada Research Chair in Law and Medi- cine. Conﬂict of interest The authors declare that they have no conﬂict of interest. Conﬂict of interest The authors declare that they have no conﬂict of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. Another important aspect of improving data access is the development of ethics tools to support the assessment of data sensitivity and therefore the risk in data sharing to better determine proportionate levels of protection (e.g., open or registered). A coherent approach involves considering both the risk of re-identiﬁcation of data and its sensitivity, along with the data sharing expectations of individuals and communities (Data Sharing Privacy Test [41]). Conclusion Foundation Flanders (FWO); MH by NIH/NHGRI 5U41HG002371- 15; SW by NIH/NHGRI R00HG008175; S Beck by the National Institute for Health Research UCLH Biomedical Research Centre (BRC369/CN/SB/101310); S Brenner by NIH/NHGRI U41 HG007346; BMK by the Canada Research Chair in Law and Medicine; and PF by WT201535/Z/16/Z and the European Mole- cular Biology Laboratory. Finally, while we have focused initially on registration criteria for researchers and clinical care professionals, many of whom have not generally had access through the controlled access system, we anticipate that data users will eventually include members of the public, including patients and citizen scientists (see e.g., mPower [40]), as well as other groups such as volunteer health-care providers and journalists. We plan to consider expanding registered access for these important and diverse groups in the near future, within the permissions of consent, and ethical stan- dards, and with broad consultation with patient advocacy groups and research participants. Funding SOMD is supported by the Canadian Institutes of Health Research (EP1-120608; EP1-120609; CEE-151618), Genome Que- bec, Genome Canada, the Government of Canada, the Ministère de l’Économie, Innovation et Exportation du Québec (Can-SHARE grant 141210), and the Canada Research Chair in Law and Medi- cine. ML, IL, JT, and TN are supported by the ELIXIR, the research infrastructure for life-science data, and the H2020 ELIXIR- EXCELERATE grant 676559. IL and GK are supported by the European Molecular Biology Laboratory; MS by Research References National Human Genome Research Institute. Workshop on sharing aggregate genomic data report. 2016. 32. Dyke SO, Philippakis AA, Rambla De Argila J, Paltoo DN, Luetkemeier ES, Knoppers BM, et al. Consent codes: upholding standard data use conditions. PLoS Genet. 2016;12: e1005772. 19. National Institutes of Health. Proposal to update data management of genomic summary results under the NIH genomic data sharing policy. 2017. 33. Internet Engineering Task Force. The OAuth 2.0 Authorization Framework, RFC6749. p y 20. Chatzimichali EA, Brent S, Hutton B, Perrett D, Wright CF, Bevan AP, et al. Facilitating collaboration in rare genetic disorders through effective matchmaking in DECIPHER. Hum Mutat. 2015;36:941–9. 34. Cabili MN, Carey K, Dyke SOM, Brookes AJ, Fiume M, Jeanson F, et al. Simplifying research access to genomics and health data with Library Cards. Sci Data. 2018;5:180039. 21. Buske OJ, Girdea M, Dumitriu S, Gallinger B, Hartley T, Trang H, et al. PhenomeCentral: a portal for phenotypic and genotypic matchmaking of patients with rare genetic diseases. Hum Mutat. 2015;36:931–40. 35. Grassi PA, Fenton JL. NIST Special Publication 800-63-3. Digital Identity Guidelines. National Institute of Standards and Technology. 2017. Available from https://pages.nist.gov/800-63- 3/sp800-63-3.html (accessed 17 January 2018). 36. Article 29 Data Protection Working Party. Guidelines on consent under Regulation 2016/679. 2018. Report no. 17/EN WP259 rev.01. 22. Hoskins RA, Repo S, Barsky D, Andreoletti G, Moult J, Brenner SE. Reports from CAGI: the Critical Assessment of Genome Interpretation. Hum Mutat. 2017;38:1039–41. 37. Global Alliance for Genomics and Health. Data Sharing Lexicon. 2016. 23. Philippakis AA, Azzariti DR, Beltran S, Brookes AJ, Brownstein CA, Brudno M, et al. The matchmaker exchange: a platform for rare disease gene discovery. Hum Mutat. 2015;36: 915–21. 38. Broeder DJB, Kelsey D, Kershaw P, Luders S, Lyall A, Nyronen T et al. Federated identity management for research collabora- tions. Report No. CERN-OPEN. 2012;006. 24. Wong KM, Langlais K, Tobias GS, Fletcher-Hoppe C, Krasne- wich D, Leeds HS, et al. The dbGaP data browser: a new tool for browsing dbGaP controlled-access genomic data. Nucleic Acids Res. 2016;45:D819–26. 39. Atherton CJ, Barton T, Basney J, Broeder D, Costa A, van Daalen M, et al. Federated Identity Management for Research Colla- borations (Version 2.0). Zenodo, 2018. https://doi.org/10.5281/ zenodo.1296031. 25. Mailman MD, Feolo M, Jin Y, Kimura M, Tryka K, Bagoutdinov R, et al. The NCBI dbGaP database of genotypes and phenotypes. Nat Genet. 2007;39:1181–6. 40. Wilbanks J, Friend SH. References ML, IL, JT, and TN are supported by the ELIXIR, the research infrastructure for life-science data, and the H2020 ELIXIR- EXCELERATE grant 676559. IL and GK are supported by the European Molecular Biology Laboratory; MS by Research 10. Ferraiole D, Kuhn, R. Role-based access control. Proceedings of the 19th National Computer Security Conference. 1992. 11. Ferraiole D, Cugini, J, Kuhn, R. Role-based access control: fea- tures and motivations. Proceedings of the 11th Conference in Computer Security Applications. 1995. 12. Sandhu R, Coyne E, Feinstein H, Youman C. Role-based access control models. IEEE Comput. 1996;29:38–47. 1730 S. O. M. Dyke et al. 13. Barkley J, Cincotta, A, Ferraiolo, D, Gavrila, S, Kuhn, D. Role based access control for the world wide web. 20th National Computer Security Conference. 1997. 27. Sobreira N, Schiettecatte F, Valle D, Hamosh A. GeneMatcher: a matching tool for connecting investigators with an interest in the same gene. Hum Mutat. 2015;36:928–30. 28. Chong JX, Yu JH, Lorentzen P, Park KM, Jamal SM, Tabor HK, et al. Gene discovery for Mendelian conditions via social networking: de novo variants in KDM1A cause developmental delay and distinctive facial features. Genet Med. 2016;18: 788–95. 14. Ferraiole D, Sandhu, R, Gavrila, S, Kuhn, D, Chandramouli, R. A proposed standard for role-based access control. National Institute of Standards and Technology. 2000. ACM Transactions on Information and System Security, Vol. 4, No. 3, August 2001, pp. 224–274. 29. Dyke SOM, Saulnier KM, Pastinen T, Bourque G, Joly Y. Evolving data access policy: the Canadian context. FACETS. 2016;1:138. 15. O’Connor AC, Loomis RJ. Economic analysis of role-based access control: ﬁnal report. 2010. 16. Homer N, Szelinger S, Redman M, Duggan D, Tembe W, Muehling J, et al. Resolving individuals contributing trace amounts of DNA to highly complex mixtures using high- density SNP genotyping microarrays. PLoS Genet. 2008;4: e1000167. 30. Moher D, Shamseer L, Cobey K. Stop this waste of people, animals and money. Nature. 2017;549:23–5. 31. Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing Directive 95/46/EC (General Data Protection Regulation), 2016. 17. Craig DW, Goor RM, Wang Z, Paschall J, Ostell J, Feolo M, et al. Assessing and managing risk when sharing aggregate genetic variant data. Nat Rev Genet. 2011;12:730. 18. References First, design for data sharing. Nat Bio- technol. 2016;34:377–9. 41. Dyke SOM, Dove ES, Knoppers BM. Sharing health-related data: a privacy test? NPJ Genom Med. 2016;1:16024. 26. Lappalainen I, Almeida-King J, Kumanduri V, Senf A, Spalding JD, Ur-Rehman S, et al. The European Genome-phenome Archive of human data consented for biomedical research. Nat Genet. 2015;47:692–5. Stephanie O. M. Dyke1,35 ●Mikael Linden 2,3 ●Ilkka Lappalainen2,3,4 ●Jordi Rambla De Argila5,6 ●Knox Carey ● David Lloyd4,7 ●J. Dylan Spalding4 ●Moran N. Cabili8 ●Giselle Kerry4 ●Julia Foreman9 ●Tim Cutts9 ● Mahsa Shabani10 ●Laura L. Rodriguez11 ●Maximilian Haeussler12 ●Brian Walsh13 ●Xiaoqian Jiang14 ● Shuang Wang14 ●Daniel Perrett9 ●Tiffany Boughtwood15 ●Andreas Matern16 ●Anthony J. Brookes 17 ● Miro Cupak18 ●Marc Fiume18 ●Ravi Pandya19 ●Ilia Tulchinsky20 ●Serena Scollen3 ●Juha Törnroos2 ●Samir Das21 Alan C. Evans21 ●Bradley A. Malin22 ●Stephan Beck23 ●Steven E. Brenner24 ●Tommi Nyrönen 2,25 ● Niklas Blomberg 3 ●Helen V. Firth9 ●Matthew Hurles9 ●Anthony A. Philippakis8 ●Gunnar Rätsch26 ● Michael Brudno27,28 ●Kym M. Boycott29 ●Heidi L. Rehm 8,30 ●Michael Baudis 31 ●Stephen T. Sherry32 ● Kazuto Kato33 ●Bartha M. Knoppers1 ●Dixie Baker 34 ●Paul Flicek 4 Afﬁliations Stephanie O. M. Dyke1,35 ●Mikael Linden 2,3 ●Ilkka Lappalainen2,3,4 ●Jordi Rambla De Argila5,6 ●Knox Carey ● David Lloyd4,7 ●J. Dylan Spalding4 ●Moran N. Cabili8 ●Giselle Kerry4 ●Julia Foreman9 ●Tim Cutts9 ● Mahsa Shabani10 ●Laura L. Rodriguez11 ●Maximilian Haeussler12 ●Brian Walsh13 ●Xiaoqian Jiang14 ● Shuang Wang14 ●Daniel Perrett9 ●Tiffany Boughtwood15 ●Andreas Matern16 ●Anthony J. Brookes 17 ● Miro Cupak18 ●Marc Fiume18 ●Ravi Pandya19 ●Ilia Tulchinsky20 ●Serena Scollen3 ●Juha Törnroos2 ●Samir Das21 ● Alan C. Evans21 ●Bradley A. Malin22 ●Stephan Beck23 ●Steven E. Brenner24 ●Tommi Nyrönen 2,25 ● Niklas Blomberg 3 ●Helen V. Firth9 ●Matthew Hurles9 ●Anthony A. Philippakis8 ●Gunnar Rätsch26 ● Michael Brudno27,28 ●Kym M. Boycott29 ●Heidi L. Rehm 8,30 ●Michael Baudis 31 ●Stephen T. Sherry32 ● Kazuto Kato33 ●Bartha M. Knoppers1 ●Dixie Baker 34 ●Paul Flicek 4 Stephanie O. M. Dyke1,35 ●Mikael Linden 2,3 ●Ilkka Lappalainen2,3,4 ●Jordi Rambla De Argila5,6 ●Knox Carey ● David Lloyd4,7 ●J. Dylan Spalding4 ●Moran N. Cabili8 ●Giselle Kerry4 ●Julia Foreman9 ●Tim Cutts9 ● Mahsa Shabani10 ●Laura L. Rodriguez11 ●Maximilian Haeussler12 ●Brian Walsh13 ●Xiaoqian Jiang14 ● Shuang Wang14 ●Daniel Perrett9 ●Tiffany Boughtwood15 ●Andreas Matern16 ●Anthony J. Brookes 17 ● Miro Cupak18 ●Marc Fiume18 ●Ravi Pandya19 ●Ilia Tulchinsky20 ●Serena Scollen3 ●Juha Törnroos2 ●Samir Das21 ● Alan C. Evans21 ●Bradley A. Malin22 ●Stephan Beck23 ●Steven E. Brenner24 ●Tommi Nyrönen 2,25 ● Niklas Blomberg 3 ●Helen V. Firth9 ●Matthew Hurles9 ●Anthony A. Philippakis8 ●Gunnar Rätsch26 ● Michael Brudno27,28 ●Kym M. Boycott29 ●Heidi L. Rehm 8,30 ●Michael Baudis 31 ●Stephen T. Sherry32 ● Kazuto Kato33 ●Bartha M. Knoppers1 ●Dixie Baker 34 ●Paul Flicek 4 y 8 ●Kym M. Boycott29 ●Heidi L. Rehm 8,30 ●Michael Baudis 31 ●Stephen T. Sherry32 ● Registered access: authorizing data access 1731 19 Microsoft, Redmond, WA, USA 1 Centre of Genomics and Policy, Faculty of Medicine, McGill University, Montreal, QC, Canada 1 Centre of Genomics and Policy, Faculty of Medicine, McGill University, Montreal, QC, Canada 19 Microsoft, Redmond, WA, USA 20 Google, Mountain View, CA, USA 2 CSC – IT Center for Science, Espoo, Finland 21 McGill Centre for Integrative Neurosciences, Montreal Neurological Institute, McGill University, Montreal, QC, Canada 3 ELIXIR Hub, Wellcome Genome Campus, Hinxton, Cambridge, UK 22 Vanderbilt University Medical Center, Nashville, TN, USA 4 European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, UK 23 UCL Cancer Institute, University College London, London, UK 24 Department of Plant & Microbial Biology, University of California, Berkeley, CA, USA 5 Centre for Genomic Regulation, Barcelona, Spain 6 Universitat Pompeu Fabra, Barcelona, Spain 25 ELIXIR Compute Platform, ELIXIR, Wellcome Genome Campus, Hinxton, Cambridge, UK 7 The Global Alliance for Genomics and Health, MaRS Centre, West Tower, 661 University Avenue, Suite 510, Toronto M5G 0A3 ON, Canada 26 Department of Computer Science, Biomedical Informatics, ETH Zurich, Zurich, Switzerland 8 Broad Institute of MIT and Harvard, Cambridge, MA, USA 27 Department of Computer Science, University of Toronto, Toronto, ON, Canada 9 Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK 28 Centre for Computational Medicine, Hospital for Sick Children, Toronto, ON, Canada 10 Center for Biomedical Ethics and Law, Department of Public Health and Primary Care, University of Leuven, Leuven, Belgium 29 Children’s Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, ON, Canada 11 National Human Genome Research Institute, NIH, Bethesda, MD, USA 30 Department of Pathology, Brigham & Women’s Hospital & Harvard Medical School, Boston, MA, USA 12 Genomics Institute, University of California at Santa Cruz, Santa Cruz, CA, USA 31 University of Zurich & Swiss Institute of Bioinformatics, Zurich, Switzerland 13 OHSU, Portland, OR, USA 13 OHSU, Portland, OR, USA 32 National Centre for Biotechnology Information, US National Library of Medicine, Bethesda, MD, USA 14 Department of Biomedical Informatics, UC San Diego, La Jolla, CA, USA 15 Australian Genomics Health Alliance, 50 Flemington Road, Parkville, VIC 3052, Australia 33 Department of Biomedical Ethics and Public Policy, Graduate School of Medicine, Osaka University, Osaka, Japan 34 Martin, Blanck & Associates, Alexandria, VA, USA 16 Bioreference Laboratories, Inc., Elmwood Park, NJ, USA 17 Department of Genetics and Genome Biology, University of Leicester, Leicester, UK 35 Present address: Montreal Neurological Institute, Faculty of Medicine, McGill University, Montreal, QC, Canada 18 DNAstack, Toronto, ON, Canada
https://openalex.org/W2146871091	https://www.scielo.br/j/rap/a/LNm8R7GCXjSPGMgBR3bgFjP/?lang=pt&format=pdf	Portuguese	null	Cursos superiores de tecnologia em gestão: reflexões e implicações da expansão de uma (nova) modalidade de ensino superior em administração no Brasil	Revista de administração pública	2,010	cc-by	12,236	Artigo recebido em out. 2008 e aceito em nov. 2009. A elaboração deste artigo foi possível graças ao suporte financeiro fornecido pelos programas de bolsa da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes). A autora é grata aos revisores do artigo por suas contribuições para este trabalho, ao debatedor prof. Pedro Lincoln C. L. De Mattos (Programa de Pós-Graduação em Administração da Universidade Federal de Pernambuco) e demais participantes da sessão, que fizeram sugestões importantes quando da sua primeira apresentação no 32o Enanpad 2008. Doutora em administração pela Faculdade de Economia e Administração da Universidade de São Paulo (FEA/USP), professora de administração na Universidade Federal do Paraná. Endereço: Av. Prefeito Lothário Meissner, 632 — Jardim Botânico — CEP 80210-170, Curitiba, PR, Brasil. E-mail: adrianarwt@terra.com.br. adriana roseli wünsch takahashi 386 O que a expansão desses cursos significa para a área de administração? Em que eles são diferentes dos tradicionais cursos de bacharelado? Qual a importância de analisar essa modalidade de ensino? De forma geral, o que representa para o cenário nacional essa mudança no ensino superior brasileiro? Mediante tais considerações e com base em pesquisa bibliográfica e documental em dados secundários, este artigo propõe um debate sobre os possíveis impactos desse crescimento para a própria modalidade de ensino, para as instituições ofertantes e para o ensino em administração no Brasil. Por fim, a partir de tais questionamentos, busca-se oferecer elementos para a orga- nização de uma agenda de pesquisas com vistas ao acompanhamento da expansão dos CSTs em administração. Higher education courses in management technology: reflections and implications of the expansion of a (new) modality of higher education in management in Brazil Since Act 9394/96, professional education has been going through great changes in Brazil. The higher education technological courses (CSTs) that exist since the seven- ties have been reformulated to answer the now existing demands of the production sector and to improve the access to higher education. Federal public policies have stimulated professional education in technology, expanding its offering of courses for both undergraduate and graduate studies, as census data can show. This brings up a few questions. What does this expansion mean to the management area? In which way are these courses different from those with a bachelor’s degree? What is the importance of analyzing this standard of education? What does this change in Brazilian higher education mean to the country? Taking the previous reflection into account and based on documentary and bibliographical research in secondary data, this article proposes a debate on the possible impacts of this increase in this kind of education itself, in the institutions that offer it and to management education in Brazil. It then presents elements for organizing a research agenda dealing with the expansion of the CSTs in managemen. Cursos superiores de tecnologia em gestão: reflexões e implicações da expansão de uma (nova) modalidade de ensino superior em administração no Brasil Adriana Roseli Wünsch Takahashi** Sumário: 1. Introdução; 2. Contexto do setor educacional profissional de nível tecnológico no Brasil; 3. Discussões e reflexões; 4. Considerações finais. Sumário: 1. Introdução; 2. Contexto do setor educacional profissional de nível tecnológico no Brasil; 3. Discussões e reflexões; 4. Considerações finais. Summary: 1. Introduction; 2. Context of the professional education sector at the technological level in Brazil; 3. Discussions and reflections; 4. Final remarks. Palavras-chave: cursos superiores de tecnologia; educação profissional; admi- nistração. Palavras-chave: cursos superiores de tecnologia; educação profissional; admi- nistração. Key words: higher education courses in technology; professional education; man- agement. A partir da Lei de Diretrizes e Bases (LDB), Lei no 9.394/96, a educação profissional tem passado por profundas mudanças no Brasil. Os cursos superiores de tecnologia (CSTs), que existem desde os anos 1970, foram reformulados a fim de atender às demandas atuais do setor produtivo e ampliar o acesso ao ensino superior. As políticas públicas federais têm fomentado o crescimento da oferta da educação profissional tecnológica superior brasileira, em nível de graduação e pós-graduação, o que pode ser observado nos dados censitários. Tal fato suscita a reflexão de algumas questões. Artigo recebido em out. 2008 e aceito em nov. 2009. A elaboração deste artigo foi possível graças ao suporte financeiro fornecido pelos programas de bolsa da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes). A autora é grata aos revisores do artigo por suas contribuições para este trabalho, ao debatedor prof. Pedro Lincoln C. L. De Mattos (Programa de Pós-Graduação em Administração da Universidade Federal de Pernambuco) e demais participantes da sessão, que fizeram sugestões importantes quando da sua primeira apresentação no 32o Enanpad 2008. *Doutora em administração pela Faculdade de Economia e Administração da Universidade de São Paulo (FEA/USP), professora de administração na Universidade Federal do Paraná. Endereço: Av. Prefeito Lothário Meissner, 632 — Jardim Botânico — CEP 80210-170, Curitiba, PR, Brasil. E-mail: adrianarwt@terra.com.br. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi cursos superiores de tecnologia em gestão cursos superiores de tecnologia em gestão 387 opções futuras das escolas e do ensino em administração no Brasil e no mundo (Fischer, 2001; Friga, Bettis e Sullivan, 2004).i opções futuras das escolas e do ensino em administração no Brasil e no mundo (Fischer, 2001; Friga, Bettis e Sullivan, 2004). Contudo, poucos estudos têm apresentado como recorte específico do ensino superior em administração os cursos de graduação situados dentro da educação profissional. Atualmente, de acordo com o Decreto no 5.154/2004, a educação profissional no Brasil consiste de três níveis, sendo um deles a edu- cação profissional tecnológica de graduação e de pós-graduação. Neste nível, estão os cursos superiores de tecnologia (CSTs), cursos de graduação também conhecidos como cursos tecnológicos ou tecnólogos. Os CSTs começaram a ser ofertados na educação profissional brasileira na década de 1970, em função da necessidade de formação e qualificação de trabalhadores para atender à demanda das empresas instaladas no período de industrialização e modernização promovido pelo governo brasileiro em mea- dos do século XX. No entanto, persistia a visão de uma educação para o tra- balho associada à formação profissional das classes menos favorecidas. Essa iniciativa não alterou a mentalidade das elites, um pensamento privilegiava, especificamente, os cursos superiores plenos. A influência histórica que mar- cou o preconceito manteve-se sobre a educação profissional. Ao longo das últimas seis décadas, a inovação nos processos produtivos passou a requerer cada vez mais dos trabalhadores uma escolaridade básica acompanhada de contínua qualificação profissional. Nos últimos 10 anos, o Brasil passou então a fomentar a educação profissional de nível superior como uma resposta estratégica tanto de escolarização quanto de atendimento ao setor produtivo. Alguns fatores parecem ter pressionado essa iniciativa. Um deles é que há um contingente expressivo de alunos formados no Ensino Mé- dio buscando a continuidade dos estudos, resultado da ampliação das vagas na educação básica nos últimos 10 anos. O segundo fator é a pressão que a chamada economia baseada no conhecimento (EBC) coloca sobre os siste- mas educacionais para qualificar os trabalhadores de forma que as organiza- cões possam inserir-se na economia globalizada. Um terceiro é a tendência educacional mundial de investimentos na educação profissional. Nos Estados Unidos e em alguns países da Europa, mais da metade dos alunos formados no Ensino Superior se forma nessa modalidade de ensino (Parecer CNE/CES no 436/2001). 1. Introdução Diversos estudos têm se voltado à reflexão sobre o ensino superior em admi- nistração no Brasil. Pesquisadores e docentes, sob diferentes enfoques, têm se mostrado preocupados com sua configuração e expansão, seja em deter- minadas localidades (Canopf, Festinalli e Ichikawa, 2005), na administração (Oliveira e Sauerbronn, 2007; Silva e Fischer, 2008) ou especificamente na administração pública (Coelho, 2008), na graduação presencial e na gradua ção a distância (Torrecillas e Miramar, 2008), ou mesmo na pós-graduação (Wood Jr. e Paula, 2004; Viana, Mantovani e Vieira, 2008). As conclusões va- riam das constatações sobre o ensino atual em administração a proposições e rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão A partir da nova LDB, conhecida pelo nome de seu idealizador — Lei Darcy Ribeiro —, que entende ser a educação profissional integrada às diferentes for- mas de educação (Parecer CNE/CP no 29/2002), esse preconceito começou a ser alterado. A partir dessa lei e das regulamentações posteriores, a educação profis- sional foi redimensionada, e o ensino tecnológico reiniciou sua trajetória no Bra- rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 388 sil. A organização e o incentivo dados pela Secretaria de Educação Profissional e Tecnológica (Setec/MEC) a essa modalidade educacional trouxeram uma nova perspectiva de formação superior para o Brasil que já existe em outros países (Pa- recer CNE/CES no 436/2001). A partir de então, instituições públicas de ensino revitalizaram seus cursos e os primeiros centros de educação tecnológica (CETs) começaram a ser credenciados pela iniciativa privada (Anet, 2003:4). i Os CSTs tiveram um significativo crescimento quanto ao número de vagas, de alunos matriculados e de instituições ofertantes, nos últimos cinco anos no Brasil. Esses cursos são graduações voltadas ao mundo do trabalho, à inovação científica e tecnológica e à gestão de produção e serviços. A principal diferença entre os cursos de graduação tecnológicos, que conferem o diploma de tecnólogo, e os cursos tradicionais de Ensino Superior, que conferem o di- ploma de licenciatura ou bacharel, está na proposta e nos propósitos de cada um. Os cursos tecnológicos vêm atender a uma demanda do mercado por es- pecialistas dentro de uma área de conhecimento e estão orientados por carac- terísticas como foco, rapidez e flexibilidade, enquanto as outras modalidades de ensino superior visam formar generalistas. Os CSTs são, portanto, cursos distintos das graduações tradicionais (Parecer CNE/CES no 436/2001), e seus concluintes ficam aptos a prosseguir seus estudos em nível de pós-graduação. No Brasil, houve um rápido crescimento dos CSTs a partir de 1999. Segundo o Censo da Educação Profissional, os cursos tecnológicos cresceram 74% entre 2000-02. Em 1999, as faculdades e os CETs ofereciam 74 cursos tecnológicos, enquanto em 2004 esse número passou para 758. Entre estes, 51,8% pertenciam ao setor privado e 48,2% eram ofertados pelo setor público (Inep, 2008). Apontado pela mídia como o “novo filão do mercado”, esses cursos tendem a continuar a crescer. Em 2006, os CSTs representavam 15% das graduações nacionais (MEC, 2006b). cursos superiores de tecnologia em gestão 389 de comércio e administração e de gerenciamento e administração, ou seja, 16,8% (Inep, 2008). O mesmo crescimento tem sido observado nos CSTs em gestão, apesar de não se dispor de informações tão detalhadas quanto as referentes ao Ensi- no Superior em geral. O predomínio dos cursos superiores em administração no Brasil traz novos desafios ao campo, pois há um contingente de alunos formados com um perfil diferenciado, de especialista nas áreas de gestão. A formação desses alunos demanda dos docentes práticas de ensino específicas e pedagogias próprias. Além disso, uma parcela dos tecnólogos formados passa a buscar continuidade nos estudos em cursos de pós-graduação, uma vez que legalmente estão habilitados para tal, inclusive nos cursos stricto sensu. Todos esses fatos afetam o grande campo da administração, de graduação e pós-gra- duação, bem como a atuação dos professores e pesquisadores. Refletir sobre essas questões é o principal desafio deste artigo. No entanto, para discutir so- bre esta realidade é necessário compreender o que são os CSTs. Para isso, com base em pesquisa realizada com dados secundários, descreve-se sua história, especificidades e crescimento para compreender sua existência e analisar sua inserção no Ensino Superior. Por fim, são apresentados questionamentos que possam auxiliar a organização de uma agenda de pesquisas sobre o tema com vistas ao acompanhamento do crescimento acadêmico (instituições e docen- tes) e profissional (alunos) relacionado aos cursos tecnológicos em gestão. cursos superiores de tecnologia em gestão A sociedade tem dado respostas ao crescimento da oferta por meio da rápida absorção do tecnólogo no mercado de trabalho. Apesar de ainda modesta, comparando ao universo de cursos superiores de graduação tradicional, a oferta dessa modalidade tem sido legi- timada nos últimos anos pela regulamentação do MEC e pela crescente acei- tação social dos cursos. O Censo da Educação Superior de 2006 do Instituto Nacional de Estu- dos e Pesquisas (Inep) demonstra que a área de administração é uma das mais procuradas. Segundo esses dados, dos 736.829 alunos que concluíram cursos de graduação presencial — bacharelados, licenciaturas e tecnológicos — em 2006, 299.246 alunos estão na grande área de ciências sociais, negócios e di- reito, ou seja, 40,61%. Entre estes, 123.816 alunos estão nas áreas específicas rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 2. Contexto do setor educacional profissional de nível tecnológico no Brasil Os cursos de educação profissional tecnológica de graduação fazem parte da oferta total de Ensino Superior brasileiro. Enquanto em anos anteriores sua participação no cenário educacional nacional era tímida e até mesmo desco- nhecida, em termos gerais, agora estes cursos vêm crescendo e se solidificando na rede de ensino, pública e privada, tornando-se reconhecidos e, gradativa- mente, aceitos no mercado de trabalho e na sociedade. Esse panorama começa a alterar-se a partir da nova LDB, quando a educação profissional começa a passar por profundas mudanças no Brasil. Com isso, o mesmo crescimento que pode ser observado na oferta de cursos bacharelados na área de administra- ção, também é visto nos cursos tecnológicos, ou CSTs, em gestão. Para explo- rar este cenário, serão enfocados os aspectos legais e os dados estatísticos que são relevantes para alcançar o objetivo do artigo. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi adriana roseli wünsch takahashi 390 cursos superiores de tecnologia em gestão 391 Surgiram 5,6 novos cursos a cada dia no Brasil em 2003, dos quais 4,5 foram criados no setor privado e 1,1 no setor público. O mesmo censo registrou a existência de 16.453 cursos presenciais de ensino superior, dos quais 10.791 (65,58%) na rede privada. O Censo 2005 apontou a existência de 20.407 cur- sos de graduação presencial, sendo 6.191 (30,33%) em IES públicas e 14.216 (69,67%) em IES privadas. O Censo 2006 identificou 22.101 cursos de gradua ção presencial, sendo 6.549 (29,6%) na rede pública e 15.552 (70,4%) na rede privada. Portanto, entre 2002 e 2006 observa-se uma variação de 14.399 cursos de graduação presencial para 22.101, um crescimento de 53,49%, com a manutenção do predomínio da rede privada. Esse cenário é confirmado pelo número de matrículas. O Censo 2006 apontou um total de 4.676.646 alunos efetivamente matriculados nos cursos de graduação presencial, sendo 25,85% na rede pública e 74,15% na rede pri- vada. Entre eles, 98.137 alunos (22%) estavam matriculados especificamente em CETs e faculdades de tecnologia (nova denominação legal para os CETs). Cabe ressaltar que não somente os CETs ofertam os cursos tecnológicos, que são também ofertados em outras categorias de IES como universidades, cen- tros universitários, faculdades integradas, faculdades, escolas e institutos. Porém, não há no censo o registro específico para a modalidade dos cursos tecnológicos, considerando-se todos os ofertantes. Cabe ressaltar que, apesar da rápida expansão dos cursos superiores no Brasil, houve também o crescimento da evasão dos alunos. Enquanto o nú- mero de matrículas nos últimos 10 anos aumentou 134% e o de ingressantes 172,6%, o número de concluintes, apesar de também aumentar, não acompa- nhou o mesmo crescimento, sendo de 114,7%. O Ensino Superior no Brasil O Censo 2006 sobre o Ensino Superior no Brasil, realizado pelo Inep, regis- trou a existência de 2.270 instituições de educação superior (IES), sendo 248 públicas (10,92%) e 2.022 privadas (89,08%). Segundo o World Education Indicators, este é um dos sistemas mais privatizado do mundo, atrás de al- guns poucos países. Entre as IES públicas, 97 são federais, 75 são estaduais e 59 são municipais. De acordo com o mesmo Censo, entre as IES privadas há dois grupos: as particulares com fins lucrativos, que somam 1.583 (78,3%), e as comunitárias, filantrópicas ou confessionais, que somam 439 (21,7%). Em relação aos censos anteriores, a comparação dos dados mostra que a educação superior no Brasil continua em expansão acelerada, principalmente nas IES privadas (Inep, 2008). O quadro 1 aponta as 10 maiores instituições brasileiras com base nas matrículas nos cursos de graduação presencial e ilustra a intensidade da ex- pansão das instituições privadas. Entre as 10, sete são privadas e três são estaduais. Q u a d r o 1 Relação das 10 maiores instituições em número de matrículas na graduação presencial (Brasil — 2003) Instituição UF Categoria administrativa Matrícula Universidade Estácio de Sá RJ Privada 100.617 Universidade Paulista SP Privada 92.023 Universidade de São Paulo SP Estadual 44.281 Universidade Luterana do Brasil RS Privada 41.450 Pontifícia Universidade Católica de Minas Gerais MG Privada 36.749 Universidade Salgado de Oliveira RJ Privada 35.719 Universidade Estadual do Piauí PI Estadual 35.683 Universidade Estadual de Goiás GO Estadual 34.113 Universidade Bandeirante de São Paulo SP Privada 32.852 Universidade do Vale do Rio dos Sinos RS Privada 31.482 Fonte: Inep/MEC. Disponível em: <www.inep.gov.br/download/superior/censo/2004/resumo_tecnico_050105. pdf>. Acesso em: set. 2005. Q u a d r o 1 Relação das 10 maiores instituições em número de matrículas na Q u a d r o 1 Q u a d r o 1 Relação das 10 maiores instituições em número de matrículas na graduação presencial (Brasil — 2003) Os dados desta década evidenciam o crescimento do ensino superior. O Censo 2003 apontou um acréscimo de 2.054 novos cursos em relação a 2002. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 O Ensino Superior em administração no Brasil No cenário de expansão do ensino superior no Brasil, observa-se também a expansão dos cursos de administração. Os primeiros cursos de administração surgiram em 1941, quando nos Estados Unidos já se formavam em torno de 50 mil bacharéis, 4 mil mestres e 100 doutores por ano nessa área (Castro, 1981). A partir da década de 1940, a profissionalização do ensino de administração destacou-se em função da necessidade de mão de obra qualificada (Martins, 1989) no processo de desenvolvimento econômico do país. A partir da década de 1960, o ensino superior intensificou-se (Couvre, 1982). De acordo com Martins (1989), duas instituições marcaram essa expan- são: a Faculdade de Economia e Administração da Universidade de São Paulo rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 392 (FEA/USP) e a Fundação Getulio Vargas (FGV). A criação e a evolução dos cursos de administração, de acordo com o Conselho Regional de Administra- ção (CRA-SP), se deram em instituições universitárias que se tornaram centros de excelência e de referência no ensino e pesquisa. Porém, a partir dos anos 1960, os cursos se expandiram em faculdades isoladas e privadas que prolife- raram na sociedade. Posteriormente, em 1965, a atividade do administrador foi regulamen- tada no nível de Ensino Superior pela Lei no 4.769, e seu currículo foi fixado pelo Conselho Federal de Educação pela Lei no 4.024 de 1961. Esses fatos ampliaram o campo de formação do administrador. Em 1966 foi fixado o pri- meiro currículo do curso de administração, ficando assim institucionalizada a profissão e a formação (CRA-SP). De acordo com Castro (1981), o ensino de administração passou de dois cursos em 1954 para 31 em 1967, para 177 em 1973, posteriormente para 244 em 1978, chegando em 454 em 1995. No início da década de 1980, o setor privado já reafirmava-se como maior ofertante dos cursos de administração, sendo responsável por aproximadamente 79% dos alunos. Quanto às regiões de oferta, no início da década de 1980 as regiões Sudeste e Sul já respondiam por 81% de todo o ensino de administração do país. Em 1995, 76% dos cursos continuavam nessas regiões (CRA-SP). Segundo o Censo 2003, dos 3.887.022 alunos de cursos de gradua- ção presencial, 27,6% estavam em dois cursos: administração com 564.681 (14,5%) matrículas e direito com 508.424 (13,1%). cursos superiores de tecnologia em gestão 393 absorção pelo mercado de trabalho, e a falta de qualidade em muitos cursos de graduação e pós-graduação. Em face dessa preocupação, algumas institui- ções concentraram esforços para analisar e melhorar a qualidade dos cursos de administração, como a Associação Nacional dos Cursos de Graduação em Administração (Angrad), o Conselho Federal de Administração (CFA), e a As- sociação Nacional de Pós-graduação e Pesquisa em Administração (Anpad). No entanto, a história do Ensino Superior em administração no Brasil não é feita somente da história dos cursos tradicionais, de bacharelado. Em paralelo, houve também a expansão dos cursos tecnológicos em gestão. O Ensino Superior em administração no Brasil O número de concluintes nesses dois cursos foi de 63.688 alunos, representando 12,1% do total de alu- nos concluintes de Ensino Superior. Os censos 2005 e 2006 confirmam o predomínio dos cursos de gradua ção nessas áreas. Segundo o Censo 2005, dos 20.407 cursos de graduação presenciais existentes, 2.484 (12,17%) eram de administração, enquanto em 2006, dos 22.101 cursos, 2.836 (12,83%) eram de administração (conside- rando a área específica de gerenciamento e administração, dentro da área geral de ciências sociais, negócios e direito). Se considerada a área geral men- cionada, estes cursos respondem por quase 30% da oferta total no Brasil. Em relação aos concluintes em 2006, dos 736.829 alunos, 40,61% são dessa área geral e 16,58% são da área específica citada. Entende-se que a predominância de determinados cursos em relação a outros se dá por diversos fatores, não es- tando somente relacionada à preferência profissional, mas também ao número de vagas ofertadas e ao custo financeiro dos cursos. Algumas preocupações decorreram da rápida expansão dos cursos de administração no Brasil, como a quantidade de alunos formados em relação à rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 394 ser destinada a todos que frequentavam o Segundo Grau, onde a habilitação para o trabalho, com conteúdos mínimos e carga horária definida, deveria predominar sobre a educação geral (Faustini, 2004). Nesse período, os CSTs foram incentivados pelo Projeto no 19, do Plano Setorial de Educação e Cul- tura para o período 1972-74, dada a constatação de que havia uma subutili- zação de profissionais formados nos cursos superiores tradicionais, e dada a necessidade de conduzir o estudante a uma rápida inserção no mercado de trabalho. Com isso, foram implantados 28 novos cursos em 19 instituições de ensino superior, principalmente em universidades e instituições federais. O di- ferencial desses cursos deveriam ser, de acordo com o Parecer CFE no 160/70, as características próprias de um curso voltado para a realidade tecnológica do mundo do trabalho. A Resolução Confea no 218/73 estabeleceu as com- petências e atribuições específicas ao técnico de nível superior ou tecnólogo das áreas de engenharia, arquitetura e agronomia, consistindo assim em um primeiro reconhecimento formal pelo mercado de trabalho do curso superior de tecnologia e dos tecnólogos. O Projeto Setorial no 15, do Segundo Plano Setorial de Educação e Cul- tura para o período 1975-79, reforçou o incentivo aos CSTs e suas melho- rias, porém, essas recomendações não foram amplamente acatadas, gerando a oferta de cursos sem os requisitos mínimos necessários para a qualidade esperada. Com isso, o Conselho Federal de Educação (CFE) passou a exigir, pela Resolução CFE no 17/77, a demonstração da necessidade do mercado de trabalho para a implantação dos cursos superiores de tecnologia, o perfil pro- fissiográfico do formando, a determinação da estrutura curricular de acordo com o perfil, e a indicação do corpo docente e suas respectivas qualificações técnicas para tal (Parecer CNE/CP no 29/02). Em 1973, o Parecer no CFE 1.060 registrou a denominação de “cursos superiores de tecnologia” e seus diplomados como “tecnólogos”. Em 1974, pelo Decreto Federal no 74.708, foram reconhecidos os cursos ofertados pela Faculdade de Tecnologia de São Paulo (Fatec/SP) e do CEETESP. A Resolução CFE no 55, de 1976, estabeleceu o currículo mínimo para cursos de tecnologia em processamento de dados, o que prejudicou a adaptação necessária à evo- lução tecnológica. Essa fixação de currículos foi superada pela atual LDB, que a delega aos estabelecimentos de ensino, ficando definido pelo Parecer CNE/ CES no 436/01 as diretrizes curriculares nacionais orientadoras dos sistemas de ensino. Os cursos superiores de tecnologia no Brasil Os cursos superiores de tecnologia nasceram na década de 1970, de forma tímida. A força de trabalho brasileira contou por muito tempo com trabalha- dores carentes de qualificação. Com o processo de industrialização do país a partir dos anos 1950, e as crescentes inovações tecnológicas, as mudanças na organização da produção passaram a demandar profissionais “com escolarida- de básica e com adequada e contínua qualificação profissional”. Nesse contex- to, o tecnólogo passou a ser requisitado (Parecer CNE/CP no 29/2002). A origem dos CSTs foi respaldada pela Lei no 4.024/61, a primeira LDB, que, em seu art. 104, contemplava “a organização de cursos ou escolas expe- rimentais, com currículos, métodos e períodos escolares próprios”. Em 1969, por meio do Decreto-lei no 547, foi autorizado o funcionamento dos cursos profissionais superiores de curta duração pelas escolas técnicas federais. Em São Paulo, nesse período, os cursos de tecnólogos ou cursos superiores de tec- nologia foram criados e implementados inicialmente pelo Centro Estadual de Educação Tecnológica Paula Souza (CEETPS — MEC/Setec, Políticas públicas para a educação profissional e tecnológica, 2004). A partir de 1972, o governo federal expandiu seu projeto em todo o país, criando em 1976 o Centro de Educação Tecnológica da Bahia (Centec/ BA) exclusivamente para a formação de tecnólogos, e, em 1978, os centros federais de educação tecnológica do Paraná, Minas Gerais e Rio de Janeiro. A Lei no 5.692, de 11 de agosto de 1971, introduziu mudanças pro- fundas na organização do sistema de ensino, no qual a educação profissio- nal (denominada formação especial) passou a ser obrigatória para todos os estudantes. Essa mudança foi tanto uma resposta às necessidades de novos arranjos sociais que demandavam força de trabalho quanto uma resposta às críticas de um sistema educacional elitista e excludente. A profissão passou a rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi cursos superiores de tecnologia em gestão 395 recer CNE/CES no 436/2001). O Decreto Federal no 97.333/88 autorizou a criação do primeiro CST em hotelaria pelo Senac de São Paulo, a partir do qual sua oferta foi se diversificando. Apesar de a Lei no 8.948 já ter instituído o Sistema Nacional de Edu- cação Tecnológica em 1994, foi somente com a LDB no 9.394/96 e o Decreto Federal no 2.208/97 que o ensino tecnológico ganhou nova dimensão e rei- niciou sua trajetória no Ensino Superior brasileiro (Anet, 2003). As opiniões foram diversas sobre essas alterações legais, variando de uma avaliação po- sitiva a negativa, como bem destacam Canopf, Festinalli e Ichikawa (2005). Para as autoras, enquanto Niskier (1997), por exemplo, analisa que o MEC, por meio da LDB, buscou pôr fim à indústria de currículos regulamentando a autorização de novos cursos superiores, outros autores, como Saviani (1997) e Demo (1998) criticam a nova lei por não ter criado um sistema nacional de educação aberto, abrangente e adequado, e por banalizar o ensino superior ao não vincular a avaliação de autorização e reconhecimento à intervenção e descredenciamento. Chauí (2001) acentuou a crítica ao associar a reforma da educação à reforma do Estado e, portanto, ao neoliberalismo (Canopf, Festi- nalli e Ichikawa, 2005). Divergências à parte, cabe ressaltar neste artigo que a nova LDB possui quatro artigos que tratam da educação profissional entre os 92 existentes: arts. 39 a 42. Outros artigos gerais também referenciam a relação com o mun- do do trabalho, como o §2o do art. 1o “a educação escolar deverá vincular-se ao mundo do trabalho e à prática social”. No entanto, a LDB não se refere à edu- cação tecnológica, e sim à educação profissional em geral. Esta lacuna vem ser preenchida somente pelo Decreto no 2.208/97, que em seu art. 10 especifica a educação tecnológica: “os cursos de nível superior, correspondentes à edu- cação profissional de nível tecnológico, deverão ser estruturados para atender aos diversos setores da economia, abrangendo áreas especializadas, e confe- rirão diploma de tecnólogo”. No mesmo ano, fica regulamentado também a criação de centros de educação tecnológica (CET), públicos ou privados. De acordo com o art. adriana roseli wünsch takahashi Em 1995, havia no Brasil 250 cursos tecnológicos, sendo a maioria ofer- tada pelo setor privado e mais da metade deles na área de computação (Pa- rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão 2o do Decreto no 2.406/97, sua finalidade é formar e qualificar profissionais, nos vários níveis e modalidades de ensino, para os diversos setores da economia e realizar pesquisa e desenvolvimento tec- nológico de novos processos, produtos e serviços, em estreita articulação com os setores produtivos e a sociedade, oferecendo mecanismos para a educação continuada. Após a publicação do Decreto no 2.208/97, houve ainda a publicação de uma série de instrumentos normativos que caracterizaram a reforma da rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 396 educação profissional: Portaria/MEC no 646/97, Portaria/MEC no 1.005/97, Portaria/MEC/MTb no 1.018/97 e Lei Federal no 9.649/98 (MEC/Setec, Políti- cas públicas para a educação profissional e tecnológica, 2004). A partir de 2004, a educação profissional em vigor no Brasil, segundo o Decreto no 5.154 de 23 de julho de 2004, passou a consistir de três níveis: I — formação inicial e continuada de trabalhadores; II — educação profissional técnica de nível médio; III — educação profissional tecnológica de graduação e de pós-graduação. Assim, a educação profissional e tecnológica vem consolidar-se como um esforço estratégico do Ministério da Educação com vistas às mudanças que vêm ocorrendo no mundo do trabalho, na economia nacional e internacional, e nos sistemas sociais: “cursos superiores de tecnologia (...) uma das princi- pais respostas do setor educacional às necessidades e demandas da socieda- de brasileira” (Parecer CNE/CES no 436/2001). O Parecer no 29/2002 desta- ca que a educação tecnológica tem um papel especial nesse contexto “como requisito de formação básica de todo cidadão que precisa de instrumental mínimo para sobrevivência na sociedade da informação, do conhecimento e das inúmeras tecnologias cada vez mais sofisticadas”. A educação tecnológica “pode ser considerada correspondente à educação profissional nos termos da atual legislação (...) pois os termos ‘técnica’ e ‘tecnologia’ estão presentes em todos os níveis da educação profissional”. Assim como os cursos técnicos estão permeados pela tecnologia, “a técnica está presente tanto no nível tecnológico quanto nas demais habilitações de nível superior”. Dessa forma, “a formação do tecnólogo requer desenvolvimento de competências mais complexas que as do nível técnico”. A seguir, algumas características dessa modalidade de ensino são des- critas, uma modalidade que cresceu 10 vezes entre 1999 e 2004. cursos superiores de tecnologia em gestão 397 Dessa forma, todos os CSTs são cursos de graduação, e seus concluintes ficam aptos a prosseguir seus estudos em nível de pós-graduação. As diretrizes curriculares nacionais gerais para a educação profissional de nível tecnológico foram definidas pelo Parecer no 29/2002 e proposta de resolução anexa, ho- mologado pelo ministro de Estado da Educação em 13 de dezembro de 2002. Além de definir os critérios e objetivos da educação tecnológica, as diretrizes também constituíram um esforço de romper com o preconceito histórico na- cional de que a educação para o trabalho destina-se à formação profissional de classes sociais menos favorecidas, ofertando uma educação profissional de nível superior fundamentada no desenvolvimento do conhecimento tecnológi- co e na realidade do mundo do trabalho. De acordo com a legislação, a principal diferença entre os cursos de gra- duação tecnológica, que conferem o diploma de tecnólogo, e os cursos tradicio- nais de Ensino Superior, que conferem o diploma de licenciatura ou bacharel, está na proposta de cada um. Os cursos tecnológicos vêm atender a uma deman- da do mercado por especialistas dentro de uma área de conhecimento, em vez dos generalistas formados pelas outras modalidades de Ensino Superior. Os principais atributos dos CSTs são o foco, a rapidez, a inserção no mercado de trabalho e a metodologia. O foco diz respeito à formação em um campo de trabalho definido, de acordo com as tendências do mercado. A rapi- dez refere-se à oferta do curso com uma carga horária menor, de dois ou três anos. Por estarem pautados em pesquisa de mercado para sua oferta e funcio- namento, visam à rápida inserção do aluno no mercado de trabalho de acordo com suas tendências. A metodologia praticada abrange técnicas, métodos e es- tratégias focadas na aprendizagem, no saber e no saber-fazer, com propostas didático-pedagógicas voltadas para a prática. Por isso, os cursos tecnológicos não constituem cursos permanentes, mas sim cursos que devem ser continua mente revistos, redesenhados e reorganizados, para garantir a adequação à mutabilidade das necessidades do mercado de trabalho (Anet, 2003). Com essas características, os cursos tecnólogos não admitem um fluxograma linear e “engessado”, requerendo assim flexibilidade de percurso e a possibilidade de certificações intermediárias para alunos que não tenham interesse ou não possam concluir o curso. O art. Características dos cursos superiores de tecnologia O Parecer CNE/CES no 436/2001, aprovado pela Câmara de Educação Superior do Conselho Nacional de Educação, define os cursos superiores de tecnologia: são cursos de graduação com características especiais, bem distintos dos tradi- cionais e cujo acesso se fará por processo seletivo, a juízo das instituições que os ministrem. Obedecerão a diretrizes curriculares nacionais a serem aprovadas pelo Conselho Nacional de Educação. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão 6o do Projeto de Resolução do Parecer no 29/2002 define que a organização curricular dos cursos superiores de tecnologia deverá contemplar o desenvolvimento de competências profissionais e será formulada em conso- nância com o perfil profissional de conclusão do curso, o qual define a identi- dade do mesmo e caracteriza o compromisso ético da instituição com os seus alunos e a sociedade. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 398 O §1o estabelece que “a organização curricular compreenderá as com- petências profissionais tecnológicas, gerais e específicas, incluindo os funda- mentos científicos e humanísticos necessários ao desempenho profissional do graduado em tecnologia”. De acordo com o Parecer no 29/2002, o objetivo é o de capacitar o estudante para o desenvolvimento de competên- cias profissionais que se traduzam na aplicação, no desenvolvimento (pesquisa aplicada e inovação tecnológica) e na difusão de tecnologias, na gestão de pro- cessos de produção de bens e serviços e na criação de condições para articular, mobilizar e colocar em ação conhecimentos, habilidades, valores e atitudes para responder, de forma original e criativa, com eficiência e eficácia, aos desafios e requerimentos do mundo do trabalho. o objetivo é o de capacitar o estudante para o desenvolvimento de competên- cias profissionais que se traduzam na aplicação, no desenvolvimento (pesquisa aplicada e inovação tecnológica) e na difusão de tecnologias, na gestão de pro- cessos de produção de bens e serviços e na criação de condições para articular, mobilizar e colocar em ação conhecimentos, habilidades, valores e atitudes para responder, de forma original e criativa, com eficiência e eficácia, aos desafios e requerimentos do mundo do trabalho. Para efeito desse parecer, alguém tem competência profissional “quando constitui, articula e mobiliza valores, conhecimentos e habilidades para a reso- lução de problemas não só rotineiros, mas também inusitados em seu campo de atuação profissional”. Esse conceito de competências, que deve proporcionar condições de laborabilidade, exige a organização de um currículo atualizado. Os currículos dos CSTs devem ter flexibilidade, interdisciplinaridade, contextualização e atualização permanente. A interdisciplinaridade evita a segmentação de conteúdos, pois nessa perspectiva os conhecimentos não são unidades isoladas. A contextualização implica relacionar conteúdo e contexto para dar significado ao aprendizado, privilegiando metodologias que integrem a vivência e a prática profissional. cursos superiores de tecnologia em gestão 399 Uma pesquisa realizada pela Associação Nacional da Educação Tecnoló- gica (Anet) identificou algumas características do perfil dos alunos de cursos tecnológicos. Essa pesquisa, denominada perfil do tecnólogo, foi aplicada aos alunos matriculados nos centros de educação tecnológica participantes da as- sociação, com o objetivo de definir o perfil do tecnólogo do Brasil. O total da amostra foi de 6.515 alunos pesquisados. Os resultados, apesar de restritos ao local de aplicação, ilustram o perfil dos alunos de cursos tecnlógicos: a idade média dos alunos é de 29 anos, sendo 64% do sexo masculino, 57% solteiros, 86% trabalham, sendo que destes 63% exercem atividades profissionais rela- cionadas com o curso que fazem, 20% atuam no comércio, 17% na tecnologia, 16% na indústria, 11% nos bancos, 5% em serviços e 5% na saúde. Desses alunos, 58% nunca começou outro curso superior. As principais expectativas quanto aos cursos são: preparo para o mercado de trabalho (29%), ascensão profissional (25%), seguir carreira na área (18%) e concluir o curso superior (15%). São provenientes do Ensino Médio público 51% dos alunos, 86% pre- tendem fazer pós-graduação, 78% possuem computador em casa e 79% se mantêm com recursos próprios durante o curso. Os CSTs podem ser ofertados por universidades, centros universitários, faculdades integradas, faculdades isoladas e institutos superiores. Da mesma forma, poderão ser ofertados por centros de educação tecnológica públicos e privados. As instituições de ensino público têm autonomia para abertura dos CSTs, mas precisam passar por um processo de avaliação para fins de reco- nhecimento de curso. Já as instituições de ensino privadas precisam passar por processos de avaliação tanto para fins de autorização de funcionamento quan- to para reconhecimento de curso. A Portaria MEC no 1.647/99, considerando o disposto na Lei no 9.131/95, na Lei no 9.394/96 e no Decreto no 2.406/97, dispõe sobre o credenciamento dos CETs e a autorização de cursos de nível tecnológico da educação profissional. Sobre a regulação da oferta de CSTs, em maio de 2004 o MEC sus- pendeu a abertura de novas instituições por seis meses a fim de garantir a qualidade no ensino. Nesse período, cerca de 5 mil pedidos de novos cursos e 520 propostas para criação de novas instituições estavam em andamento. Em agosto do mesmo ano, o MEC adotou novas regras para autorizar novos cursos. cursos superiores de tecnologia em gestão A atualização envolve a adequação da or- ganização curricular às demandas sociais, do mercado, das peculiaridades lo- cais e regionais, da vocação e da capacidade institucional, e, por isso, deverá enfocar as competências profissionais do tecnólogo e o perfil de conclusão pretendido (Parecer no 29/2002). Uma das formas previstas de flexibilizar o currículo é a modularização, cujos módulos devem ser organizados sistematicamente para o desenvolvi- mento de competências. Dessa forma, pode haver entradas e saídas inter- mediárias, e certificação de qualificação profissional em módulos com termi- nalidade que permitam ao indivíduo algum tipo de exercício profissional. A instituição deve também prever mecanismos de avaliação das competências desenvolvidas em atividades fora da escola, no mundo do trabalho e na práti- ca social, bem como de aproveitamento para fins de continuidade de estudos, considerando a “perspectiva de educação permanente e de contínuo desenvol- vimento da capacidade de aprender e de aprender a aprender, com crescente grau de autonomia intelectual” (Parecer no 29/2002). rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão Para o pedido de credenciamento, as instituições precisam atender às necessidades da região e ofertar um número de vagas correspondente à infra- estrutura apresentada no momento do pedido. Muitas instituições de ensino solicitavam abertura de vários cursos para criar uma reserva de vagas, apesar de conseguir manter apenas alguns. A Portaria MEC no 3.643/2004 determinou que os pedidos de autoriza- ção para cursos tecnológicos devem ser protocolados por meio do Sistema de rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 400 Acompanhamento de Processos das Instituições de Ensino Superior (Sapiens). Os novos processos puderam ser protocolados a partir de 2 de janeiro de 2005. Dessa forma, a partir de 3 de janeiro, todos os processos referentes ao creden- ciamento/recredenciamento de faculdades de tecnologia (nova denominação dos CETs), assim como todos os processos referentes aos CSTs (autorização, reconhecimento, renovação de reconhecimento, aumento de vagas, mudança de endereço e outros) passaram a ser protocolados pelo Sapiens. A sistemática de avaliação também foi alterada. Todos os processos de avaliação passaram a ser coordenados pelo Inep. O preenchimento dos dados resultantes da ava- liação, feita por especialistas do MEC, passou a ser feito eletronicamente. En- quanto anteriormente os especialistas emitiam um parecer consultivo, agora eles registram apenas os resultados da avaliação. Novas regulamentações foram elaboradas normatizando as funções de regulação, supervisão e avaliação de cursos e instituições de graduação, como o Decreto no 5.773, de 9 de maio de 2006, e o Decreto no 5.840, de 13 de julho de 2006. Mais recentemente, a Portaria Normativa MEC no 40, de 12 de dezembro de 2007, instituiu o e-MEC, um sistema eletrônico de fluxo de trabalho e gerenciamento de informações relativas aos processos de regulação da educação superior no sistema federal de educação. ç p ç Sobre as áreas de oferta e denominação dos cursos tecnológicos, confor- me o Parecer CNE/CES no 436/2001, os CSTs poderiam ser ofertados dentro de 20 áreas profissionais. No entanto, devido à grande diversidade de CSTs ofertados, as possibilidades foram repensadas e circunscritas a 10 áreas. Estas são atualmente definidas pelo Catálogo Nacional dos Cursos Superiores de Tecnologia, publicado em 2006. São elas: produção alimentícia; recursos na- turais; produção cultural e design; gestão e negócios; infraestrutura; controle e processos industriais; produção industrial; hospitalidade e lazer; informação e comunicação; e ambiente, saúde e segurança. Cada área tem suas diretrizes e a carga horária mínima obrigatória. cursos superiores de tecnologia em gestão Dentro da área de gestão e negócios, podem ser ofertados cursos superiores de tecnologia em: comércio exterior, gestão comercial, gestão da qualidade, gestão de cooperativas, gestão de re- cursos humanos, gestão financeira, gestão pública, logística, marketing, ne- gócios imobiliários, processos gerenciais e secretariado. No entanto, cabe a todos os CSTs promover o desenvolvimento da capacidade empreendedora e de competências profissionais e tecnológicas para a gestão de processos e a produção de bens e serviços (Parecer no 29/2002).i Dadas as alterações e constantes revisões legais da educação profissio- nal, em geral, e da educação profissional tecnológica, em específico, a oferta de CSTs apresentou um rápido crescimento da oferta e da absorção dos tecnó- logos pelo mercado de trabalho. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão 401 Crescimento dos cursos superiores de tecnologia Em 1998, o Brasil contava com 104 mil alunos em 554 CSTs. Deste total, 32% eram de processamento de dados; 14% de turismo; 11% de secretaria- do executivo; 7% de análise de sistemas; 5% de zootecnia e 31% de outras modalidades. Ao todo, existiam 70 modalidades diferentes em todas as áreas profissionais (Parecer CNE/CES no 436/2001). A nova dimensão dada aos CSTs pela Lei no 9.394/96 e pelo Decreto Federal no 2.208/97 proveu organização e incentivo a essa modalidade edu- cacional. As políticas públicas do MEC nos últimos anos trouxeram uma nova perspectiva de formação superior para o Brasil que já existe em outros países. O aumento da oferta dos CSTs em CETs e faculdades de tecnologia ilustra o crescimento desses cursos no Brasil. Conforme dados do Inep, em 1999 somente essas instituições ofertavam 74 cursos no país. Em 2001, esse número passou para 183, um crescimento de 147,3%. Em 2003, havia 495 cursos representando 170,5% de crescimento em relação a 2001 e 568,9% em relação a 1999. De acordo com o Censo da Educação Superior de 2004, reali- zado em 2005, essas IES ofertavam 758 cursos, o que indica que, em seis anos, o Brasil decuplicou o número de cursos oferecidos por essa modalidade de organização acadêmica. O crescimento continuou em 2005, quando o Censo da Educação Superior de 2006 identificou a oferta de 959 cursos tecnológicos presenciais. Contudo, há outras instituições de ensino que ofertam tais cursos, além dos CETs. A figura ilustra esses dados. Evolução dos cursos nos centros de educação tecnológica no Brasil Fonte: Inep/MEC. Disponível em: <www.inep.gov.br> (2005). 800 700 600 500 400 300 200 100 0 1999 2001 2003 2004 74 183 495 758 Evolução dos cursos nos centros de educação tecnológica no Brasil Fonte: Inep/MEC. Disponível em: <www.inep.gov.br> (2005). rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 402 Nesses números observa-se a participação do setor privado de ensino, que em 2001 ofertava 30 cursos e em 2003, 181; um crescimento de 503,3%. Em 2004 foram criados mais 209 cursos na rede privada. Dos 758 CSTs ofertados nos CETs em 2004, 51,8% pertenciam ao setor privado e 48,2% ao setor público. A partir de 2004, as escolas agrotécnicas federais também obtiveram a permissão pelo CNE/MEC, em caráter experimental, para abrir cursos tecno- lógicos. Crescimento dos cursos superiores de tecnologia Conforme a reportagem do Paraná on-line – PR, de 12 de fevereiro de 2005, a coordenadora geral de Educação Profissional e Tecnológica da Setec/MEC, Andréa de Faria Barros Andrade, aponta como principais motivos da expansão desses cursos o fato de estarem muito próximos do mercado de trabalho e de refletirem as características das regiões onde estão inseridos: “desde a sua denominação, eles tentam mostrar a que demanda estão vincu- lados. Acompanham a tendência mundial”. Exemplos desta contextualização são os CSTs de viticultura e enologia, em Bento Gonçalves (Rio Grande do Sul), de alimentos, em Chapecó (Santa Catarina), de produção moveleira, em Votuporanga (São Paulo), de irrigação e drenagem em Iguatu (Ceará), e o de produção de cachaça em Salinas (Minas Gerais). O Censo da Educação Superior 2004 apontou a distribuição dos CSTs presenciais segundo o tipo de instituição de ensino. As universidades, públicas ou privadas, ficaram em primeiro lugar sendo responsáveis pela maioria dos cursos de educação tecnológica (38,14%). Os CETs e as faculdades tecnoló- gicas vêm logo a seguir, concentrando 36,70% do total de 1.804 cursos desse tipo existentes no Brasil. Já as faculdades, escolas e institutos, que em 1998 promoviam 55,81% dos cursos de educação tecnológica, atualmente respon- dem por 8,92%. A tabela mostra essa distribuição. A educação superior tecnológica diplomou um total de 11.759 estudantes, representando 1,9% dos 626.160 graduados da educação superior em 2004. O Censo 2006 não aponta os dados sobre os CSTs considerando-se todos os tipos de instituições de ensino. Nos CETs e faculdades tecnológicas, o número de ingressos em CSTs presenciais foi de 50.682 em 2005, sendo 37,24% no setor público e 62,76% no setor privado. Ao total, foram ofertadas 90.894 vagas nes- sas instituições, o que significa que apenas 40.212 (55,76%) foram preenchidas. Ao analisar a distribuição por setor, foi possível verificar que 95,4% das vagas no setor público foram preenchidas, enquanto no setor privado foram preenchi- das 43,63%. O total dos concluintes nessas instituições em 2005 foi de 18.253 alunos. Cabe ressaltar que, apesar de esses dados fornecerem informações re- levantes sobre a oferta e o crescimento dos cursos tecnológicos no Brasil, seria necessário ter dados atuais sobre a oferta dessa modalidade de ensino em todas as instituições de ensino para uma análise mais precisa. O mesmo ocorre na análise de dados sobre os cursos na área de gestão e negócios. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão 403 e Janeiro 44(2):385-414, MAR./ABR. 2010 Distribuição percentual do número de cursos de graduação presenciais com grau acadêmico de tecnólogo, por forma de organização acadêmica (Brasil — 1998-2004) Ano Total geral Centros de educação tecnológica e faculdades tecnológicas % Universidades % Centros universitários % Faculdades integradas % Faculdades, escolas e institutos % 1998 258 0 0,00 107 41,47 7 2,71 36 0,00 108 55,81 1999 317 48 15,14 117 36,91 24 7,57 29 9,15 99 31,23 2000 364 75 20,60 130 35,71 35 9,62 33 9,07 91 25,00 2001 447 143 31,99 129 28,86 46 10,29 36 8,05 93 20,81 2002 636 268 42,14 164 25,79 69 10,85 34 5,35 101 15,88 2003 1.142 389 34,06 441 38,62 142 12,43 46 4,03 124 1,86 2004 1.804 662 36,70 668 38,14 239 13,25 54 2,99 161 8,92 Fonte: Inep/MEC Disponível em: <www inep gov br> (2005) adriana roseli wünsch takahashi 404 Os cursos superiores de tecnologia na área de gestão Pesquisas realizadas nos sites oficiais do Ministério da Educação, Censo do En- sino Superior (Inep), revistas especializadas em educação, revistas da área de administração e internet não permitiram identificar o número exato de insti- tuições, cursos e alunos dos CSTs na área de gestão. O último Censo da Educa- ção Profissional foi realizado em 1999, não retratando, portanto, a expansão recente da educação tecnológica. Sabe-se, no entanto, que, nas estatísticas sobre o ensino superior de forma geral, a área de administração tem predomi- nado. De acordo com a participação da autora como avaliadora dos CSTs na área de gestão desde 2003, esse predomínio também pode ser percebido. g p p p Alguns dados do Censo 2006 podem ser analisados com as mesmas li- mitações apontadas anteriormente. Eles identificam dados dos cursos tecnoló- gicos nos CETs e faculdades tecnológicas, mas não apontam dados que consi- derem todas as instituições ofertantes. Entre os 18.253 concluintes em 2005, a maioria está na grande área de ciências sociais, negócios e direito: 8.866 alunos. Destes, 6.568 concluintes estão na área específica de administração, 36% do total. Os CSTs na área de gestão apresentam claras diferenças em relação aos cursos de bacharelado em administração. A organização curricular dos bacharelados privilegia o conhecimento em suas diversas áreas, abrangendo inúmeros focos de forma generalista, enquanto currículos de cursos tecnoló- gicos privilegiam unidades curriculares dentro de uma determinada área de conhecimento. Dessa forma, um exemplo de organização curricular de CST em gestão financeira poderia ser: Q u a d r o 2 Grade curricular de um CST em gestão financeira Planejamento financeiro t Informatização empresarial \| 80h t Fundamentos de contabilidade \| 120h t Fundamentos de finanças \| 80h t Gestão em empresa moderna \| 120h t Fundamentos de gestão \| 80h t Planejamento estratégico financeiro \| 120h Análise de crédito t Matemática financeira \| 80h t Análise dos demonstrativos financeiros \| 120h t Economia e mercado \| 80h t Análise de risco e crédito \| 120h Continua Q u a d r o 2 Grade curricular de um CST em gestão financeira rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão 405 Investimentos t Administração do circulante \| 120h t Elaboração de orçamento \| 80h t Análise e decisão de investimentos \| 120h t Legislação tributária \| 80h t Desenvolvimento de projetos financeiros \| 120h Fonte: Grupo Opet. Disponível em: <www.opet.com.br/superior/reboucas_cursos.asp>. 3. Discussões e reflexões A educação superior no Brasil consolidou-se nas últimas décadas e, em espe- cial, na área de administração, nos últimos 50 anos. Atualmente, ela represen- ta o campo de conhecimento mais ofertado para formação superior, o que lhe traz benefícios e restrições. Os cursos superiores de tecnologia na área de gestão Acesso em: abr. 2006. Neste caso, pode-se observar que os três módulos estão organizados em torno de um eixo comum, no caso gestão financeira, e as disciplinas são agrupadas em módulos segundo as áreas de atividade. Por isso, o projeto pe- dagógico do curso, em geral, e a organização curricular, em específico, devem estar em sintonia com a justificativa da oferta (demanda dos profissionais no mercado de trabalho, novas formas de trabalho e de produção, características de avanço tecnológico do setor, postos de trabalho e atividades corresponden- tes, e dados socioeconômicos da região), a finalidade e os objetivos do curso, e o perfil profissional de conclusão esperado do aluno. Em suma, apesar da escassez de informações existentes sobre os cursos tecnológicos específicos da área de gestão, em parte justificável pelo seu cres- cimento recente nos últimos anos, pode-se perceber que os CSTs em geral têm se consolidado no setor educacional brasileiro, não representando um modis- mo como foram, por exemplo, os cursos sequenciais. Dada essa inserção e ex- pansão no setor educacional, busca-se então refletir sobre possíveis impactos no cenário da formação superior em administração no Brasil, tanto em nível de graduação como de pós-graduação. adriana roseli wünsch takahashi 406 tação e legitimação. Apesar de existirem desde os anos 1970, somente na última década é que eles realmente se consolidaram. A partir de tal fato, o panorama do ensino superior é profundamente alterado. Mesmo represen- tando uma porcentagem pequena diante dos cursos tradicionais, a taxa de crescimento dos CSTs tem sido superior à taxa de crescimento dos cursos de bacharelado. Esse cenário, entretanto, não se modifica por acaso. Ele coincide com os esforços de expansão da educação profissional no Brasil por meio das estratégias governamentais, acompanhando algumas tendências mundiais. De acordo com Stern (1996), a educação profissional está sendo refor- mulada em diversos países de forma combinada com a educação acadêmica, o que reflete a convergência do trabalho e da aprendizagem no local de tra- balho. Nos Estados Unidos há uma proliferação de novos programas delinea- dos para integrar a educação acadêmica e profissional, como, por exemplo, o teach prep, que combina o currículo acadêmico e profissional e também liga os dois últimos anos do secondary school com os dois primeiros anos do post- secondary educational. O Japão tem desenvolvido um novo currículo integrado acadêmico-profissional para a high school, que até 1994 oferecia somente um currículo geral como preparação para a universidade ou um currículo profis- sional especializado. A França tem criado uma variedade de diplomas secun- dários superior: geral, técnico e profissional. A partir de 1985, ela inseriu o diploma profissional, oferecendo aos graduados de programas profissionais de dois anos a opção de receber um diploma secundário-superior após um adicional de dois anos. Na Alemanha, um sistema duplo de aprendizagem tem sido amplamente aceito como um modelo de sucesso para iniciar a educação profissional. O Reino Unido introduziu um novo conjunto de qualificações profissionais denominado general national vocational qualifications (GNVQs), procurando remover barreiras para equalizar o status entre o percurso acadê- mico e profissional. Diversos outros exemplos podem ser citados na busca do novo enfoque de educação e trabalho (Stern, 1996). A Suécia requer que estudantes do novo programa secundário superior de três anos passem 15% do seu tempo em locais de trabalho. A Austrália está criando o student traineeships para permitir que estudantes combinem os estudos da escola com a experiência do trabalho e o treinamento fora do local de trabalho. Para o ensino em administração no Brasil No âmbito nacional, enquanto os cursos tradicionais de administração expan- diram-se a partir dos anos 1980, principalmente no setor privado, os cursos profissionais de nível tecnológico foram marcados pela dificuldade de implan- rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi Esse quadro de princípios orientadores, apresentado nesse documento que tem como objetivo estabelecer diretrizes e definir políticas públicas para a educação profissional e tecnológica, re- sultou do Documento-base e do Relatório Final do Seminário Nacional de Educação Profissional (2003). adriana roseli wünsch takahashi O Reino Unido lançou uma iniciativa para criar modern apprenticeships para estudantes de 16-17 anos que terminam a escola, onde programas foram desenvolvidos em 1994 em 12 seto- res. A Coreia tem reestruturado seu currículo de high school profissional para incluir um ano em empresas nos programas de três anos. Nos Estados Unidos, o School-to-Work Opportunities Act, em 1994, forneceu recursos federais para rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão 2010 adriana roseli wünsch takahashi 408 Apesar de os resultados da expansão da educação profissional, no âm- bito nacional, parecerem positivos, embora haja também o debate sobre sua qualidade, deve-se ainda questionar sobre o efeito dessa expansão em áreas específicas do conhecimento. É o caso desse estudo no campo da administra- ção. Ou seja, trata-se de questionar em que medida a expansão e consolidação dos CSTs em gestão importam ou trazem preocupações para a área de admi- nistração, e que implicações o surgimento dos CSTs têm para os acadêmicos e práticos, para os docentes e para os alunos. A administração tem se destacado nas estatísticas tanto pela liderança na oferta de cursos, bacharelados e tecnológicos, quanto pela liderança na preferência de alunos. Respondendo à demanda, as instituições têm ampliado a gama de opções de cursos dentro da área de administração, trazendo pro- fundas implicações para os profissionais do campo. cursos superiores de tecnologia em gestão 407 os estados implementarem novos sistemas de escola para o trabalho, onde a aprendizagem baseada no trabalho é o componente necessário. Mediante tal comparativo, a estratégia de fomento da educação profis- sional parece ser positiva para a inserção e qualificação de trabalhadores no sistema educacional e, por conseguinte, para a inserção do Brasil na chamada economia baseada em conhecimento (Dahlman, 2002; Castells, 1999; OCDE, 1996), uma vez que nela a educação recebe especial destaque. Em alguns países, a maioria dos alunos é formada em cursos superiores de tecnologia. De acordo com o Parecer CNE/CES no 436/2001, nos Estados Unidos e em alguns países da Europa, essa modalidade educacional abrange metade dos alunos do Ensino Superior. Essa realidade ainda é recente no Brasil quando vista sob a ótica da nova LDB e suas posteriores regulamentações. Comparados aos cur- sos tradicionais, de licenciatura e bacharelado, ainda são a minoria. Porém, um rápido crescimento tem sido observado nos últimos cinco anos. Em abril de 2004, o MEC/Setec publicou o documento Políticas públicas para a educação profissional e tecnológica, reafirmando o papel estratégico des- sa modalidade dentro de sua política econômica nacional. Neste documento, o sistema educacional é apontado como historicamente localizado e circuns- tanciado, no qual circulam movimentos de construção e reconstrução, deter- minados por fatores de ordem econômico-social e político-cultural. Com isso, o objetivo maior deste documento é o de estabelecer diretrizes e definir políti- cas públicas para a educação profissional e tecnológica visando à consolidação de ações efetivas que redundem no aperfeiçoamento da democracia, na melhor qualificação do cidadão, jovem ou trabalhador, na redução das desigualdades sociais e na sua participação como agente de transformação para construir o desenvolvimento do Brasil. o objetivo maior deste documento é o de estabelecer diretrizes e definir políti- cas públicas para a educação profissional e tecnológica visando à consolidação de ações efetivas que redundem no aperfeiçoamento da democracia, na melhor qualificação do cidadão, jovem ou trabalhador, na redução das desigualdades sociais e na sua participação como agente de transformação para construir o desenvolvimento do Brasil. Os efeitos de tais mudanças têm sido a gradual inserção dos tecnólogos no mercado de trabalho. Essa inserção tem mudado lentamente o preconceito histórico associado aos cursos de formação profissional, conferindo reconheci- mento social aos mesmos. Trata-se de uma caminhada, um processo que vem ocorrendo na sociedade nestes últimos anos. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. cursos superiores de tecnologia em gestão 409 em cursos tecnológicos podem, legalmente, prosseguir seus estudos em cursos de pós-graduação lato sensu e stricto sensu, cabe questionar como os cursos de pós-graduação têm tratado os candidatos que são tecnólogos. Se eles têm sido selecionados, como tem sido seu desempenho? Qual tem sido sua con- tribuição para a produção acadêmica em administração? No caso dos cursos de mestrado, em especial, pode-se ainda investigar se há congruência entre o crescimento dos cursos de mestrado profissional e o crescimento do número de concluintes em CSTs. Esses cursos de pós-graduação seriam o caminho mais provável para formados em cursos tecnológicos? Se sim, poderia-se espe- rar, em contrapartida, um crescimento acelerado nos cursos de mestrado pro- fissional? Por sua vez, esse crescimento demandaria que perfil de docentes de pós-graduação? Que impacto esses (novos) cursos terão para os atuais cursos de pós-graduação em administração que se congregam na Anpad? Uma terceira questão no nível acadêmico diz respeito à qualidade dos cursos tecnológicos, muito embora essa preocupação tangencie todos os níveis de ensino e tipos de cursos de administração. Dessa forma, caberia investigar de que maneira esses cursos têm sido avaliados e que resultados têm apresen- tado como resposta às políticas públicas de regulamentação (autorização, re- conhecimento e pós-reconhecimento). Essa preocupação com a qualidade está expressa na crítica que autores como Saviani (1997), Demo (1998) e Chauí (2001), citados por Canopf, Festinalli e Ichikawa (2005), fizeram a respeito das reformas legais na educação a partir da LDB. Para as instituições de ensino ofertantes de CSTs e docentes No âmbito acadêmico, tal fato chama a atenção para uma reflexão: se essa modalidade de ensino tem se consolidado como mostram as pesquisas, pro- fessores e pesquisadores de administração precisam conhecer melhor sua es- trutura, compreender sua proposta pedagógica e curricular, entender as de- mandas discentes, e, principalmente, adaptar suas práticas de ensino. No caso de CSTs, privilegiam-se as dinâmicas e estratégias didáticas que permitam vincular teoria e prática. Esse é o caso, por exemplo, de metodologias como estudos de caso, redes como ambiente de aprendizagem, visitas técnicas, uso de laboratórios, entre outras. As características desses cursos, apresentadas anteriormente, e as demandas dos alunos, que têm um perfil diferenciado dos que frequentam cursos de bacharelado, requerem outra postura do professor de educação profissional de nível superior. Assim, uma questão a ser investi- gada é sobre a existência de docentes com tal perfil no grupo total de docentes em administração. Se sim, cabe verificar como esses docentes têm atuado no processo de ensino-aprendizagem; se não, cabe analisar como tem sido o en- sino nos CSTs. Ou seja: estariam esses cursos sendo realmente diferenciados conforme a proposta apresentada na legislação e nas diretrizes, ou seriam uma adaptação dos cursos tradicionais de bacharelado? Em suma, que compe- tências os cursos tecnológicos demandam dos coordenadores e docentes para atuar nos cursos tecnológicos de graduação? Ainda na esfera acadêmica, uma segunda questão a ser levantada refere- se à continuidade dos estudos de tecnólogos. Ou seja, se os alunos formados rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 Para a própria modalidade de ensino e para os alunos Uma reflexão relevante é sobre o impacto dessa realidade no campo prático da administração. É sabido que há um contingente significativo de alunos con- cluintes em administração (bacharelado) todos os anos, principalmente nas grandes capitais. Reunindo-se esse número ao número de concluintes de CSTs em gestão, emerge uma questão: há paralelismo entre a quantidade total de profissionais formados e de vagas no mercado de trabalho? Há sobreposição de funções nas organizações entre os tecnólogos e os administradores com es- pecialidade adquirida nos cursos de pós-graduação lato sensu? Essas questões parecem conduzir a uma reflexão sobre a própria validade e adequação de cursos tecnológicos na área de administração. Para alimentar esse debate, há de se levar em conta as pesquisas que mostram a significativa falta de qualifi- cação dos trabalhadores em geral para ocupar os postos de trabalho disponí- veis no Brasil, e a falta de profissionais com formação em nível superior. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 410 Outras questões podem ser formuladas nessa categoria, que permane- cem por investigar: como o mercado de trabalho vem recebendo ou receberá estes profissionais? Os CSTs têm qualificado profissionais especialistas para atender às demandas específicas do setor produtivo? O crescimento dos CSTs promove outra vertente, prática, da administração não atendida anteriormen- te? A expansão da educação tecnológica de nível superior deverá acelerar a produção, difusão e aplicação do conhecimento em administração? Os cursos tecnológicos poderão ser extintos ou reduzidos significativamente no Brasil nos próximos anos? Haverá um crescimento prejudicial ao campo de adminis- tração pelo excesso de “mão de obra” qualificada? Os CSTs estão crescendo pelo apelo mercadológico de curta duração e, por isso, podem estar prejudi- cando a qualidade dos profissionais? Certamente, todas essas questões me- recem atenção e acompanhamento, uma vez que os estudos têm apontado para a continuidade e consolidação dos cursos superiores de administração no Brasil. cursos superiores de tecnologia em gestão 411 avaliados. Para isso, foi necessário descrever as características de uma (nova) modalidade de ensino que vem crescendo “vertiginosamente” no Brasil. Esse objetivo teve o papel de esclarecer suas especificidades e sua inserção no siste- ma educacional. Em um segundo momento, verificou-se a posição da área de administração na oferta e procura dos cursos superiores, tanto de bacharelado quanto de tecnologia. Diversas pesquisas são ainda necessárias para explorar os questiona- mentos levantados. Alguns futuros estudos que podem contribuir envolvem pesquisar a comparação entre currículos e práticas pedagógicas de cursos tec- nológicos e de bacharelado; a concepção de currículos para os cursos tecno- lógicos em gestão; a construção e compreensão de estratégias metodológicas; a avaliação apropriada para as metodologias de ensino desses cursos; a ava- liação das políticas públicas brasileiras na educação profissional e tecnológica de graduação e pós-graduação em administração; o grau de conhecimento e concordância dos docentes e pesquisadores de administração sobre esses cur- sos; as competências necessárias das instituições de ensino ofertantes de CSTs e dos docentes. Em suma, neste artigo, a contribuição a ser dada é divulgar a realida- de apresentada e inseri-la na agenda de debates sobre ensino e pesquisa em administração de forma a acompanhar os impactos da expansão dos cursos tecnológicos em gestão para a própria modalidade, para a academia, para as instituições de ensino, para os docentes e para os alunos. De forma mais am- pla, também para as políticas públicas voltadas para essa área, uma vez que estas necessitam de contínuo acompanhamento e adequação. 4. Considerações finais O crescimento dos cursos superiores de tecnologia em gestão está contido no cenário de expansão do ensino superior em administração. O aumento na oferta dessa modalidade de ensino de graduação está vinculado, por sua vez, às reformas ocorridas no sistema educacional e, num âmbito maior, às confi- gurações socioeconômicas e políticas do país, que busca alinhar suas estraté- gias educacionais ao contexto internacional e às pressões exercidas pela EBC para inserção na economia chamada globalizada. Tal crescimento, acelerado, incita reflexões sobre os possíveis impactos para o ensino em adminsitração no Brasil, para as instituições de ensino e docentes, para os alunos e para a própria modalidade de ensino. Com base nas referências consultadas e nos dados secundários analisa- dos, este artigo não teve a intenção de fornecer respostas para tais reflexões, muitas vezes apresentadas na forma de perguntas de pesquisa. Teve sim o propósito de jogar luz em um fenômeno atual no campo da administração e apresentar questionamentos que possam subsidiar a composição de uma agenda de pesquisas com vistas ao acompanhamento do crescimento da oferta dos CSTs em administração e de seu impacto na atuação de docentes, pesqui- sadores e tecnólogos da comunidade acadêmica e prática. As implicações da expansão da oferta dessa modalidade de ensino no Brasil, a exemplo do que ocorre em outros países, podem trazer diferentes resultados que merecem ser rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 Referências ANET (Associação Nacional de Educação Tecnológica). Educação profissional de nível tecnológico. Maio 2003. BRASIL. Ministério da Educação. Parecer n. 160, 1970. BRASIL. Ministério da Educação. Parecer n. 160, 1970. BRASIL. Ministério da Educação. Parecer n. 160, 1970. ______. Ministério da Educação. Resolução Confea n. 218/73. ______. Ministério da Educação. Resolução Confea n. 218/73. ______. Lei n. 9.394/96, de 20 de dezembro de 1996. Estabelece as diretrizes e bases da educação nacional. ______. Lei n. 9.394/96, de 20 de dezembro de 1996. Estabelece as diretrizes e bases da educação nacional. ______. Decreto n. 2.208, de 17 de abril de 1997. Regulamenta o §2o do art. 36 e os arts. 39 a 42 da Lei Federal n. 9.394/96, que estabelece as Diretrizes e Bases da Educação Nacional. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 412 ______. Decreto n. 2.406, de 27 de novembro de 1997. Regulamenta a Lei Federal n. 8.948/94 (trata de centros de educação tecnológica). cursos superiores de tecnologia em gestão cursos superiores de tecnologia em gestão 413 INEP (Instituto Nacional de Estudos e Pesquisas). Diretoria de Estatísticas e Ava liação da Educação Superior. Censo da Educação Profissional, 1999. Disponível em <www.inep.gov.br>. Acesso em: abr. 2008. q liação da Educação Superior. Censo da Educação Profissional, 1999. Disponível em: <www.inep.gov.br>. Acesso em: abr. 2008. MARTINS, C. B. Surgimento e expansão dos cursos de administração no Brasil (1952-1983). Ciência e Cultura, São Paulo, v. 41, n. 7, p. 663-676, jul. 1989. MINISTÉRIO DA EDUCAÇÃO. Instituto Nacional de Estudos e Pesquisas. Direto- ria de Estatísticas e Avaliação da Educação Superior. Censo da educação superior, 2003a. p g MARTINS, C. B. Surgimento e expansão dos cursos de administração no Brasil (1952-1983). Ciência e Cultura, São Paulo, v. 41, n. 7, p. 663-676, jul. 1989. MINISTÉRIO DA EDUCAÇÃO. Instituto Nacional de Estudos e Pesquisas. Direto- ria de Estatísticas e Avaliação da Educação Superior. Censo da educação superior, 2003 MARTINS, C. B. Surgimento e expansão dos cursos de administração no Brasi (1952-1983). Ciência e Cultura, São Paulo, v. 41, n. 7, p. 663-676, jul. 1989. MINISTÉRIO DA EDUCAÇÃO. Instituto Nacional de Estudos e Pesquisas. Direto- ria de Estatísticas e Avaliação da Educação Superior. Censo da educação superior, 2003a. ______. Secretaria de Educação Profissional e Tecnológica. Subsídios para a discus- são de proposta de anteprojeto de lei orgânica da educação profissional e tecnológica. 2003b. ______. Secretaria de Educação Profissional e Tecnológica. Pacto pela valorização da educação profissional e tecnológica — por uma profissionalização sustentável. Proposta de Agenda Mínima Pactuada MEC/Setec, 2003c. ______. Instituto Nacional de Estudos e Pesquisas. Diretoria de Estatísticas e Ava- liação da Educação Superior. Censo da educação superior, 2004a. ______. Secretaria de Educação Profissional e Tecnológica. Políticas públicas para a educação profissional e tecnológica. Brasília, abr. 2004b. ______. Instituto Nacional de Estudos e Pesquisas. Diretoria de Estatísticas e Ava- liação da Educação Superior. Censo da educação superior, 2005. ______. Instituto Nacional de Estudos e Pesquisas. Diretoria de Estatísticas e Ava- liação da Educação Superior. Censo da educação superior, 2006a. ______. Catálogo Nacional dos Cursos superiores de tecnologia. 31 jul. 2006b. ______. Educação profissional — legislação básica. 2001. Disponível em: <http:// portal.mec.gov.br/setec/arquivos/pdf/LegisBasica.pdf>. Acesso em: mar. 2008. OCDE (Organização para a Cooperação e Desenvolvimento Econômico). The kno- wledge-based economy. Paris: OCDE, 1996. OLIVEIRA, F. B.; SAUERBRONN, F. F. Trajetória, desafios e tendências no ensino superior de administração e administração pública no Brasil: uma breve contribui- ção. ______. Ministério da Educação. Parecer n. 436, 2001. ___. Ministério da Educação. Parecer n. 436, 2001. ______. Ministério da Educação. Parecer n. 436, 2001. ______. Ministério da Educação. Parecer n. 436, 2001. ______. Ministério da Educação. Parecer n. 29, 2002. BRASIL. Ministério da Educação. Decreto n. 5.154, 2004. CANOPF, L.; FESTINALLI, R. C.; ICHIKAWA, E. Y. A expansão do ensino superior em administração no sudoeste do Paraná: reflexões introdutórias. Revista de Ad- ministração Contemporânea, v. 9, n. 3, p. 79-97, jul./set. 2005. CASTELLS, M. A era da informação: economia, sociedade e cultura. A sociedade em rede. São Paulo: Paz e Terra, v. 1, p. 86-221, 1999. CASTRO, C. de M. O ensino da administração e seus dilemas: notas para debates. Revista de Administração de Empresas, Rio de Janeiro, v. 21, n. 3, p. 58-61, jul./set. 1981. COELHO, F. S. Uma radiografia do ensino de graduação em administração pública no Brasil (1995-2006). In: ENCONTRO ANUAL DA ANPAD, 32., Rio de Janeiro, 2008. Anais... Rio de Janeiro: Anpad, 2008. COUVRE, M. L. M. A formação e a ideologia do administrador de empresas. Petró- polis: Vozes, 1982. CRA-SP (CONSELHO REGIONAL DE ADMINISTRAÇÃO). Histórico do ensino de ad- ministração. Disponível em: <www.crasp.com.br/biblioteca/historico_ensino_adm. html>. Acesso em: abr. 2006. DAHLMAN, C. J. A economia do conhecimento: implicações para o Brasil. In: VELLOSO, J. P. R. (Org.). O Brasil e a economia do conhecimento. Rio de Janeiro: José Olympio, 2002. p. 161-196. FAUSTINI, L. A. Estrutura administrativa da educação básica. In: MENESES, J. G. C. et al. (Orgs.). Estrutura e funcionamento da educação básica. 2. ed. São Paulo: Pioneira Thomson Learning, 2004. p. 137-151. FISCHER, T. M. D. A difusão do conhecimento sobre organizações e gestão no Bra- sil: seis propostas de ensino para o decênio 2000/2010. Revista de Administração Contemporânea, Edição Especial, p. 123-139, 2001. FRIGA, P. N.; BETTIS, R. A.; SULLIVAN, R. S. Mudanças no ensino em adminis- tração: novas estratégias para o século XXI. Revista de Administração de Empresas, v. 44, n. 1, p. 96-115, jan./mar. 2004. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 cursos superiores de tecnologia em gestão RAP, v. 41, Edição Especial Comemorativa, p. 149-170, 2007. SEMINÁRIO NACIONAL DE EDUCAÇÃO PROFISSIONAL. Documento base. Brasília, 2003. Disponível em: <www.bvseps.epsjv.fiocruz.br/html/pt/seminarioeducacaoprofis- sional.htm>. Acesso em: maio 2010. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010 adriana roseli wünsch takahashi 414 SILVA; M. R.; FISCHER, T. Ensino de administração: um estudo da trajetória cur- ricular de cursos de graduação. In: ENCONTRO ANUAL DA ANPAD, 32., Rio de Janeiro, 2008. Anais... Rio de Janeiro: Anpad, 2008. SILVA; M. R.; FISCHER, T. Ensino de administração: um estudo da trajetória cur- ricular de cursos de graduação. In: ENCONTRO ANUAL DA ANPAD, 32., Rio de Janeiro, 2008. Anais... Rio de Janeiro: Anpad, 2008. STERN, D. Human resource development in the knowledge-based economy: roles of firms, schools, and governments. In: Employment and growth in the knowledge- based economy. Paris: OECD Documents, p. 189-206, 1996. TORRECILLAS, G. L. S.; MIRAMAR, R. M. V. Educação a distância na adminis- tração e em outras graduações: a experiência de uma IES do Distrito Federal. In: ENCONTRO ANUAL DA ANPAD, 32., Rio de Janeiro, 2008. Anais... Rio de Janeiro: Anpad, 2008. VIANA, A. B. N.; MANTOVANI, D. M. N.; VIEIRA, A. R. Análise dos programas de pós-graduação avaliados pela Capes: relação entre conceitos dos programas e ín- dice de publicação. In: ENCONTRO ANUAL DA ANPAD, 32., Rio de Janeiro, 2008. Anais... Rio de Janeiro: Anpad, 2008. WOOD JR., T.; PAULA, A. P. P. O fenômeno dos MPAs brasileiros: hibridismo, di- versidade e tensões. Revista de Administração de Empresas, v. 44, n. 1, p. 116-229, jan./mar. 2004. rap — Rio de Janeiro 44(2):385-414, MAR./ABR. 2010
https://openalex.org/W2897495776	https://www.nature.com/articles/s41467-018-06530-5.pdf	English	null	Neurokinin-3 receptor activation selectively prolongs atrial refractoriness by inhibition of a background K+ channel	Nature communications	2,018	cc-by	18,812	1 Department of Clinical and Experimental Cardiology, Heart Center, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 2 Department of Cardiothoracic Surgery, RadboudUMC, Geert Grooteplein Zuid 10, 6525 GA Nijmegen, The Netherlands. 3 Department of Medical Biology, Academic Medical Center Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. 4 Biomedical Sciences VU University Medical Center, De Boelelaan 1105, 1081 HV Amsterdam, The Netherlands. 5 Department of Cardiothoracic Surgery, Heart Center, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 6 L’Institut de RYthmologie et de Modélisation Cardiaque (LIRYC), Fondation Université Bordeaux, Avenue du Haut Lévêque-33604 Pessac cedex, Bordeaux, France. These authors contributed equally: Marieke W. Veldkamp, Guillaume S. C. Geuzebroek, Antonius Baartscheer, Arie O. Verkerk. Correspondence and requests for materials should be addressed to M.W.V. (email: m.w.veldkamp@amc.uva.nl) NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications ARTICLE The increase in APD90 was statistically signiﬁcant at a concentration of 10 nM Sub-P and higher, and exceeded 20% at a concentration of 1 µM (Fig. 1e, dotted lines). Sub-P caused a signiﬁcant AP prolongation at all tested stimulation frequencies (1, 2, 3 and 4 Hz, Fig. 1f). Effects of Sub-P on atrial membrane currents. We next inves- tigated the effects of Sub-P on the major membrane currents controlling atrial AP duration in rabbit atrial cardiomyocytes, including steady-state and transient outward currents, L-type calcium currents, and calcium-dependent currents. Steady-state and transient outward currents. Representative examples of current tracings recorded at −120 and +40 mV (see protocol, Fig. 2a) show a decrease in steady-state outward current in the presence of Sub-P (10 µM), which was conﬁrmed by the average current–voltage (I–V) relationships in Fig. 2b. A statis- tically signiﬁcant decrease in steady-state outward current was induced at all voltages positive to −50 mV. At +50 mV the outward current decreased by 20% from 6.9 ± 0.5 pA/pF (Control) to 5.5 ± 0.6 pA/pF (Sub-P, p < 0.05). In addition, the inward component of steady-state current at −120 mV was moderately decreased by Sub-P (Control: −6.8 ± 1.1 vs Sub-P: −6.0 ± 1.3 pA/pF, p < 0.05). p Substance-P14 belongs to the tachykinin peptide family. It exerts its biological effects by binding to one or more of three distinct types of neurokinin receptors (NK-1R, NK-2R and NK- 3R), with a preferential binding for NK-1R15,16. Sub-P is widely distributed in the cardiovascular system and is involved in the regulation of many physiological functions, including heart rate (through stimulation of cholinergic neurons) and blood pressure17,18. Reduced Sub-P content has been associated with AF incidence19,20. Speciﬁcally, the occurrence of post-operational AF was associated with decreased Sub-P serum levels in patients who had undergone coronary artery bypass surgery19. Furthermore, degeneration of Sub-P immuno-reactive nerves has been reported in a dog model of AF20. These observations imply that intact Sub- P levels protect against the development of AF. However, a direct action of Sub-P on atrial myocardial electrophysiology has thus far not been identiﬁed. p p p The resultant I–V curve of the Sub-P sensitive current (Fig. 2c, grey ﬁlled circles) is virtually linear and has a reversal potential (dotted line) close to the calculated Nernst potential for K+ (−85 mV), indicating that it concerns a background K+ current. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 N N ormal atrial electrophysiology is critically controlled by the cardiac autonomic nervous system (ANS), which modulates heart rate, excitability, conduction and refractoriness1,2. The cardiac ANS plays a key role in the genesis of malignant atrial arrhythmias, particularly atrial ﬁbrillation (AF)3–7. The major neurotransmitters that mediate the control of cardiac electrophysiological properties are the classical neu- rotransmitters acetylcholine (ACh) and noradrenalin (NA). However, the intracardiac ganglia located in fat pads on the heart produce a variety of neuropeptides including substance-P (Sub-P)8–11, in addition to NA and ACh. While the actions of these neuropeptides on the cardiac neuron as effector cell are subject of ongoing investigation, their direct effects on atrial cardiomyocyte electrophysiology are largely unknown. This is relevant considering the potential role of neuropeptides in arrhythmogenesis as they may constitute targets for anti- arrhythmic therapy. Current therapeutic approaches in AF are aimed at prolonging action potential duration and consequently increasing refractoriness of atrial tissue, thereby reducing the susceptibility for re-entrant based arrhythmias12,13. However, clinical success rate is often diminished by the limited selectivity for atrial tissue, potentially leading to ventricular pro-arrhythmia secondary to action potential prolongation in this cardiac compartment12,13. were statistically signiﬁcantly increased following Sub-P applica- tion. Sub-P furthermore reduced the resting membrane potential (RMP) (Control: −81.0 ± 1.5 vs. Sub-P: −75.0 ± 2.1 mV, p < 0.05, Fig. 1c, Table 1) and the action potential amplitude (APA) (Control: 108.6 ± 3.4 mV vs. Sub-P: 97.0 ± 6.1 mV, p < 0.05, Fig. 1c, Table 1). A trend towards a concomitant reduction in AP upstroke velocity (Vmax) was observed following Sub-P applica- tion (Fig. 1d, Table 1), but this did not reach statistical sig- niﬁcance due to a large variation between cells. were statistically signiﬁcantly increased following Sub-P applica- tion. Sub-P furthermore reduced the resting membrane potential (RMP) (Control: −81.0 ± 1.5 vs. Sub-P: −75.0 ± 2.1 mV, p < 0.05, Fig. 1c, Table 1) and the action potential amplitude (APA) (Control: 108.6 ± 3.4 mV vs. Sub-P: 97.0 ± 6.1 mV, p < 0.05, Fig. 1c, Table 1). A trend towards a concomitant reduction in AP upstroke velocity (Vmax) was observed following Sub-P applica- tion (Fig. 1d, Table 1), but this did not reach statistical sig- niﬁcance due to a large variation between cells. g Sub-P prolonged APD90 in a dose-dependent manner (Fig. 1e). ARTICLE For accurate measurement of the transient outward current (Ito), blockers of the Na+ current (INa), the L-type calcium current (ICa,L), and of the slow and rapid components of the delayed rectiﬁer currents (IKr and IKs) were included in the various solutions (see Methods section). Figure 2d shows representative Ito currents upon depolarization to +50 mV, demonstrating no change in peak currents following application of Sub-P (10 µM). On average, Sub-P did not affect Ito peak densities (Fig. 2e) or the time course of current decay at +50 mV (Table 2), but it caused a small, but signiﬁcant shift in V1/2 of inactivation by −3.5 mV (Table 2, p < 0.05), as well as a steepening of the slope of the voltage dependency of activation (Table 2, p < 0.01). Figure 2f depicts the average I–V relation- ships, obtained by current amplitude measurement at the end of the 500 ms voltage steps. Despite the presence of the various ion channel blockers, Sub-P causes a decrease in the steady-state inward current from −6.9 ± 1.6 pA/pF to −5.8 ± 1.5 pA/pF at −120 mV (p < 0.05), and a 25% reduction in steady-state outward current at +50 mV (Control: 6.1 ± 0.85 vs. Sub-P: 4.6 ± 0.46 pA/ pF, p < 0.05). The Sub-P-sensitive current under these experi- mental conditions is plotted in Fig. 2c (grey ﬁlled triangles). It shows an I–V relation similar to the sub-P-sensitive current in the absence of the various ion channel blockers (Fig. 2c, grey ﬁlled circles). These data demonstrate that the Sub-P effects on background current are not mediated by changes in the delayed rectiﬁer potassium currents IKr and IKs. In this study, we investigated the electrophysiological effects of the neuropeptide Sub-P in isolated rabbit atrial myocytes, and in Langendorff-perfused and in situ rabbit hearts. We show that Sub-P produces a selective prolongation of atrial AP duration and refractoriness in a dose-dependent manner. Importantly, these effects were also present at high pacing rates (typically observed in AF), and were absent in ventricular myocardium, indicating atrial-speciﬁc efﬁcacy. We demonstrate that these effects are mediated through activation of the neurokinin-3 receptor (NK- 3R) and involve inhibition of a background K+ current. In a rabbit isolated heart model of AF, NK-3R stimulation produced a strong anti-arrhythmic effect by reducing AF duration and inci- dence. We therefore propose NK-3R as a therapeutic target for re- entry-based atrial arrhythmias, including AF. Neurokinin-3 receptor activation selectively prolongs atrial refractoriness by inhibition of a background K+ channel Marieke W. Veldkamp1, Guillaume S.C. Geuzebroek2, Antonius Baartscheer1, Arie O. Verkerk3, Cees A. Schumacher1, Gedeon G. Suarez4, Wouter R. Berger1, Simona Casini1, Shirley C.M. van Amersfoorth1, Koen T. Scholman3, Antoine H.G. Driessen5, Charly N.W. Belterman1, Antoni C.G. van Ginneken3, Joris R. de Groot1, Jacques M.T. de Bakker1, Carol Ann Remme1, Bas J. Boukens3 & Ruben Coronel1,6 The cardiac autonomic nervous system (ANS) controls normal atrial electrical function. The cardiac ANS produces various neuropeptides, among which the neurokinins, whose actions on atrial electrophysiology are largely unknown. We here demonstrate that the neurokinin substance-P (Sub-P) activates a neurokinin-3 receptor (NK-3R) in rabbit, prolonging action potential (AP) duration through inhibition of a background potassium current. In contrast, ventricular AP duration was unaffected by NK-3R activation. NK-3R stimulation lengthened atrial repolarization in intact rabbit hearts and consequently suppressed arrhythmia duration and occurrence in a rabbit isolated heart model of atrial ﬁbrillation (AF). In human atrial appendages, the phenomenon of NK-3R mediated lengthening of atrial repolarization was also observed. Our ﬁndings thus uncover a pathway to selectively modulate atrial AP duration by activation of a hitherto unidentiﬁed neurokinin-3 receptor in the membrane of atrial myocytes. NK-3R stimulation may therefore represent an anti-arrhythmic concept to sup- press re-entry-based atrial tachyarrhythmias, including AF. 1 1 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 All values shown are mean ± * P < 0.05 a 25 ms 25 pA Control 1 μM Sub-P 1 Hz 0 mV 0 20 40 60 80 100 120 140 APD50 APD90 APD20 * * Duration (ms) Control Sub-P (1 μM) n = 6, 1 Hz b 0 mV 50 0 –50 –100 100 150 Potential (mV) RMP APA * * c Vmax (V/s) d 0 100 200 300 400 500 600 700 d c Frequency (Hz) 4 2 1 70 80 90 100 110 120 130 140 APD90 (ms) * * * * Control Sub-P (1 μM) n = 6 f 0 3 100 Control Control Sub-P n = 5, 2 Hz * * * 100 110 120 130 140 150 160 10–9 10–8 10–7 10–6 10–5 APD90 (ms) Concentration (M) e * f e Fig. 1 Effect of substance-P on action potential parameters of rabbit atrial cardiomyocytes. a APs elicited at 1 Hz from an atrial cardiomyocyte under control conditions and in the presence of 1 µM Sub-P. b–d Bar graph showing average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) b, resting membrane potential (RMP) and action potential amplitude (APA) c, and maximal upstroke velocity (Vmax) d before (Control) and after application of 1 µM Sub-P. (N = 4, n = 6, paired t-test.) e Concentration-dependence of APD90 before (Control) and after application of increasing concentrations of Sub-P. Dotted lines: concentration of Sub-P at which the increase of APD90 exceeded 20%. (N = 4, n = 5, one-way RM ANOVA.) f Frequency-dependence of APD90 before (Control) and after application of 1 µM Sub-P (N = 4, n = 6, two-way RM ANOVA). All values shown are mean ± SEM * P < 0 05 L-type calcium current and calcium-dependent currents. The representative ICa,L recordings upon a depolarizing pulse to 0 mV (Fig. 3a, top: voltage protocol) show that peak ICa,L is reduced following application of Sub-P (10 µM). The average I–V rela- tionships of ICa,L (Fig. 3b) conﬁrm a statistically signiﬁcant decrease in peak ICa,L by about 20% from −21 ± 3.3 pA/pF in Control to –16.6 ± 2.3 pA/pF (p < 0.05) in the presence of Sub-P at 0 mV, expected to decrease AP duration (contrary to the observed AP prolonging effect of Sub-P). Results Sub-P prolongs the action potential in atrial cardiomyocytes. Figure 1 illustrates that application of substance-P (Sub-P; 1 µM) resulted in signiﬁcant action potential (AP) prolongation (Fig. 1a) in an isolated rabbit atrial cardiomyocyte. The average effects (mean ± SEM) of Sub-P (1 µM) on AP duration at 20%, 50% and 90% of repolarization (APD20, APD50 and APD90) in cardio- myocytes stimulated at 1 Hz are summarized in Fig. 1b. APD50 (Control: 21.2 ± 7.5 vs. Sub-P: 43.4 ± 14.6 ms, p < 0.05) and APD90 (Control: 89.0 ± 2.0 vs. Sub-P: 125.2 ± 2.5 ms, p < 0.001) NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 50 0 –50 –100 100 150 Potential (mV) RMP APA * 0 20 40 60 80 100 120 140 APD50 APD90 APD20 * * Duration (ms) Control Sub-P (1 μM) n = 6, 1 Hz * Frequency (Hz) 4 2 1 70 80 90 100 110 120 130 140 APD90 (ms) * * * * Control Sub-P (1 μM) n = 6 Control Control Sub-P n = 5, 2 Hz * * * 100 110 120 130 140 150 160 10–9 10–8 10–7 10–6 10–5 APD90 (ms) Concentration (M) Vmax (V/s) a 25 ms 25 pA Control 1 μM Sub-P 1 Hz 0 mV b c d e f 0 100 200 300 400 500 600 700 3 * Effect of substance-P on action potential parameters of rabbit atrial cardiomyocytes. a APs elicited at 1 Hz from an atrial cardiomyocyte under control ions and in the presence of 1 µM Sub-P. b–d Bar graph showing average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 PD90) b, resting membrane potential (RMP) and action potential amplitude (APA) c, and maximal upstroke velocity (Vmax) d before (Control) and pplication of 1 µM Sub-P. (N = 4, n = 6, paired t-test.) e Concentration-dependence of APD90 before (Control) and after application of increasing ntrations of Sub-P. Dotted lines: concentration of Sub-P at which the increase of APD90 exceeded 20%. (N = 4, n = 5, one-way RM ANOVA.) uency-dependence of APD90 before (Control) and after application of 1 µM Sub-P (N = 4, n = 6, two-way RM ANOVA). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Neither the voltage dependencies of (in)activation, nor the time course of current decay at 0 mV (Table 2) were changed. ﬁeld stimulation at 5 Hz, before and after application of Sub-P (10 µM). On average, no differences in diastolic Ca2+i, systolic Ca2+i or transient amplitude occurred (Supplementary Figure 1b). Figure 3c shows a representative example of the Na+–Ca2+ exchange current (INCX) recorded during a descending voltage ramp protocol (top) before and after application of Sub-P (10 µM). On average, Sub-P had no direct effect on the forward (inward) or inverse (outward) mode of the INCX (Fig. 3d). The Ca2+-activated Cl−current (ICl(Ca)) was deﬁned as the early transient peak amplitude upon depolarizing steps to +30, +40 and +50 mV (Fig. 3e). Average ICl(Ca) peak amplitudes were similar before (Control) and after application of Sub-P (Fig. 3f). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Supplementary Figure 1a shows representative examples of intracellular Ca2+i transients in atrial cardiomyocytes following 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Table 1 Effects of substance-P and selective NK-1, NK-2 and NK-3 receptor agonists on action potential parameters in atrial and ventricular myocytes RMP (mV) APA (mV) Vmax (V/s) APD20 (ms) APD50 (ms) APD90 (ms) Atrial myocyte Control −81.0 ± 1.5 108.6 ± 3.5 290.5 ± 57.9 5.1 ± 1.4 21.2 ± 7.5 89.0 ± 2.0 Sub-P (1 µM, n = 6) −75.0 ± 2.1a 97.0 ± 6.1a 236.7 ± 88.7 16.0 ± 7.0 43.4 ± 14.6a 125.2 ± 2.5a Control −82.4 ± 1.1 106.0 ± 2.4 238.6 ± 35.2 4.8 ± 0.6 14.2 ± 3.1 73.7 ± 3.1 [Sar9,Met(O2)11]-SP (100 nM, n = 7) −79.9 ± 1.7 101.8 ± 5.4 252.1 ± 72.9 5.0 ± 0.3 18.8 ± 3.9 72.7 ± 4.3 Control −81.6 ± 1.1 111.5 ± 2.3 281.6 ± 57.6 4.1 ± 1.2 25.0 ± 13.3 98.4 ± 10.3 (b-Ala)-NKA (4-10) (100 nM, n = 4) −79.0 ± 2.2 100.9 ± 6.1 255.1 ± 87.0 6.2 ± 1.4 35.1 ± 14.7 105.2 ± 10.4 Control −82.7 ± 1.2 109.9 ± 2.6 259.9 ± 47.8 6.7 ± 1.3 27.9 ± 7.2 95.8 ± 11.3 Senktide (100 nM, n = 16) −80.6 ± 1.5 104.0 ± 4.3 259.7 ± 39.2 11.3 ± 2.9 64.5 ± 7.5a 143.9 ± 9.0a Ventricular myocyte Control −85.9 ± 1.2 121.6 ± 1.6 260.4 ± 55.3 67.0 ± 9.4 140.4 ± 11.6 176.0 ± 10.6 Sub-P (1 µM, n = 9) −86.5 ± 1.2a 122.3 ± 1.8 300.0 ± 53.0a 65.9 ± 9.4 137.5 ± 11.3 171.8 ± 10.1 Control −86.4 ± 0.4 120.8 ± 1.3 267.7 ± 45.9 59.8 ± 7.9 134.3 ± 8.3 170.4 ± 7.4 Senktide (100 nM, n = 10) −86.7 ± 0.6 121.1 ± 1.4 298.0 ± 41.6a 56.8 ± 9.4 128.9 ± 10.4 166.2 ± 8.6 All values are mean ± SEM RMP: resting membrane potential, APA: action potential amplitude, Vmax: upstroke velocity, APD20: action potential duration at 20% repolarization, APD50: action potential duration at 50% repolarization, APD90: action potential duration at 90% repolarization Paired t-test = p < 0.05; Compared to control = a ce-P and selective NK-1, NK-2 and NK-3 receptor agonists on action potential parameters in atrial and All values are mean ± SEM RMP: resting membrane potential, APA: action potential amplitude, Vmax: upstroke velocity, APD20: action potential duration at 20% repolarization, APD50: action potential duration at 50% repolarization, APD90: action potential duration at 90% repolarization Paired t-test = p < 0.05; Compared to control = a d 4 6 8 10 Ito (pA/pF) Membrane potential (mV) –80 0 2 12 14 –60 Control Sub-P (10 μM) n = 5 8 6 –2 –8 0 2 4 –4 –6 –10 Steady state currenI (pA/pF) Membrane potential (mV) * * * * * * * Control Sub-P (10 μM) n = 5 10 –60 mV 50 mV –120 mV 500 ms, ΔV = 10 mV 8 6 –2 0 2 4 –4 –6 Membrane potential (mV) Steady state current (pA/pF) –8 Control Sub-P (10 μM) n = 6 * * * * * * * * * * * * a –120 mV –120 mV –120 mV 40 mV Control Sub-P (10 μM) 0 pA 250 pA 100 ms 40 mV 50 mV 50 mV Membrane potential (mV) Sub-P sensitive current (pA/pF) No blockers Blockers for INa, ICa,L, IKr, IKs Blockers for INa, Ito1, IKur 50 mV ΔV = 10 mV –80 mV –120 mV 500 ms 250 pA 100 ms 0 pA Control b c f e –2 –1 0 1 2 3 –120 –120 mV 500 ms Sub-P (10 μM) –80 –40 40 0 –120 –80 –40 40 0 –120 –80 –40 40 0 –40 –20 0 20 40 60 Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 P < 0.05 8 6 –2 0 2 4 –4 –6 Membrane potential (mV) Steady state current (pA/pF) –8 Control Sub-P (10 μM) n = 6 * * * * * * * * * * * b –120 –80 –40 40 0 M b t ti l ( V) Sub-P sensitive current (pA/pF) No blockers Blockers for INa, ICa,L, IKr, IKs Blockers for INa, Ito1, IKur c –2 –1 0 1 2 3 –120 –80 –40 40 0 –60 mV 50 mV –120 mV 500 ms, ΔV = 10 mV a –120 mV 40 mV Control Sub-P (10 μM) 0 pA 250 pA 100 ms 40 mV –120 mV b a Control Membrane potential (mV) 4 6 8 10 Ito (pA/pF) Membrane potential (mV) –80 0 2 12 14 –60 Control Sub-P (10 μM) n = 5 8 6 –2 –8 0 2 4 –4 –6 –10 Steady state currenI (pA/pF) Membrane potential (mV) * * * * * * * Control Sub-P (10 μM) n = 5 10 f e –120 –80 –40 40 0 –40 –20 0 20 40 60 d 50 mV ΔV = 10 mV –80 mV –120 mV 500 ms 500 ms d e –120 mV –120 mV 50 mV 50 mV 500 ms 250 pA 100 ms 0 pA Control 500 ms Sub-P (10 μM) Membrane potential (mV) Membrane potential (mV) Fig. 2 Effect of substance-P on steady-state and transient outward currents. a Voltage protocol and current tracings recorded at −120 and +40 mV before (Control) and after application of 10 µM Sub-P. b Average current–voltage relationships of the steady-state current in Control and after application of Sub-P (10 µM) (N = 2, n = 6, two-way RM ANOVA). c Average current–voltage relationships of the Sub-P sensitive current. Sub-P sensitive currents were derived from b (grey ﬁlled circles), f (grey ﬁlled triangles) and Supplementary Figure 2 (black ﬁlled squares), as differential current. d Voltage protocol and transient outward K+ current (Ito) current tracings recorded at −120 and +50 mV before (Control) and after application of Sub-P (10 µM). e Average current–voltage (I–V) relationships of Ito peak currents before (Control) and after application of Sub-P (10 µM) (N = 4, n = 5, two-way RM ANOVA). f Average I–V relationships of steady-state current before (Control) and after application of Sub-P (10 µM) (N = 4, n = 5, two-way RM ANOVA). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 2 Effect of substance-P on steady-state and transient outward currents. a Voltage protocol and current tracings recorded at −120 and +40 mV before (Control) and after application of 10 µM Sub-P. b Average current–voltage relationships of the steady-state current in Control and after application of Sub-P (10 µM) (N = 2, n = 6, two-way RM ANOVA). c Average current–voltage relationships of the Sub-P sensitive current. Sub-P sensitive currents were derived from b (grey ﬁlled circles), f (grey ﬁlled triangles) and Supplementary Figure 2 (black ﬁlled squares), as differential current. d Voltage protocol and transient outward K+ current (Ito) current tracings recorded at −120 and +50 mV before (Control) and after application of Sub-P (10 µM). e Average current–voltage (I–V) relationships of Ito peak currents before (Control) and after application of Sub-P (10 µM) (N = 4, n = 5, two-way RM ANOVA). f Average I–V relationships of steady-state current before (Control) and after application of Sub-P (10 µM) (N = 4, n = 5, two-way RM ANOVA). Blockers for Na+, L-type Ca2+, rapid and slow delayed rectiﬁer K+ currents were present. All values shown are mean ± SEM. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Two-way repeated measures ANOVA followed by pairwise comparison using the Student–Newman–Keuls method = p < 0.05; Compared to control = a Sub-P: substance-P, V1/2: voltage of half-maximal (in)activation, k: slope factor, tfast and tslow: fast and slow time constants of inactivation Table 2 Effects of substance-P (10 µM) on biophysical properties of the L-type calcium current (ICa,L) and the transient outward current (Ito) peated measures ANOVA followed by pairwise comparison using the Student–Newman–Keuls method = p < 0.05; Compared to control = a f-maximal (in)activation, k: slope factor, tfast and tslow: fast and slow time constants of inactivation antagonist Osanetant (300 nM) (Fig. 4d). As the AP prolonging effect of Sub-P is entirely mediated through the NK-3R, we maintained the use of Senktide—a potent analogue of the endogenous agonist of the NK-3R (neurokinin B)—in all following experiments. However, Sub-P reduced steady-state outward currents at the end of the 250 ms depolarizing steps by ~25% (Supplementary Figure 2; Control: 7.2 ± 0.8 pA/pF vs. Sub-P: 5.7 ± 0.5, Vm = + 50 mV, p < 0.05). Data points for the Sub-P-sensitive current at +30, +40 and +50 mV are included in the I–V relation of Fig. 2c (black ﬁlled squares), and show complete overlap with the Sub-P- sensitive current in the absence of ion channel blockers. Since ICl (Ca) measurements were performed in the presence of 2 mM 4-AP, the effect of Sub-P is not mediated by a reduction in sustained component of Ito or IKur. However, Sub-P reduced steady-state outward currents at the end of the 250 ms depolarizing steps by ~25% (Supplementary Figure 2; Control: 7.2 ± 0.8 pA/pF vs. Sub-P: 5.7 ± 0.5, Vm = + 50 mV, p < 0.05). Data points for the Sub-P-sensitive current at +30, +40 and +50 mV are included in the I–V relation of Fig. 2c (black ﬁlled squares), and show complete overlap with the Sub-P- sensitive current in the absence of ion channel blockers. Since ICl (Ca) measurements were performed in the presence of 2 mM 4-AP, the effect of Sub-P is not mediated by a reduction in sustained component of Ito or IKur. Nature of the background K+ current. We next explored the nature of the background K+ current affected by NK-3 receptor stimulation. Two-pore-domain potassium (K2P) channels have been often shown to underlie the background K+ currents in excitable tissues21. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 The NK-3 receptor is coupled to the Gq/11 subgroup of G-proteins22, and several members of the K2P family are known to be potently inhibited by receptors that signal through Gq/1123–25. Of those, TREK-1, TASK-1 and TASK-3, have been shown to be functionally relevant for atrial AP dura- tion26–28. We therefore considered these 3 channels likely can- didates for the AP prolonging effect of NK-3 receptor stimulation. We investigated mRNA expression levels of the different mem- bers of the K2P channel family as well as the various NK receptor types in rabbit left atrial myocardium using RNA sequencing. As shown in Fig. 5a, mRNA expression levels of TACR3 (encoding NK-3R) were clearly higher than those of TACR1 and TACR2 (encoding NK-1R and NK-2R, respectively). We did not ﬁnd differences in mRNA expression levels for the different genes between the left atrial free wall and the left atrial appendage (Fig. 5a, upper right inset). The presence of NK-3R on the plasma membrane of rabbit atrial cardiomyocytes was conﬁrmed by immunohistochemistry (Fig. 5b). The RNA sequencing results furthermore demonstrated higher expression levels of KCNK3 (encoding TASK-1) in rabbit atrial tissue as compared to KCNK2 (encoding TREK-1) and KCNK9 (encoding TASK-3) (Fig. 5a). p Taken together, these data indicate that Sub-P acts through inhibition of a K+ background current in rabbit atrial myocytes. Neurokinin-3 receptor mediates action potential prolongation. To deﬁne the neurokinin receptor (NK-R) subtype mediating the AP prolonging effect produced by Sub-P, we examined the effect of various NK receptor subtype selective agonists on AP duration in isolated rabbit atrial cardiomyocytes. Fig. 4a, b (left panels) show representative APs at 2 Hz before (Control) and after application of the NK-1R agonist [Sar9,Met(O2)11]-SP (100 nM), and the NK-2R agonist (β-Ala)-NKA(4-10) (100 nM), respec- tively. Administration of NK-1R and NK-2R agonists did not signiﬁcantly alter AP morphology or duration (Fig. 4a, b (right panels), Table 1). In contrast, application of 100 nM of the NK-3R agonist Senktide (Fig. 4c) signiﬁcantly prolonged APD50 (Con- trol: 27.9 ± 7.2 vs. Senktide: 64.5 ± 7.5 ms, p < 0.001) and APD90 (Control: 95.8 ± 11.3 vs. Senktide: 143.9 ± 9.0 ms, p < 0.001). Senktide (100 nM) prolonged APD50 and APD90 by 130% and 50%, respectively, but did not alter other AP parameters (Table 1). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Blockers for Na+, L-type Ca2+, rapid and slow delayed rectiﬁer K+ currents were present. All values shown are mean ± SEM. P < 0.05 Fig. 2 Effect of substance-P on steady-state and transient outward currents. a Voltage protocol and current tracings recorded at −120 and +40 mV before (Control) and after application of 10 µM Sub-P. b Average current–voltage relationships of the steady-state current in Control and after application of Sub-P (10 µM) (N = 2, n = 6, two-way RM ANOVA). c Average current–voltage relationships of the Sub-P sensitive current. Sub-P sensitive currents were derived from b (grey ﬁlled circles), f (grey ﬁlled triangles) and Supplementary Figure 2 (black ﬁlled squares), as differential current. d Voltage protocol and transient outward K+ current (Ito) current tracings recorded at −120 and +50 mV before (Control) and after application of Sub-P (10 µM). e Average current–voltage (I–V) relationships of Ito peak currents before (Control) and after application of Sub-P (10 µM) (N = 4, n = 5, two-way RM ANOVA). f Average I–V relationships of steady-state current before (Control) and after application of Sub-P (10 µM) (N = 4, n = 5, two-way RM ANOVA). Blockers for Na+, L-type Ca2+, rapid and slow delayed rectiﬁer K+ currents were present. All values shown are mean ± SEM. P < 0.05 NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications 4 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Table 2 Effects of substance-P (10 µM) on biophysical properties of the L-type calcium current (ICa,L) and the transient outward current (Ito) ICa,L-Control ICa,L-Sub-P n Ito-Control Ito-Sub-P n Current density (pA/pF) −21.3 ± 3.3 −16.6 ± 2.3a 6 9.7 ± 2.9 8.9 ± 2.8 5 Activation V1/2, mV −15.4 ± 0.8 −17.2 ± 1.4 6 −7.0 ± 7.4 −15.1 ± 3.3 5 k, mV 6.8 ± 0.5 7.0 ± 0.7 6 14.2 ± 0.9 9.8 ± 0.1a 5 Inactivation V1/2, mV −28.9 ± 1.3 −31.2 ± 2.3 6 −32.5 ± 3.4 −35.9 ± 2.8a 5 k, mV 4.4 ± 0.3 4.9 ± 0.3 6 −5.9 ± 0.9 −5.4 ± 0.2 5 Inactivation time course τfast, ms 4.4 ± 0.6 4.9 ± 0.7 6 39.6 ± 13.2 41.3 ± 8.1 5 τslow, ms 28.0 ± 3.5 28.2 ± 3.4 6 131.1 ± 33.7 114.6 ± 16.2 5 All values are mean ± SEM. NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 P < 0.05 V Membrane potential (mV) ICa,L (pA/pF) –25 –20 –15 –10 –5 5 0 –60 * * * * * 20 Control Sub-P (10 μM) n = 6 b –40 –20 0 40 a –60 mV 50 mV P1 P2 300 ms ΔV = 10 mV 0 mV 200 ms Control 0 pA –60 mV 0 mV 50 ms 200 pA Sub-P (10 μM) b 50 mV Control Control 0 pA –60 mV 0 mV 50 ms 200 pA Sub-P (10 μM) Membrane potential (mV) Membrane potential (mV) 80 mV –40 mV 2 s 100 ms –120 mV 0 pA Control 200 pA 500 ms c Sub-P (10 μM) Membrane potential (mV) INCX (pA/pF) –100 –12 12 8 4 0 –4 –8 0 Control Sub-P (10 μM) n = 6 d –50 50 c d –60 mV 50 mV 250 ms ΔV = 10 mV e Sub-P (10 μM) Control 200 pA 50 ms 30 mV 0 pA 30 mV Membrane potential (mV) ICl(Ca) (pA/pF) 50 40 30 0 5 10 15 20 25 Control Sub-P (10 μM) n = 6 f f e Membrane potential (mV) Fig. 3 Effect of substance-P on L-type Ca2+ current and calcium-dependent currents. a Voltage protocol and ICa,L tracings recorded at 0 mV under control conditions (Control) and in the presence of Sub-P (10 µM). b Average current–voltage (I–V) relationships of ICa,L during Control and after application of Sub-P (10 µM) (N = 3, n = 6, two-way RM ANOVA). c Voltage protocol and Na+–Ca2+ exchange current (INCX) tracings recorded in Control and in the presence of Sub-P (10 µM). d Average I–V relationships of INCX before (Control) and after application of Sub-P (10 µM) (N = 2, n = 6, two-way RM ANOVA). e Voltage protocol and Ca2+ activated Cl−current (ICa(Cl)) recordings at +30 mV in Control and in the presence of Sub-P (10 µM). f Average I–V relationships of peak ICa(Cl) before (Control) and after application of Sub-P (10 µM) (N = 2, n = 6, two-way RM ANOVA). Blockers for Na+-, transient outward and ultra-rapid delayed rectiﬁer K+ currents were continuously present. All values shown are mean ± SEM. P < 0.05 increase in APD90 from 95.6 ± 10.5 ms to 100.4 ± 10.9 ms (Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 To rule out the possibility of a time-dependent increase in APD90 contributing to the observed Senktide effect, we per- formed a set of time-matched controls. Supplementary Figure 3a shows in a representative example that the APD90 remained stable during a period of 15 min, whereafter the application of 50 nM Senktide caused a steep increase in APD90. On average, APD90 remained unchanged at 1, 5 and 10 min (common dura- tion of the experiment) (Supplementary Figure 3b). To test the potential contribution of these K2P channels to the observed effects of NK-3 receptor stimulation, we assessed the effects of selective blockers for these channels on steady-state current and AP duration. We ﬁrst examined the effects of ZnCl2 on steady-state outward currents in individual rabbit atrial cardiomyocytes. Zinc has been reported to be a selective blocker of TASK-3 channels, but may also inhibit TASK-1 and TREK-1 channels, albeit with lower sensitivities.29–31 As expected, the average I–V relation in Fig. 6b shows a signiﬁcant decrease in steady-state outward current at all voltages positive to −60 mV following Senktide application (50 nM), similar to the effects observed for Sub-P (Fig. 2b). At +30 mV the outward current decreased by 44% from 4.6 ± 0.4 pA/pF (Control, n = 8) to 3.2 ± 0.3 pA/pF (Senktide, n = 8, p < 0.05). The Senktide-sensitive I–V relation (Fig. 6c) is similar to the Sub-P-sensitive current (Fig. 2c), Figure 4d shows the dose-response relationship of Senktide and atrial APD90. At a concentration of ~10 nM, Senktide induced a 20% increase in APD90 (dotted lines), indicating that it is 100 times more potent than Sub-P (cf. Fig. 1e). Taken together these data indicate that AP prolongation by Sub-P is consequent to a reduction in a background K+ current which is mediated through stimulation of the NK-3 receptor. This was further supported by the observation that the Senktide-induced AP prolongation was largely prevented by pre-incubation with the competitive NK-3R NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunication 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Membrane potential (mV) INCX (pA/pF) –100 –12 12 8 4 0 –4 –8 0 Control Sub-P (10 μM) n = 6 80 mV –40 mV 2 s 100 ms –120 mV 0 pA Control 200 pA 500 ms Membrane potential (mV) ICl(Ca) (pA/pF) 50 40 30 0 5 10 15 20 25 Control Sub-P (10 μM) n = 6 –60 mV 50 mV 250 ms ΔV = 10 mV e Sub-P (10 μM) Control 200 pA 50 ms 30 mV 0 pA 30 mV a –60 mV 50 mV P1 P2 300 ms ΔV = 10 mV 0 mV 200 ms Control 0 pA –60 mV 0 mV 50 ms c Membrane potential (mV) ICa,L (pA/pF) –25 –20 –15 –10 –5 5 0 –60 * * * * * 20 Control Sub-P (10 μM) n = 6 200 pA f b d Sub-P (10 μM) –40 –20 0 –50 50 40 Sub-P (10 μM) Fig. 3 Effect of substance-P on L-type Ca2+ current and calcium-dependent currents. a Voltage protocol and ICa,L tracings recorded at 0 mV under control onditions (Control) and in the presence of Sub-P (10 µM). b Average current–voltage (I–V) relationships of ICa,L during Control and after application of Sub-P (10 µM) (N = 3, n = 6, two-way RM ANOVA). c Voltage protocol and Na+–Ca2+ exchange current (INCX) tracings recorded in Control and in the presence of Sub-P (10 µM). d Average I–V relationships of INCX before (Control) and after application of Sub-P (10 µM) (N = 2, n = 6, two-way RM ANOVA). e Voltage protocol and Ca2+ activated Cl−current (ICa(Cl)) recordings at +30 mV in Control and in the presence of Sub-P (10 µM). f Average –V relationships of peak ICa(Cl) before (Control) and after application of Sub-P (10 µM) (N = 2, n = 6, two-way RM ANOVA). Blockers for Na+-, transient outward and ultra-rapid delayed rectiﬁer K+ currents were continuously present. All values shown are mean ± SEM. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Bar graph shows average values for APD20, APD50 and APD90 before (Control) and after application of 100 nM Senktide (N = 7, n = 16, paired t-test). d Concentration-response curve for increase of APD induced by Senktide in the absence (N = 13, n = 9–16, one-way RM ANOVA) and presence of 300 nM Osanetant (N = 3, n = 3–5, two-way RM ANOVA). Dotted lines: concentration of Senktide at which APD90 prolongation exceeded 20%. All values shown are mean ± SEM. P < 0.05 tive neurokinin receptor agonists on action potential duration. a APs elicited at 2 Hz from an atrial cardiomyocyte un Fig. 4 Effects of different selective neurokinin receptor agonists on action potential duration. a APs elicited at 2 Hz from an atrial cardiomyocyte under control conditions (Control) and in the presence of 100 nM NK-1R agonist [Sar9,Met(O2)11]-SP. Bar graph shows average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 100 nM [Sar9,Met(O2)11]-SP (N = 6, n = 7, paired t-test). b Atrial APs elicited at 2 Hz in Control and in the presence of the NK-2R agonist (β-Ala)-NKA(4-10) (100 nM). Bar graph shows average values for APD20, APD50, and APD90 before (Control) and after application of 100 nM (β -Ala)-NKA(4-10) (N = 3, n = 4, paired t-test). c Atrial APs elicited at 2 Hz in Control and in the presence of the NK-3R agonist Senktide (100 nM). Bar graph shows average values for APD20, APD50 and APD90 before (Control) and after application of 100 nM Senktide (N = 7, n = 16, paired t-test). d Concentration-response curve for increase of APD induced by Senktide in the absence (N = 13, n = 9–16, one-way RM ANOVA) and presence of 300 nM Osanetant (N = 3, n = 3–5, two-way RM ANOVA). Dotted lines: concentration of Senktide at which APD90 prolongation exceeded 20%. All values shown are mean ± SEM. P < 0.05 Ventricular APD is unaffected by NK-3 receptor stimulation. We also explored the potential atrial selectivity of NK-3R sti- mulation, a prerequisite for safe anti-arrhythmic efﬁcacy. We therefore tested the effects of Sub-P and Senktide on isolated rabbit ventricular cardiomyocytes. Figure 7a, b shows repre- sentative ventricular APs at 2 Hz in the absence and in the pre- sence of the NK-3R agonists Sub-P (1 µM) and Senktide (100 nM), respectively. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Neither Sub-P nor Senktide prolonged the ventricular AP. Overall, no relevant alterations were observed for the other ventricular AP characteristics on NK-3R stimulation, except for an increase in Vmax (Table 1, Fig. 7a, b). showed a similar increase in AERP in response to 20 nM Senktide (Supplementary Figure 5a, b). We next studied the in vivo response to intravenous Senktide administration on AERP in open-chested rabbits. Supplementary Figure 5c shows unipolar electrograms recorded from the left atrium during programmed stimulation, showing an increased AERP following infusion of a single bolus of Senktide (11 nmol/ kg). On average, AERP increased from 96.7 ± 13.3 ms to 130.0 ± 20.8 ms after application of Senktide (34% increase; Supplemen- tary Fig. 5d, p < 0.01). Similarly, we tested the effect of Senktide on ERP in human isolated left atrial appendages (LAA) resected from patients with persistent AF undergoing thoracoscopic surgery for AF. Similar to rabbit atrial tissue, robust cardiomyocyte plasma membrane labelling of NK-3R was observed in human LAA tissue (Fig. 5b). As shown in the example depicted in Fig. 8b (left panel), superfusion of the LAA preparation (stimulated at BCL 600 ms) with 100 nM Senktide led to an increase in ERP. Prolongation of the ERP occurred in all ﬁve human LAAs tested. On average, Senktide caused an 18.0 ± 0.1% increase of LAA ERP from 201.8 ± 6.1 ms (Control) to 237.8 ± 7.5 ms (Senktide) (Fig. 8b, right panel, n = 5, p = 0.06). NK-3 receptor stimulation prevents atrial ﬁbrillation. To assess the potential impact of selective NK-3R activation on the in vivo, whole heart and tissue level, we tested whether NK-3R mediated AP prolongation is accompanied by an increase in effective refractory period (ERP) in rabbit intact hearts and human atrial tissue. Figure 8a (left panel) shows atrial unipolar electrograms recorded from a Langendorff-perfused rabbit heart in response to programmed stimulation at the right atrium, showing a prolongation of right atrial effective refractory period (AERP) after addition of the NK-3R agonist (Senktide 20 nM). On average, right AERP increased from 68.1 ± 5.1 ms at baseline (Control) to 89.3 ± 6.3 ms after application of 20 nM Senktide (32% increase; p = 0.00006) (Fig. 8a, right panel). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 6j, n = 8, p < 0.05), PK-THPP application induced an 8% increase in APD90 from 73.5 ± 3.2 ms to 79.0 ± 3.1 ms (Fig. 6k, n = 7, p < 0.05), and Spadin application resulted in a 3% increase in APD90 from 80.1 ± 6.7 ms to 82.1 ± 6.6 ms (Fig. 6l, n = 7, p = NS) (Supplementary Table 1). Subsequent addition of 50 nM Senktide in the continued presence of the respective blockers, resulted in a 41% (ML-365), 53% (PK-THHP) and 42% (Spadin) increase in APD90 (Fig. 6g–l, Supplementary Table 1). These percentages increase in APD90 induced by Senktide in the presence of the selective blockers, is in the same order of magnitude as the increase in APD90 by Senktide alone (Fig. 4d). This implies that the background current inhibited by NK-3 receptor stimulation is likely not carried by one of these K+ channels alone. both in shape and magnitude of current density. The application of ZnCl2 signiﬁcantly reduced steady-state outward current (Fig. 6d,e). At +30 mV, the average steady-state outward current (Fig. 6e) was reduced by 39% from 7.8 ± 0.9 pA (Control, n = 5) to 5.6 ± 0.8 (ZnCl2, n = 5, p < 0.05). The ZnCl2-sensitive current (Fig. 6f) was not critically different from the Senktide-sensitive current (Fig. 6c). In the continued presence of ZnCl2, additional application of 50 nM Senktide did not further reduce steady-state outward current (Fig. 6f). These ﬁndings imply that the ZnCl2- sensitive current and the Senktide-sensitive current are the same, and possibly carried by either TASK-1, TASK-3 or TREK-1 channels. The selective blockers for TASK-1 (ML-365), TASK-3 (PK-THPP) and TREK-1 (Spadin), all gave a minor prolongation of the APD90 (Fig. 6g–i). Application of ML-365 gave rise to a 5% NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications 6 6 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 P < 0.05 0 Duration (ms) b Control [Sar9,Met(O2)11]-SP (100 nM) n = 7, 2 Hz ms ist 0 20 40 60 80 100 APD20 APD50 APD90 0 mV 0 mV Duration (ms) b a Control [Sar9,Met(O2)11]-SP (100 nM) n = 7, 2 Hz Control 100 nM (β-Ala)-NKA (4–10) Control 100 nM [Sar9,Met(O2)11]-SP 25 ms 10 mV NK1R-agonist NK2R-agonist Control (β-Ala)-NKA (4–10) (100 nM) n = 4, 2 Hz Duration (ms) 0 20 40 60 80 100 0 20 40 60 80 100 120 140 APD20 APD50 APD90 APD20 APD50 APD90 0 mV 0 m Duration (ms) b a Control [Sar9,Met(O2)11]-SP (100 nM) n = 7, 2 Hz Control 100 nM [Sar9,Met(O2)11]-SP 25 ms 10 mV NK1R-agonist 0 20 40 60 80 100 APD20 APD50 APD90 mV b 100 nM (β-Ala)-NKA (4–10) Control NK2R-agonist Control (β-Ala)-NKA (4–10) (100 nM) n = 4, 2 Hz Duration (ms) 0 20 40 60 80 100 120 140 APD20 APD50 APD90 b b a Control Senktide n = 9-16, 2Hz d agonist Control Concentration (M) * * * * * * 100 120 140 160 180 Control 300 nM osanetant + senktide n = 3–5, 2 Hz APD90 (ms) 10–6 10–7 10–8 10–9 0 mV Duration (ms) Control Senktide (100 nM) n = 16, 2 Hz c Control 100 nM senktide NK3R-a 0 50 100 150 200 250 * * APD20 APD50 APD90 c Concentration (M) Fig. 4 Effects of different selective neurokinin receptor agonists on action potential duration. a APs elicited at 2 Hz from an atrial cardiomyocyte under control conditions (Control) and in the presence of 100 nM NK-1R agonist [Sar9,Met(O2)11]-SP. Bar graph shows average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 100 nM [Sar9,Met(O2)11]-SP (N = 6, n = 7, paired t-test). b Atrial APs elicited at 2 Hz in Control and in the presence of the NK-2R agonist (β-Ala)-NKA(4-10) (100 nM). Bar graph shows average values for APD20, APD50, and APD90 before (Control) and after application of 100 nM (β -Ala)-NKA(4-10) (N = 3, n = 4, paired t-test). c Atrial APs elicited at 2 Hz in Control and in the presence of the NK-3R agonist Senktide (100 nM). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 0 mV 0 mV 0 mV Duration (ms) Duration (ms) Control Senktide (100 nM) n = 16, 2 Hz Control Senktide n = 9-16, 2Hz b a c d Control [Sar9,Met(O2)11]-SP (100 nM) n = 7, 2 Hz Control 100 nM (β-Ala)-NKA (4–10) Control Control 100 nM senktide 100 nM [Sar9,Met(O2)11]-SP 25 ms 10 mV NK1R-agonist NK2R-agonist NK3R-agonist Control (β-Ala)-NKA (4–10) (100 nM) n = 4, 2 Hz Duration (ms) Control Concentration (M) * * * * * * * 100 120 140 160 180 Control 300 nM osanetant + senktide n = 3–5, 2 Hz 0 20 40 60 80 100 0 20 40 60 80 100 120 140 0 50 100 150 200 250 * * APD20 APD50 APD90 APD20 APD50 APD90 APD20 APD50 APD90 APD90 (ms) 10–6 10–7 10–8 10–9 Fig. 4 Effects of different selective neurokinin receptor agonists on action potential duration. a APs elicited at 2 Hz from an atrial cardiomyocyte under control conditions (Control) and in the presence of 100 nM NK-1R agonist [Sar9,Met(O2)11]-SP. Bar graph shows average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 100 nM [Sar9,Met(O2)11]-SP (N = 6, n = 7, paired t-test). b Atrial APs elicited at 2 Hz in Control and in the presence of the NK-2R agonist (β-Ala)-NKA(4-10) (100 nM). Bar graph shows average values for APD20, APD50, and APD90 before (Control) and after application of 100 nM (β -Ala)-NKA(4-10) (N = 3, n = 4, paired t-test). c Atrial APs elicited at 2 Hz in Control and in the presence of the NK-3R agonist Senktide (100 nM). Bar graph shows average values for APD20, APD50 and APD90 before (Control) and after application of 100 nM Senktide (N = 7, n = 16, paired t-test). d Concentration-response curve for increase of APD induced by Senktide in the absence (N = 13, n = 9–16, one-way RM ANOVA) and presence of 300 nM Osanetant (N = 3, n = 3–5, two-way RM ANOVA). Dotted lines: concentration of Senktide at which APD90 prolongation exceeded 20%. All values shown are mean ± SEM. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Left AERP, measured during programmed stimulation at the left atrium, In a rabbit isolated heart model of AF based on atrial dilatation, we ﬁnally examined whether the increase in AERP resulting from NK-3 receptor stimulation by Senktide, suppresses AF induci- bility. Fig. 8c shows representative electrograms recorded from the left atrium following a burst pacing protocol to provoke AF. 7 NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 0 100 200 300 400 500 600 700 800 KCNK1 KCNK10 KCNK12 KCNK13 KCNK15 TACR1 TACR2 TACR3 LAFW LAA Normalized tag counts KCNK6 KCNK5 KCNK4 KCNK3 KCNK2 KCNK18 KCNK17 KCNK16 KCNK9 0 0 1 2 3 4 5 LAFW (log (normalized counts)) LAA (log (normalized counts)) 1 2 3 4 5 6 –1 –1 6 Rabbit Human NK3R NK3R α-actinin nucleus a b α-actinin ression of the NK-3 receptor and KCNK gene family in left atrium of rabbit and human. a Bar graph showing the normalized tag counts from RNA- e TACR gene and KCNK gene family in the left atrial free wall (LAFW) and the left atrial appendage (LAA) of rabbit (n = 1). Right inset: Scatter plot he relation between expression proﬁles of all genes in LAWF and LAA in rabbit. The blue and red dots indicate TACR and KCNK genes, ly. b Sections of rabbit (upper panel) and human (lower panel) left atrium showing labelling of the NK-3R (green) in the peripheral sarcolemma myocytes, co-stained for the intracellular myocardial marker a-actinin (red). Scale bar 25 µM. Negative controls for immunohistochemistry are Supplementary Figure 4 0 100 200 300 400 500 600 700 800 KCNK1 KCNK10 KCNK12 KCNK13 KCNK15 TACR1 TACR2 TACR3 LAFW LAA Normalized tag counts KCNK6 KCNK5 KCNK4 KCNK3 KCNK2 KCNK18 KCNK17 KCNK16 KCNK9 0 0 1 2 3 4 5 LAFW (log (normalized counts)) LAA (log (normalized counts)) 1 2 3 4 5 6 –1 –1 6 a Rabbit Human NK3R NK3R α-actinin nucleus b α-actinin b Human Fig. 5 Expression of the NK-3 receptor and KCNK gene family in left atrium of rabbit and human. a Bar graph showing the normalized tag counts from RNA- seq for the TACR gene and KCNK gene family in the left atrial free wall (LAFW) and the left atrial appendage (LAA) of rabbit (n = 1). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Right inset: Scatter plot showing the relation between expression proﬁles of all genes in LAWF and LAA in rabbit. The blue and red dots indicate TACR and KCNK genes, respectively. b Sections of rabbit (upper panel) and human (lower panel) left atrium showing labelling of the NK-3R (green) in the peripheral sarcolemma of cardiomyocytes, co-stained for the intracellular myocardial marker a-actinin (red). Scale bar 25 µM. Negative controls for immunohistochemistry are shown in Supplementary Figure 4 All electrograms were recorded from the same heart with and without increased left atrial pressure, and in the absence and presence of Senktide. In the non-dilated left atrium (0 cm H2O), burst pacing incidentally elicited short runs of premature atrial activations (Fig. 8c, upper left). However, in the dilated atrium where intra-atrial pressure was increased to 20 cm H2O, burst pacing regularly resulted in episodes of AF (Fig. 8c, lower left). On average, the total duration of arrhythmia episodes following each of the 10 consecutive runs of burst pacing, increased from 4.0 ± 0.6 s at 0 cm H2O to 20.6 ± 5.3 s at 20 cm H2O (Fig. 8d, n = 7, p < 0.05, Supplementary Table 2). In the presence of 20 nM Senktide, however, burst pacing failed to evoke any arrhythmias in the undilated atrium (Fig. 8c, upper right), and the duration of the arrhythmia episode was strongly reduced in the dilated atrium (Fig. 8c, lower right). On average, the total duration of arrhythmia episodes in the presence of 20 nM Senktide decreased by ~65% from 4.0 ± 0.6 s to 1.5 ± 0.2 s at 0 cm H2O, and from 20.6 ± 5.3 s to 6.6 ± 2.5 s at 20 cm H2O (Fig. 8d, n = 7, p < 0.05, Supplemen- tary Table 2). Senktide not only reduced the duration of the arrhythmia episodes, but also reduced the AF incidence. Figure 8e shows that AF incidence in the presence of Senktide was reduced from 0.08 ± 0.04 to 0.0 ± 0.0 (n = 7, p < 0.05) at 0 cm H2O and from 0.59 ± 0.09 to 0.17 ± 0.08 at 20 cm H20 (n = 7, p < 0.05), representing a 70% reduction in AF vulnerability. All effects were reversible upon wash-out of Senktide, and on normalization of pressure (Fig. 8d, e, Supplementary Table 2). Thus, by selectively increasing AERP, NK-3R stimulation strongly reduces AF inducibility and duration. NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 I (pA/pF) Control –15 –10 –5 0 5 10 * ***** # # * ZnCl2 + senktide n = 5 ZnCl2 100 μM I (pA/pF) ZnCl2 – sensitive current ZnCl2 + senktide sensitive current –2 –1 0 1 2 3 4 Vm (mV) Control Senktide 50 nM n = 8 –10 –8 –6 –4 –2 0 2 4 6 –150 –100 –50 0 50 –150 –100 –50 0 50 * * * * * * * * * * I (pA/pF) Vm (mV) Vm (mV) –150 –100 –50 0 50 –150 –100 –50 0 50 Vm (mV) I (pA/pF) Senktide-sensitive current –2 –1 0 1 2 3 4 200 pA 100 ms Control Senktide 50 nM 200 pA 100 ms Control ZnCl2 100 μM n = 7, 2 Hz Control Spadin (100 nM) + senktide (50 nM) Spadin (100 nM) 50 ms 25 mV 0 mV 0 mV Control ML-365 Spadin Spadin + senktide Control Spadin ML-365 + senktide ML-365 n = 8, 2 Hz Control ML-365 (100 nM) + senktide (50 nM) ML-365 (100 nM) Duration (ms) 0 20 40 60 80 100 120 140 n = 7, 2 Hz Control PK-THPP (100 nM) + senktide (50 nM) PK-THPP (100 nM) 0 mV Control PK-THPP PK-THHP + senktide PK-THHP 0 40 80 120 160 200 240 * * * * * * * * * * * 0 30 60 90 120 150 180 APD90 APD50 APD20 APD90 APD50 APD20 APD90 APD50 APD20 a b c d e f g h i j k l ig. 6 Effects of Senktide and different K2P channel blockers on atrial electrophysiology. a–c Current tracings recorded at +40 mV before (Contro fter application of 50 nM Senktide (a), associated average current–voltage I–V relationships of the steady-state current (b), and I–V relationship enktide-sensitive current (c) (N = 4, n = 8, two-way RM ANOVA). d–f Current tracings recorded at +40 mV before (Control) and after applica nCl2 (100 µM), followed by additional Senktide (50 nM) (d), associated average I–V relationships of the steady-state current (e), and average elationships of ZnCl2-sensitive current (f). (N = 4, n = 5, two-way RM ANOVA). g–i Atrial action potentials (AP) elicited at 2 Hz under control con Control) and in the presence of 100 nM ML-365 (g), PK-THPP (h) and Spadin (i). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 6 Effects of Senktide and different K2P channel blockers on atrial electrophysiology. a–c Current tracings recorded at +40 mV before (Control) and after application of 50 nM Senktide (a), associated average current–voltage I–V relationships of the steady-state current (b), and I–V relationships of Senktide-sensitive current (c) (N = 4, n = 8, two-way RM ANOVA). d–f Current tracings recorded at +40 mV before (Control) and after application of ZnCl2 (100 µM), followed by additional Senktide (50 nM) (d), associated average I–V relationships of the steady-state current (e), and average I–V relationships of ZnCl2-sensitive current (f). (N = 4, n = 5, two-way RM ANOVA). g–i Atrial action potentials (AP) elicited at 2 Hz under control conditions (Control) and in the presence of 100 nM ML-365 (g), PK-THPP (h) and Spadin (i). j–l Bar graphs showing average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 100 nM ML-365 (j, N = 4, n = 8), PK-THPP (k, N = 3, n = 7), and Spadin (l, N = 2, n = 7). One-way RM ANOVA. All values shown are mean ± SEM. P < 0.05 vs. Control nktide and different K2P channel blockers on atrial electrophysiology. a–c Current tracings recorded at +40 mV befor el blockers on atrial electrophysiology. a–c Current tracings recorded at +40 mV before (Control) and Fig. 6 Effects of Senktide and different K2P channel blockers on atrial electrophysiology. a–c Current tracings recorded at +40 mV before (Control) and after application of 50 nM Senktide (a), associated average current–voltage I–V relationships of the steady-state current (b), and I–V relationships of Senktide-sensitive current (c) (N = 4, n = 8, two-way RM ANOVA). d–f Current tracings recorded at +40 mV before (Control) and after application of ZnCl2 (100 µM), followed by additional Senktide (50 nM) (d), associated average I–V relationships of the steady-state current (e), and average I–V relationships of ZnCl2-sensitive current (f). (N = 4, n = 5, two-way RM ANOVA). g–i Atrial action potentials (AP) elicited at 2 Hz under control conditions (Control) and in the presence of 100 nM ML-365 (g), PK-THPP (h) and Spadin (i). Discussion This study provides biophysical and pharmacological evidence that the neuropeptide Sub-P prolongs the atrial action potential in rabbit myocytes by activation of the neurokinin-3 receptor (NK-3R) and consequent inhibition of a background K+ current. We further demonstrate that the NK-3R mediated AP pro- longation is maintained at high pacing rates and results in increased atrial effective refractory period (ERP) in Langendorff- perfused and in situ rabbit hearts. Also in human atrial appen- dages the latter phenomenon was observed. Ventricular action potential does not change following NK-3R stimulation, indi- cating atrial-speciﬁc efﬁcacy. In a rabbit isolated heart model of AF, NK-3R stimulation exerts a potent anti-arrhythmic action by strongly reducing AF duration and incidence. The AP prolonging effect by Sub-P was dose-dependent and occurred at nanomolar concentrations, supporting the view that NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications 8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Control ATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 ARTIC Vm (mV) Control Senktide 50 nM n = 8 –10 –8 –6 –4 –2 0 2 4 6 –150 –100 –50 0 50 * * * * * * * * * I (pA/pF) b c 200 pA 100 ms Control Senktide 50 nM a Vm (mV) Control Senktide 50 nM n = 8 –10 –8 –6 –4 –2 0 2 4 6 –150 –100 –50 0 50 –150 –100 –50 0 50 * * * * * * * * * * I (pA/pF) Vm (mV) I (pA/pF) Senktide-sensitive current –2 –1 0 1 2 3 4 b c –150 –100 –50 0 50 Vm (mV) I (pA/pF) Senktide-sensitive current –2 –1 0 1 2 3 4 c a 200 pA 100 ms Control ZnCl2 100 μM d I (pA/pF) Control –15 –10 –5 0 5 10 * ***** # # * ZnCl2 + senktide n = 5 ZnCl2 100 μM Vm (mV) –150 –100 –50 0 50 e I (pA/pF) ZnCl2 – sensitive current ZnCl2 + senktide sensitive current –2 –1 0 1 2 3 4 –150 –100 –50 0 50 Vm (mV) f f d e 50 0 mV ML-365 Control ML-365 + senktide ML-365 g ( ) 25 mV 0 mV Control Spadin Spadin + senktide Spadin i 0 mV Control PK-THPP PK-THHP + senktide PK-THHP h i g h n = 7, 2 Hz Control Spadin (100 nM) + senktide (50 nM) Spadin (100 nM) 50 ms 25 mV 0 20 40 60 80 100 120 140 n = 7, 2 Hz Control PK-THPP (100 nM) + senktide (50 nM) PK-THPP (100 nM) * * * * * * * 0 30 60 90 120 150 180 APD90 APD50 APD20 APD90 APD50 APD20 k l n = 7, 2 Hz Control Spadin (100 nM) + senktide (50 nM) Spadin (100 nM) 50 ms n = 8, 2 Hz Control ML-365 (100 nM) + senktide (50 nM) ML-365 (100 nM) Duration (ms) 0 20 40 60 80 100 120 140 n = 7, 2 Hz Control PK-THPP (100 nM) + senktide (50 nM) PK-THPP (100 nM) 0 40 80 120 160 200 240 * * * * * * * * * * * 0 30 60 90 120 150 180 APD90 APD50 APD20 APD90 APD50 APD20 APD90 APD50 APD20 k l n = 8, 2 Hz Control ML-365 (100 nM) + senktide (50 nM) ML-365 (100 nM) Duration (ms) 0 40 80 120 160 200 240 * * * * APD90 APD50 APD20 j j l k Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 j–l Bar graphs showing average values for AP duration at 20, 5 0% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 100 nM ML-365 (j, N = 4, n = 8), PK-THPP (k, N = 3, nd Spadin (l, N = 2, n = 7). One-way RM ANOVA. All values shown are mean ± SEM. P < 0.05 vs. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 The exact nature of the effects of NKB/ Sub-P on in vivo atrial electrophysiology are difﬁcult to assess, as matters are further complicated by the fact that also (cholinergic) intracardiac neurons possess NK receptors and may release ACh upon stimulation.40 the electrophysiological actions of Sub-P on atrial cardiomyocytes are receptor-mediated involving the neurokinin cell-surface receptors15,16. We established that the AP prolongation by Sub- P was mediated through neurokinin-3 receptor (NK-3R) stimu- lation, as the selective NK-3R agonist Senktide32 increased APD90 with a 100 times higher potency than Sub-P, while NK-1R and NK-2R agonists at concentrations that maximally activate their respective receptors33,34, did not change AP duration. Moreover, the AP prolongation by NK-3R stimulation was antagonized by the speciﬁc NK-3R antagonist Osanetant35. The presence of NK- 3R on the surface of atrial cardiomyocytes has thus far not been demonstrated18. Hoover and Hancock36 were unable to detect Sub-P binding sites in atria and ventricle of guinea-pig heart. Also Walsh et al.37 did not ﬁnd evidence for Sub-P binding sites in rat heart. However, neonatal rat ventricular cardiomyocytes express mRNA for NK-1R and NK-3R38. RNA-sequencing data and immunohistochemical analysis in our study further supports the presence of NK-3R in the sarcolemmal membrane of rabbit and human atrial cardiomyocytes. Thus, this demonstrates the exis- tence of NK-3R on adult cardiomyocytes from rabbit and human atrium and to provide evidence of their functional signiﬁcance. The RNA sequencing data indicate that the NK-3 receptor is the dominant neurokinin receptor in atrial cardiomyocytes as mRNA levels of NK-1R and NK-2R were relatively low. The endogenous ligand for the NK-3R is neurokinin B (NKB). However, neuro- kinin A (NKA) and Sub-P, as conﬁrmed in the present study, also bind to the NK-3R, albeit with different afﬁnities.15,16 All three neurokinins are produced by neurons of the intrinsic cardiac nervous system.19,39 The preferential afﬁnity of NK-3R for the endogenous ligands has a potency order of NKB>NKA>Sub-P, p To our knowledge no data are available concerning the actions of Sub-P or Senktide on membrane currents in atrial cardio- myocytes. The main ﬁnding of the present study concerned the reduction in steady-state outward current in the presence of Sub- P and Senktide, which is consistent with the observed AP pro- longation. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 APD50 APD90 APD20 APD50 APD90 APD20 n = 9, 2 Hz Control Sub-P (1 μM) 0 mV Control 100 nM senktide 0 mV Control 1 μM Sub P Duration (ms) n = 10, 2 Hz Control Senktide (100 nM) 50 ms 20 mV 50 ms 20 mV Substance P Senktide 0 60 120 180 240 0 60 120 180 240 a b Fig. 7 Effect of NK-3 receptor stimulation on action potentials of ventricular myocytes. (a–b, left panel) Ventricular APs elicited at 2 Hz under control conditions (Control) and in the presence of 1 µM Sub-P a or 100 nM Senktide b. (a–b, right panel) Bar graph showing average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 1 µM Sub-P (a, N = 5, n = 9) or 100 nM Senktide (b, N = 5, n = 10). Paired t-test. All values shown are mean ± SEM. P < 0.05 0 mV Control 1 μM Sub P 50 ms 20 mV Substanc a APD50 APD90 APD20 n = 9, 2 Hz Control Sub-P (1 μM) 0 mV Control 1 μM Sub P 50 ms 20 mV Substance P 0 60 120 180 240 a APD50 APD90 APD20 n = 9, 2 Hz Control Sub-P (1 μM) e P 0 60 120 180 240 0 mV Control 100 nM senktide 50 ms 20 mV Senkt b APD50 APD90 APD20 Duration (ms) n = 10, 2 Hz Control Senktide (100 nM) ide 0 60 120 180 240 b Fig. 7 Effect of NK-3 receptor stimulation on action potentials of ventricular myocytes. (a–b, left panel) Ventricular APs elicited at 2 Hz under control conditions (Control) and in the presence of 1 µM Sub-P a or 100 nM Senktide b. (a–b, right panel) Bar graph showing average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 1 µM Sub-P (a, N = 5, n = 9) or 100 nM Senktide (b, N = 5, n = 10). Paired t-test. All values shown are mean ± SEM. P < 0.05 which implies that NKB may be more relevant for atrial elec- trophysiology than Sub-P. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 j–l Bar graphs showing average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 100 nM ML-365 (j, N = 4, n = 8), PK-THPP (k, N = 3, n = 7), and Spadin (l, N = 2, n = 7). One-way RM ANOVA. All values shown are mean ± SEM. P < 0.05 vs. Control ( ) ( , , y ) g + ( ) pp ZnCl2 (100 µM), followed by additional Senktide (50 nM) (d), associated average I–V relationships of the steady-state current (e), and average I–V relationships of ZnCl2-sensitive current (f). (N = 4, n = 5, two-way RM ANOVA). g–i Atrial action potentials (AP) elicited at 2 Hz under control conditions (Control) and in the presence of 100 nM ML-365 (g), PK-THPP (h) and Spadin (i). j–l Bar graphs showing average values for AP duration at 20, 50 and 90% of repolarization (APD20, APD50 and APD90) before (Control) and after application of 100 nM ML-365 (j, N = 4, n = 8), PK-THPP (k, N = 3, n = 7), and Spadin (l, N = 2, n = 7). One-way RM ANOVA. All values shown are mean ± SEM. P < 0.05 vs. Control NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Control Senktide 20 nM 0 cm 20 cm 1 s Arrhythmia episode Burst protocol Burst protocol Burst protocol Burst protocol AF episode Arrhythmia episode * * 0 0.2 0.4 0.6 0.8 1 AF incidence Control Senktide 20 nM Control Senktide 100 nM 194 ms 195 ms A A A 228 ms A A 227 ms A 5 mV 100 ms * * * * * * * * 88 ms 250 ms * A A V V * * * A A A 119 ms V V A A A V * * * V A A V * * * V 87 ms 250 ms V 250 ms 118 ms 250 ms * * 100 ms AERP (ms) * AERP (ms) 0 50 100 150 200 250 300 # # Rabbit Human 0 20 40 60 80 100 120 H2O H2O 10 20 30 40 50 60 * * # * # Total duration arrhythmia episodes (s) Senktide Control Senktide 20 nM Washout Control Senktide 50–200 nM a b c d e Wash out CTRL Senktide Wash out CTRL 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O Fig. 8 Effect of NK-3R stimulation on ERP and AF inducibility in atria of human and rabbit. a Unipolar electrograms recorded from the left atrium of a Langendorff-perfused rabbit heart before (Control) and after application of 20 nM of Senktide (left). Bar graph showing average values for AERP in Control, in the presence of 20 nM Senktide, and after wash-out (right, N = 7, one-way RM ANOVA). b Unipolar electrograms recorded from human LAA before (Control) and after application of 100 nM of Senktide (left). Bar graph showing average values for AERP in Control and in the presence of 50–200 nmol/L Senktide (right, N = 5, paired t-test, p = 0.06). c Unipolar electrograms recorded from the left atrium of a rabbit Langendorff-perfused heart following a burst pacing protocol in the absence (Control, left) and in the presence of 20 nM of the NK-3R agonist Senktide (right), at baseline (0 cm H2O, upper tracings) and during atrial dilatation (20 cm H2O, lower tracings). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 d, e Bar graph showing average values for total duration of arrhythmia episodes d and AF incidence e in Control, after application of 20 nM Senktide, and after wash-out (N = 7, two-way RM ANOVA). All values shown are mean ± SEM. P < 0.05. Electrograms: A atrial activation; V ventricular activation; stimulus AERP (ms) * 0 20 40 60 80 100 120 Control Senktide 20 nM Washout Control Senktide 20 nM 88 ms 250 ms * A A V V * * * A A A 119 ms V V A A A V * * * V A A V * * * V 87 ms 250 ms V 250 ms 118 ms 250 ms * * 100 ms AERP (ms) * Rabbit 0 20 40 60 80 100 120 Control Senktide 20 nM Washout a Control Senktide 100 nM 194 ms 195 ms A A A 228 ms A A 227 ms A 5 mV 100 ms * * * * * * * * AERP (ms) 0 50 100 150 200 250 300 Human Control Senktide 50–200 nM b Control Senktide 20 nM 88 ms 250 ms * A A V V * * * A A A 119 ms V V A A A V * * * V A A V * * * V 87 ms 250 ms V 250 ms 118 ms 250 ms * * 100 ms AERP ( ) Rabbit a b a Control Senktide 20 nM 0 cm 20 cm 1 s Arrhythmia episode Burst protocol Burst protocol Burst protocol Burst protocol AF episode Arrhythmia episode H2O H2O c 10 20 30 40 50 60 * * # * # Total duration arrhythmia episodes (s) Senktide d Wash out CTRL 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O Control 0 cm Arrhythmia episode Burst protocol AF episode H2O c * * * 0 0.2 0.4 0.6 0.8 1 AF incidence # # e Senktide Wash out CTRL 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O 20 cm H2O 0 cm H2O d d e c e c 20 cm 1 s Burst protocol Burst protocol AF episode Arrhythmia episode H2O Fig. 8 Effect of NK-3R stimulation on ERP and AF inducibility in atria of human and rabbit. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 a Unipolar electrograms recorded from the left atrium of a Langendorff-perfused rabbit heart before (Control) and after application of 20 nM of Senktide (left). Bar graph showing average values for AERP in Control, in the presence of 20 nM Senktide, and after wash-out (right, N = 7, one-way RM ANOVA). b Unipolar electrograms recorded from human LAA before (Control) and after application of 100 nM of Senktide (left). Bar graph showing average values for AERP in Control and in the presence of 50–200 nmol/L Senktide (right, N = 5, paired t-test, p = 0.06). c Unipolar electrograms recorded from the left atrium of a rabbit Langendorff-perfused heart following a burst pacing protocol in the absence (Control, left) and in the presence of 20 nM of the NK-3R agonist Senktide (right), at baseline (0 cm H2O, upper tracings) and during atrial dilatation (20 cm H2O, lower tracings). d, e Bar graph showing average values for total duration of arrhythmia episodes d and AF incidence e in Control, after application of 20 nM Senktide, and after wash-out (N = 7, two-way RM ANOVA). All values shown are mean ± SEM. P < 0.05. Electrograms: A atrial activation; V ventricular activation; stimulus observed effects of Sub-P, since the reduction in steady-state outward current persists in the presence of the respective block- ers. A candidate underlying the Sub-P mediated effect, is the background or leakage K+ current, which was also inhibited by Sub-P in neurons43–45. Recent evidence suggests that members of the two-pore-domain K+ (K2P) channel family generate the K+ background conductance in excitable tissues21. Neurokinin receptors are known to be coupled to the Gq/G11 subgroup of G- proteins22 and several members of the K2P family have been shown to be potently inhibited by receptors that signal through Gq/11, including TREK-1, TREK-2, TASK-1 and TASK-323,25. As TASK-1, TASK-3 and TREK-1 channels have been functionally identiﬁed in atrial cardiomyocytes to contribute to AP dura- tion26–28, we considered these channels as candidates for the action potential prolonging effect. Nevertheless, selective inhibi- tion of these channels scarcely affected AP duration, which makes their involvement in isolation unlikely. Application of the non- speciﬁc channel blocker ZnCl242,46 abolished the inhibition of the background K+ current by Senktide, and revealed the pre- sence of a ZnCl2-sensitive component with electrophysiological characteristics comparable to the Sub-P-sensitive and Senktide- sensitive currents. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 Other studies have shown that a reduction in sustained outward current of similar amplitude, has important implications for atrial AP duration41,42 Changes in outward ICa(Cl), INCX and Ito do not contribute to AP prolongation, as their current amplitudes were unchanged in the presence of Sub-P, despite minor kinetic alterations in the latter. The reduction of ICa,L is not compatible with AP prolongation by NK-3R stimulation. On the contrary, a decrease in inward current would shorten the AP. Evidently, the reduction in steady-state outward current by far outweighs the effect of ICa,L reduction on AP duration. Detailed examination of the Sub-P sensitive current indicated that an outward K+ current is involved, since its reversal potential is close to the calculated Nernst potential for K+ ions. The steady-state outward current of atrial myocytes is composed of different types of K+ currents, including the sustained component of the tran- sient outward current (Ito), and the slow, rapid and ultra-rapid components of the delayed rectiﬁer K+ current (IKs, IKr and IKur, respectively)12,13. Our experiments exclude a decrease in any of these current types as a possible mechanism underlying the NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications 10 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 ARTICLE Methods During this last period, the heart was perfused at a constant ﬂow in a recirculating manner. When perfusion pressure dropped from an initial value of 50 to <2 mmHg (usually after about 30 min), the left ventricular wall and the left atrium were cut into small pieces and further fractionated in the enzyme-containing dissociation solution using a gyrotory water bath shaker (25 min). During the last 10 min, 1% albumin (fatty acid free, Roche 10775835001) was added to the enzyme-containing dissociation solution. All dis- sociation solutions were saturated with 100% O2 and the temperature was main- tained at 37 °C. Cells were allowed to sediment and were resuspended in (enzyme- free) dissociation solution to which 1% albumin and 1.3 mM CaCl2 was added. Small aliquots of single cell suspension were introduced into a recording chamber on the stage of an inverted microscope. Cells were allowed to adhere for 5 min after which superfusion was started. Single quiescent rod-shaped myocytes with clear cross-striations and smooth surfaces were selected for measurements. Atrial refractoriness in the Langendorff rabbit heart. Bipolar stimulation- and unipolar recording electrodes were placed on the left atrial myocardium. The heart was stimulated at a basic cycle length of 200 ms (cathodal stimulation, 1 ms current pulse duration, twice the diastolic stimulation threshold). After an equilibrium period of 30 min, the effective refractory period (ERP) was determined by a train of 16 stimuli at 200 ms followed by a single stimulus with progressively shortened coupling interval until loss of capture. The ERP was deﬁned as the longest S1–S2 interval without capture. After an equilibrium period of 30 min, wash-in of 20 nM NK-3R-agonist Senktide was started and ERP measurements were repeated 10 min following wash-in. Rabbit isolated heart model of AF based on atrial dilatation. After mounting of the heart on the Langendorff perfusion setup, a distendable balloon (volume 4–5 ml) connected to a manometer, was inserted through the pulmonary vein openings into the left atrium, Subsequently, ±0.5 ml saline was injected into the balloon to yield an intra-atrial pressure of 0 cm H2O (baseline condition). A bipolar pacing electrode was positioned at the right atrium free wall and the heart was stimulated at a basic cycle length of 250 ms. Unipolar recording electrodes were positioned on the left atrium free wall and the left ventricle wall to record atrial and ventricular electrograms, respectively. Methods Animals and human tissue. The study conformed to the ‘Guide for the Care and Use of Laboratory Animals’ published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and was approved by the local animal experiments committee of the Academic Medical Center, Amsterdam, The Neth- erlands. The study on human atrial appendages was in accordance with the declaration of Helsinki and was approved by the Institutional Review Board of the Academic Medical Center, Amsterdam, The Netherlands. All patients gave written informed consent. The ERP was determined by a train of 8 stimuli (at 600 ms interval) followed by a single stimulus with progressively shortened coupling interval until loss of capture. After an equilibrium period of 30 min, Senktide superperfusion (50–200 nM) was started, and the ERP protocol was repeated. The Langendorff-perfused rabbit heart. Male New Zealand White rabbits (2.5–3.5 kg) were anaesthetized with 20 mg xylazine and 100 mg ketamine (intra- muscularly) and heparinized with a bolus of 1000 IU heparin (intravenously). Subsequently, the animals were killed by 200 mg pentobarbital intravenously, the thorax was opened and the heart was quickly removed and submerged in ice-cold perfusion solution (composition see below). After cannulation of the aorta, the heart was mounted on a Langendorff perfusion setup, and perfused at 37 °C with a modiﬁed Tyrode’s solution containing (in mmol/L) 128 NaCl, 4.7 KCl, 1.45 CaCl2, 0.6 MgCl2, 27 NaHCO3, 0.4 NaH2PO4 and 11 glucose (pH maintained at 7.4 by equilibration with a mixture of 95% O2 and 5% CO2)49. Single cardiomyocyte preparation. After excision of the heart from male New Zealand White rabbits49, single left atrial and ventricular cells were isolated as described previously, with minor modiﬁcations51,52. In short, hearts were mounted on a Langendorff perfusion apparatus and retrogradely perfused at 37 °C through the aorta with a modiﬁed Tyrode’s solution (see above) at a constant pressure (50 mmHg) for 15 min. Next, perfusion (50 mmHg) was changed to a nominally calcium-free dissociation solution containing in mM: HEPES 16.8, NaCl 146.5, KHCO3 3.3, KH2PO4 1.4, NaHCO3 1.0, MgC12 2.0, CaCl2 0.01, glucose 11.0, pH 7.3 (NaOH). After 15 min, collagenase type B (0.15 mg/ml, Roche 11088815), collagenase type P (0.05 mg/ml, Roche 11213865), trypsine inhibitor (0.1 mg/ml, Roche 10109878), 0.2 mg/ml hyaluronidase (Sigma H-3506), protease XIV (Sigma H-3506) and creatine (10 mM) were added. Methods The heart was then allowed to equilibrate for a 20-min period of continuous stimulation, after which right atrial effective refractory period (AERP) was determined by a train of 8 stimuli at 250 ms followed by a single stimulus with progressively shortened coupling interval until loss of capture. The ERP was deﬁned as the longest S1–S2 interval without capture. Subsequently, after 20 stimuli at the basic cycle length (250 ms), a burst pacing protocol consisting of 20 stimuli at 20 Hz (2 ms current pulse duration) was delivered to provoke AF, followed by a 5 s pause. The protocol to evoke AF was repeated ten times. Next, the left atrial balloon was distended by injection of 2–4 ml saline up to an intra-atrial pressure of 20 cm H2O to dilate the atrium, and AERP measurements and burst pacing protocol to provoke AF were repeated. After a 5 min. recovery period at 0 cm H2O, drug infusion was started and the complete procedure was repeated in the presence of 20 nM Senktide. Then, a 30 min wash-out period (at intra-atrial pressure 0 cm H2O) followed, and AERP and AF inducibility were determined again at intra-atrial pressures of 0 and 20 cm H2O, respectively. Rabbit isolated heart model of AF based on atrial dilatation. After mounting of the heart on the Langendorff perfusion setup, a distendable balloon (volume 4–5 ml) connected to a manometer, was inserted through the pulmonary vein openings into the left atrium, Subsequently, ±0.5 ml saline was injected into the balloon to yield an intra-atrial pressure of 0 cm H2O (baseline condition). A bipolar pacing electrode was positioned at the right atrium free wall and the heart was stimulated at a basic cycle length of 250 ms. Unipolar recording electrodes were positioned on the left atrium free wall and the left ventricle wall to record atrial and ventricular electrograms, respectively. The heart was then allowed to equilibrate for a 20-min period of continuous stimulation, after which right atrial effective refractory period (AERP) was determined by a train of 8 stimuli at 250 ms followed by a single Cellular electrophysiology. Action potentials (APs) and membrane currents were recorded at 36.5 °C with the amphotericin-B-perforated or ruptured patch clamp technique51, using an Axopatch 200B Clamp ampliﬁer (Molecular Devices Cor- poration, Sunnyvale, CA, USA). Voltage control, data acquisition, and analysis were performed using custom-made software. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 by neural tissue, but was hitherto unidentiﬁed in cardiac atrial muscle. Stimulation of NK-3R produces signiﬁcant prolongation of the atrial but not the ventricular AP through inhibition of a background K+ channel, possibly a member of the K2P family. The AP-prolonging action of NK-3R stimulation in the atrium was also present at high heart rates and greatly reduced AF inducibility. Hence, we propose NK-3R as a potential anti- arrhythmic drug target for treatment of AF. (0.8–1.2%). Catheters for drug infusions and blood pressure recording were inserted into marginal ear veins and arteries, respectively. Following thoracotomy, the pericardium was opened and the heart was instrumented as above. When the rabbit was hemodynamically and electrophysiologically stable, the atrial ERP was determined by a train of 8 stimuli at 220 ms followed by a premature stimulus with progressively shortened coupling interval until loss of capture. Subsequently, a single bolus injection (1 ml i.v.) of 11 nmol/kg Senktide was administered and the measurement protocol was repeated. Refractory periods in human left atrial appendages. Left atrial appendages (LAA) were routinely removed from patients with atrial ﬁbrillation undergoing minimally invasive surgical pulmonary vein isolation with an endoscopic stapling device (Endo Gia stapler, Tyco Healthcare Group)50. The tissue samples were transported in 100-ml cooled solution I (see modiﬁed Tyrode’s solution above) containing 1000 IE heparin, and submerged in a tissue bath. The superfusion ﬂuid was kept at a stable temperature of 36.5–37.5 °C and gassed with a mixture of 95% O2 and 5% CO2 (pH 7.4). LAAs were stimulated at 100 beats per minute at 2–3 times diastolic threshold with a pulse width of 2 ms using a bipolar epicardial electrode. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 However, as zinc modulates the activity of a broad range of ion channels, the nature of this background K+ current remains unsure. Future studies dedicated to exact channel identiﬁcation, are required to designate a particular K+ channel type. The cardiac autonomic nervous system plays a signiﬁcant role in the genesis and maintenance of AF. Particularly, alterations in the sympatho-vagal balance and excessive intrinsic cardiac nerve activity have been shown to trigger atrial arrhythmias47,48. The precise role of the various neuropeptides therein is still poorly understood. In this study, AP prolongation by NK-3R stimulation in isolated atrial cardiomyocytes (by Sub-P or Senktide) was reﬂected by an increased atrial effective refractory period (ERP) in rabbit Langendorff hearts and in situ anaesthetized rabbits, as well as in human isolated left atrial tissue. An increased ERP forms an anti-arrhythmic mechanism which may prevent or terminate AF12,13. To date however, pharmacological interven- tion aimed at lengthening of atrial repolarization has a limited applicability due to pro-arrhythmic ventricular side effects and reverse rate-dependence (i.e., a loss of action at high heart rates)12,13. As NK-3R stimulation does not prolong the ven- tricular AP, and prolongs the atrial AP also at high heart rates, it meets the important prerequisites for a potential atrial-speciﬁc, anti-ﬁbrillatory action. Indeed we show that NK-3R stimulation by Senktide in a rabbit isolated heart model of AF based on atrial dilatation, reduces AF incidence and duration by 70%. Thus, we demonstrate that the electrophysiological effects of NK-3R sti- mulation are of potent anti-ﬁbrillatory nature. In conclusion, this study provides evidence for the functional expression of the neurokinin-3 receptor in the sarcolemma of atrial cardiomyocytes. This receptor was known to be expressed 11 NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications Methods Series resistance was compensated for and potentials were corrected for liquid junction potential53. Signals were low-pass ﬁltered (cutoff frequency: 5 kHz) and digitized at 40 kHz. Cell membrane capacitance (Cm) was estimated by dividing the time constant of the capacitive transient in response to 5 mV hyperpolarizing voltage clamp steps from a holding potential of −40 mV, by the series resistance. 20 stimuli at 20 Hz (2 ms current pulse duration) was delivered to provoke AF, followed by a 5 s pause. The protocol to evoke AF was repeated ten times. Next, the left atrial balloon was distended by injection of 2–4 ml saline up to an intra-atrial pressure of 20 cm H2O to dilate the atrium, and AERP measurements and burst pacing protocol to provoke AF were repeated. After a 5 min. recovery period at 0 cm H2O, drug infusion was started and the complete procedure was repeated in the presence of 20 nM Senktide. Then, a 30 min wash-out period (at intra-atrial pressure 0 cm H2O) followed, and AERP and AF inducibility were determined again at intra-atrial pressures of 0 and 20 cm H2O, respectively. Current-clamp experiments. For AP measurements, a standard superfusion solution was used containing (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0, pH 7.4 (NaOH). The patch-pipettes (borosilicate glass; 1–3 MΩ) were ﬁlled with a standard pipette solution containing (in mM): K-gluconate 110, KCl 30, NaCl 5, MgCl2 1, amphotericin-B 0.22, HEPES 10, pH 7.3 (KOH). APs were elicited at 1–4 Hz by 2-ms (1.5× diastolic stimulation threshold) current pulses applied through the patch pipette. Resting membrane potential (RMP), maximal upstroke velocity (Vmax), AP amplitude (APA) and AP duration (APD) at 20%, 50% and 90% repolarization (APD20, APD50 and APD90, respectively, were obtained from 10 consecutive APs and averaged. Cardiomyocytes were allowed to equilibrate for a 5-min period of continuous stimulation (1 Hz or 2 Hz) after which Sub-P (1 µM) or one of the speciﬁc neurokinin receptor agonists (100 nM) was administered. Agonists for the NK-1R, NK-2R and NK-3R were respectively: [Sar9,Met(O2)11]-SP33, (β-Ala)-NKA(4-10)34 and Senktide32. APs were recorded just before, and 3–5 min after application of the neuropeptides. In dose-response experiments, Sub-P or Senktide was applied in a cumulative sequence with 5 min intervals between increments in concentration Current-clamp experiments. ARTICLE The superfusion solution consisted of a K+-free solution to which 1 mM BaCl2, 2 mM CsCl, 5 µM nifedipine, 100 µM ouabain and 200 µM DIDS was added to suppress membrane currents other than INCX. INCX58 was measured as 10 mM Ni2+-sensitive current during a descending voltage ramp. As the effects of Ni2+ on INCX are reversible58, INCX measurements in the absence and presence of Sub-P analogues were carried out in the same cell. DIDS was freshly prepared as a 0.5 M and chromanol 293B as a 0.1 M stock solution in DMSO. Nifedipine was prepared as a 5 mM stock solution in ethanol. E-4031 and TTX were prepared as a 5 and 30 mM stock solution in distilled water. Senktide was prepared as a 1 mM stock solution in ethanol. PK-THPP and ML-365 were prepared as a 1 mM stock solutions in DMSO. Spadin was prepared as a 1 mM Stock solution in distilled water. ZnCl2 was prepared as a 100 mM stock solution in distilled water. All stock solutions were diluted appropriately before use. Sub-P was freshly dissolved at its ﬁnal concentration. DIDS and nifedipine were stored in the dark. 4,4’diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS; Sigma-Aldrich, MO, USA) to block ICl(Ca)57. INCX58 was measured with pipette solution containing (in mM): CsCl 145, NaCl 5, Mg-ATP 10, TEA-Cl 10, EGTA 20, CaCl2 10, HEPES 10, pH 7.2 (NMDG-OH). The superfusion solution consisted of a K+-free solution to which 1 mM BaCl2, 2 mM CsCl, 5 µM nifedipine, 100 µM ouabain and 200 µM DIDS was added to suppress membrane currents other than INCX. INCX58 was measured as 10 mM Ni2+-sensitive current during a descending voltage ramp. As the effects of Ni2+ on INCX are reversible58, INCX measurements in the absence and presence of Sub-P analogues were carried out in the same cell. Statistics. The data are presented as mean ± SEM. Normality and equal variance assumptions were tested with the Kolmogorov–Smirnov and the Levene median test, respectively. Groups were compared using a paired Student's t-test (two- sided), one-way repeated measures (RM) analysis of variance (ANOVA), or two- way RM ANOVA followed by post hoc comparison using the Student–Newman–Keuls method, where appropriate. Factor correction was applied on APD (in Figs. 1e and 4d) and ERP (in Fig. 8 and Supplementary Figure 5) using day of experiment and the experimental animal as session factor, respectively67. ARTICLE p y p g To assess the contribution of TASK-1, TASK-3 and TREK-1 currents to the action potential, APD90 was measured after application of the selective blockers ML-36554 (100 nM), PK-THPP (100 nM)55 and Spadin56 (100 nM), respectively. APs were recorded just before, and 3–5 min after application of the channel blockers. y g To assess the contribution of TASK-1, TASK-3 and TREK-1 currents to the action potential, APD90 was measured after application of the selective blockers ML-36554 (100 nM), PK-THPP (100 nM)55 and Spadin56 (100 nM), respectively. APs were recorded just before, and 3–5 min after application of the channel blockers. Voltage-clamp experiments. Steady-state currents, L-type Ca2+ current (ICa,L), transient outward K+ current (Ito), Ca2+-activated Cl−current (ICl(Ca)) and Na+–Ca2+ exchange current (INCX) were measured with solutions speciﬁed below and using voltage-clamp protocols depicted in the corresponding ﬁgures. Steady- state currents (current at the end of the 500-ms voltage step), Ito and ICl(Ca) were measured using the amphotericin-B-perforated patch clamp technique with stan- dard pipette- and superfusion-solution (see current-clamp experiments above). To accurately determine Ito, measurements were performed in the presence of blockers of Na+ channels (30 µM tetrodotoxin (TTX)), Ca2+ channels (0.25 mM CdCl2) and of delayed rectiﬁer K+ channels (5 µM E-4031, 100 µM chromanol 293B).51 For ICl(Ca) measurements, 10 µM TTX and 2 mM 4-aminopyridine (4-AP) was added to the standard superfusion solution.57 Drugs. All drugs were obtained from Sigma-Aldrich (MO, USA), except for E- 4031, PK-THPP, ML-365 (Tocris, MN, USA), Sub-P (Enzo Life Sciences, NY, USA), and TTX (Abcam Biomedicals, Cambridge, UK). Drugs. All drugs were obtained from Sigma-Aldrich (MO, USA), except for E- 4031, PK-THPP, ML-365 (Tocris, MN, USA), Sub-P (Enzo Life Sciences, NY, USA), and TTX (Abcam Biomedicals, Cambridge, UK). DIDS f hl d 0 5 M d h l 293B 0 1 M t k ICa,L and INCX were measured using the ruptured patch clamp technique. ICa,L57 was measured with pipette solution containing (in mM): CsCl 145, K2-ATP 5, EGTA 10, HEPES 10, pH 7.2 (NMDG-OH). The superfusion solution contained (mM): TEA-Cl 145, CsCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0; pH 7.4 (NMDG-OH). ICa, was measured in the presence of 0.25 mM 4,4’diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS; Sigma-Aldrich, MO, USA) to block ICl(Ca)57. INCX58 was measured with pipette solution containing (in mM): CsCl 145, NaCl 5, Mg-ATP 10, TEA-Cl 10, EGTA 20, CaCl2 10, HEPES 10, pH 7.2 (NMDG-OH). ARTICLE N is number of hearts, n is number of cells. P < 0.05 was deﬁned as statistically signiﬁcant. g In voltage clamp experiments the effect of Sub-P and Senktide on the various membrane currents was assessed at a concentration of 10 µM and 50 nM, respectively, 3–5 min after application. p y pp To test for the functional presence of members of the K2P family, steady-state currents were also measured after application of the ion channel blocker ZnCl2 (100 µM).29–31 Voltage dependencies of (in)activation were determined by ﬁtting a Boltzmann function (y = A/[1 + exp{(V–V1/2)/k}]) to the individual curves, yielding a half- maximal voltage V1/2 (mV) and a slope factor k (mV). Time constants of inactivation were obtained by ﬁtting current decay with a bi-exponential function y = y0 + Afexp(−t/τf) + Asexp(–t/τs), where Af and As are the amplitudes of the fast and slow inactivating components, and τf and τs their respective inactivation time constants. Current densities were calculated by dividing the current amplitude by Cm. Data availability The data supporting the ﬁndings of this manuscript are available from the corresponding authors upon reasonable request. RNA sequencing data that support the ﬁndings of this study have been deposited in Geo with the accession code GSE117801. Cytosolic Ca2+ transients. Intracellular Ca2+ (Ca2+i) was measured in indo-1-am loaded atrial cardiomyocytes as described previously59. Dual wavelength emission of indo-1 was recorded ((405–440)/(505–540) nm, excitation at 340 nm) and free Ca2+i was calculated. Ca2+i transients were elicited at 5 Hz using ﬁeld stimulation. For determination of diastolic and systolic Ca2+i concentrations, and Ca2+i tran- sient amplitudes, data from 10 consecutive Ca2+i transients were averaged. The effect of Sub-P on Ca2+i was assessed at a concentration of 10 µmol/L, 3 min after application. Received: 22 December 2017 Accepted: 7 September 2018 References 1. Kapa, S., Venkatachalam, K. L. & Asirvatham, S. J. The autonomic nervous system in cardiac electrophysiology. Cardiol. Rev. 18, 275–284 (2010). 1. Kapa, S., Venkatachalam, K. L. & Asirvatham, S. J. The autonomic nervous system in cardiac electrophysiology. Cardiol. Rev. 18, 275–284 (2010). Immunohistochemistry. Rabbit left atrium and human left atrial appendages were snap-frozen in liquid nitrogen, and stored at −80 °C. Cryosections (7 µm) were mounted on 3-aminopropyltriethoxysilane (AAS)-coated glass slides and per- meabilized in 0.2% Triton X-100 in PBS for 20 min, where after they were blocked in 2% bovine serum albumin for 30 min. Human cryosections were incubated overnight with the following primary antibodies: polyclonal anti-NK3R (Immu- nostar 20061; 1:50 dilution) and monoclonal anti-α-actinin (Sigma T7811; 1:1000). Next, they were incubated for 90 min with secondary antibodies Alexa-conjugated goat anti-mouse and goat anti-rabbit antibodies (1:250, Molecular Probes, Invi- trogen) in 10% Normal Goat Serum (at room temperature). For double labelling, sections were incubated with a mixture of primary antibodies, followed by an appropriate mixture of secondary antibodies60. Rabbit cryosections were incubated overnight with the polyclonal primary antibody anti-NK3R (Abcam ab123303, 1:5 dilution) directly labelled with ATTO-488 ﬂuorophore using Lightning-Link® Rapid Conjugation System (Innova Biosciences, Cambridge, UK), to eliminate background staining on rabbit tissue. Confocal imaging was performed using a confocal laser scanning microscope (BioRad MRC1024) equipped with a 15-mV 2. Ripplinger, C. M., Noujaim, S. F. & Linz, D. The nervous heart. Prog. Biophys. Mol. Biol. 120, 199–209 (2016). 2. Ripplinger, C. M., Noujaim, S. F. & Linz, D. The nervous heart. Prog. Biophys. Mol. Biol. 120, 199–209 (2016). 3. Coumel, P. Autonomic inﬂuences in atrial tachyarrhythmias. J. Cardiovasc. Electrophysiol. 7, 999–1007 (1996). 4. Shen, M. J. et al. Neural mechanisms of atrial arrhythmias. Nat. Rev. 9, 30–39 (2011). 5. Wickramasinghe, S. R. & Patel, V. V. Local innervation and atrial ﬁbrillation. Circulation 128, 1566–1575 (2013). 6. Chen, P. S., Chen, L. S., Fishbein, M. C., Lin, S. F. & Nattel, S. Role of the autonomic nervous system in atrial ﬁbrillation: pathophysiology and therapy. Circ. Res. 114, 1500–1515 (2014). 6. Chen, P. S., Chen, L. S., Fishbein, M. C., Lin, S. F. & Nattel, S. Role of the autonomic nervous system in atrial ﬁbrillation: pathophysiology and therapy. Circ. Res. 114, 1500–1515 (2014). 7. Krul, S. P. J. et al. Treatment of atrial and ventricular arrhythmias through autonomic modulation. JACC Clin. Electrophysiol. 1, 496–508 (2015). 7. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 (Sub-P: 1, 10, 100 nM, 1, 10 µM; Senktide: 1, 2, 5 nM and 10, 20, 50 nM). The concentration of 100 nM Senktide was applied as a singular dose. (Sub-P: 1, 10, 100 nM, 1, 10 µM; Senktide: 1, 2, 5 nM and 10, 20, 50 nM). The concentration of 100 nM Senktide was applied as a singular dose. (Sub-P: 1, 10, 100 nM, 1, 10 µM; Senktide: 1, 2, 5 nM and 10, 20, 50 nM). The concentration of 100 nM Senktide was applied as a singular dose. Krypton/Argon laser, using the 568 and 488 excitation lines and 605DF32 and 522DF35 emission ﬁlters. Krypton/Argon laser, using the 568 and 488 excitation lines and 605DF32 and 522DF35 emission ﬁlters. Cardiomyocytes were pre-incubated with the speciﬁc NK-3R antagonist Osanetant35 ((R)-N-{{3-[1-Benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]prop-1- yl}-4-phenylpiperidin-4-yl}-N-methylacetamine) (300 nM) for 10 min to study th speciﬁcity of the Senktide-mediated APD prolongation. Cardiomyocytes were pre-incubated with the speciﬁc NK-3R antagonist Osanetant35 ((R)-N-{{3-[1-Benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]prop-1- yl}-4-phenylpiperidin-4-yl}-N-methylacetamine) (300 nM) for 10 min to study the speciﬁcity of the Senktide-mediated APD prolongation. RNA-sequencing. After excision of the heart from a male New Zealand White rabbit, tissue samples from the left atrial free wall and left atrial appendage were isolated (4 mm3) and snap frozen in liquid nitrogen. RNA was isolated using TruSeq (Illumina) and sequenced using Illumina Hiseq 4000 (sequence length 50, single end). Reads are subjected to quality control (FastQC, Picard Tools), trimmed using Trimmomatic v0.3261 and aligned to the genomes using HISAT2 (v2.0.4)62. The genome and GTF were obtained from Ensembl (v0.89): OryCun2.0 (assembly GCA_000003625.1) and Oryctolagus_cuniculus.OryCun2.0.89.asFasta.gtf, respec- tively. Counts are obtained using HTSeq (v0.6.1)63. Statistical analyses are per- formed using the edgeR64 and limma/voom65 R packages. All genes with no counts in any of the samples are removed whilst genes with more than ﬁve reads in at least two of the samples are kept. Count data are transformed to log2-counts per million (logCPM), normalized by applying the trimmed mean of M-values method64 and precision weighted using voom66. Differential expression is assessed using an empirical Bayes moderated t-test within limma’s linear model framework, including the precision weights estimated by voom. Resulting P-values are cor- rected for multiple testing using the Benjamini-Hochberg false discovery rate. Genes are re-annotated using biomaRt using the Ensembl genome databases (v91). For ~65% of Ensembl IDs an Entrez Gene ID was retrieved using biomaRt.. Methods For AP measurements, a standard superfusion solution was used containing (in mM): NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1.0, glucose 5.5, HEPES 5.0, pH 7.4 (NaOH). The patch-pipettes (borosilicate glass; 1–3 MΩ) were ﬁlled with a standard pipette solution containing (in mM): K-gluconate 110, KCl 30, NaCl 5, MgCl2 1, amphotericin-B 0.22, HEPES 10, pH 7.3 (KOH). APs were elicited at 1–4 Hz by 2-ms (1.5× diastolic stimulation threshold) current pulses applied through the patch pipette. Resting membrane potential (RMP), maximal upstroke velocity (Vmax), AP amplitude (APA) and AP duration (APD) at 20%, 50% and 90% repolarization (APD20, APD50 and APD90, respectively, were obtained from 10 consecutive APs and averaged. For data analysis we considered three groups: (1) no arrhythmia, (2) an arrhythmia episode consisting of short runs of premature atrial activations with a duration Δt <1 s, and (3) an irregular arrhythmia episode with Δt >1 s in duration, which was deﬁned as AF. To assess the anti-arrhythmic effect of Senktide we determined the total duration of arrhythmias episodes, which was deﬁned as the sum of durations of arrhythmia episodes following 10 consecutive burst pacing protocols, and the AF incidence, which was deﬁned as the fraction of the 10 consecutive burst pacing protocols imposed, that evoked AF. Left atrial refractoriness in the anaesthetized rabbit. Male New Zealand White rabbits (2.5–3.5 kg) were anaesthetized with ketamine 50 mg/kg and xylazine 8 mg/kg, and received 0.09 mg/kg Temgesic subcutaneously. The rabbits were intubated and artiﬁcially ventilated. Anaesthesia was maintained by isoﬂurane 12 NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications ARTICLE g y p p p y 24. Wilke, B. U. et al. Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors. Nat. Commun. 5, 1–11 (2014). 52. Verkerk, A. O., Geuzebroek, G. S. C., Veldkamp, M. W. & Wilders, R. Effects of acetylcholine and noradrenalin on action potentials of isolated rabbit sinoatrial and atrial myocytes. Front. Physiol. 3, 174 (2012). y 25. Feliciangeli, S., Chatelain, F. C., Bichet, D. & Lesage, F. The family of K2P channels: salient structural and functional properties. J. Physiol. 593, 2587–2603 (2015). 53. Barry, P. H. & Lynch, J. W. Liquid junction potentials and small cell effects in patch-clamp analysis. J. Membr. Biol. 121, 101–117 (2012). l h l d l h b f h patch-clamp analysis. J. Membr. Biol. 121, 101–117 (2012) 26. Zhang, H., Shepherd, N. & Creazzo, T. L. Temperature-sensitive TREK currents contribute to setting the resting membrane potential in embryonic atrial myocytes. J. Physiol. 586, 3645–3656 (2008). 54. Flaherty, D. P. et al. Potent and selective inhibitors of the TASK-1 potassium channel through chemical optimization of a bis-amide scaffold. Bioorg. Med. Chem. Lett. 24, 3968–3973 (2014). y y y 27. Limberg, S. H. et al. TASK-1 channels may modulate action potential duration of human atrial cardiomyocytes. Cell Physiol. Biochem. 28, 613–624 (2011). 55. Coburn, C. A. et al. Discovery of a pharmacologically active antagonist of the two-pore-domain potassium channel K2P9.1 (TASK-3). Chem. Med. Chem. 7, 123–133 (2011). 28. Rinné, S. et al. TASK-1 and TASK-3 may form heterodimers in human atrial cardiomyocytes. J. Mol. Cell Cardiol. 81, 71–80 (2015). 56. Mazella, J. et al. Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design. PLoS Biol. 8, e1000355 (2010). 29. Clarke, C. E., Veale, E. L., Green, P. J., Meadows, H. J. & Mathie, A. Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IK SO, by zinc. J. Physiol. 560, 51–62 (2004). 57. Zygmunt, A. C. & Gibbons, W. R. Properties of the calcium-activated chloride current in heart. J. Gen. Physiol. 99, 391–414 (1992). p y y 30. Leonoudakis, D. et al. An open rectiﬁer potassium channel with two pore domains in tandem cloned from rat cerebellum. J. Neurosci. 18, 868–877 (1998). 58. Hinde, A. K., Perchenet, L., Hobai, I. A., Levi, A. J. & Hancox, J. C. ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 microscopical and immunocytochemical study. J. Auton. Nerv. Syst. 17, 21–32 (1986). 38. Church, D. J. et al. Stimulation of atrial natriuretic peptide release by neurokinins in neonatal rat ventricular cardiomyocytes. Am. J. Physiol. 270, H935–H944 (1996). microscopical and immunocytochemical study. J. Auton. Nerv. Syst. 17, 21–32 (1986). 9. Wharton, J. et al. Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage. Cell Tissue Res. 254, 155–166 (1988). 39. Onuoha, G. N., Alpar, E. K., Chukwulobelu, R. & Nicholls, D. P. Distributions of VIP, substance P, neurokinin A and neurotensin in rat heart: an immunocytochemical study. Neuropeptides 33, 19–25 (1999). 10. Pauza, D. H., Pauziene, N., Pakeltyte, G. & Stropus, R. Comparative quantitative study of the intrinsic cardiac ganglia and neurons in the rat, guinea-pig, dog and human as revealed by histochemical staining for acetylcholinesterase. Ann. Anat. 184, 125–136 (2002). 40. Chang, Y., Hoover, D. B. & Hancock, J. C. Endogenous tachykinins cause bradycardia by stimulating cholinergic neurons in the isolated guinea pig heart. Am. J. Physiol., Reg. Integr. Comp. Physiol. 278, R1483–R1489 (2000). y 11. Hoover, D. B. et al. Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia. Neuroscience 164, 1170–1179 (2009). y g g p y 41. Schmidt, C. et al. Upregulation of K(2P)3.1 K+current causes action potential shortening in patients with chronic atrial ﬁbrillation. Circulation 132, 82–92 (2015). 12. Dorian, P. & Newman, D. Rate dependence of the effect of antiarrhythmic drugs delaying cardiac repolarization: an overview. Europace 2, 277–285 (2000). 42. Nygren, A. Mathematical model of an adult human atrial cell. the role of K currents in repolarization. Circ. Res. 82, 63–81 (1998). 43. Vanner, S., Evans, R. J., Matsumoto, S. G. & Surprenant, A. Potassium currents and their modulation by muscarine and substance P in neuronal cultures from adult guinea pig celiac ganglia. J. Neurophysiol. 69, 1632–1644 (1993). 13. Ravens, U. Antiarrhythmic therapy in atrial ﬁbrillation. Pharmacol. Ther. 128, 129–145 (2010). 14. Von Euler, U. S. & Gaddum, J. H. An unidentiﬁed depressor substance in certain tissue extracts. J. Physiol. 72, 74–87 (1931). 44. Ishimatsu, M. Substance P produces an inward current by suppressing voltage-dependent and -independent K+currents in bullfrog primary afferent neurons. Neurosci. Res. 19, 9–20 (1994). y 15. Almeida, T. A. et al. Tachykinins and tachykinin receptors: structure and activity relationships. Curr. Med. Chem. 11, 2045–2081 (2004). ARTICLE 45. Shen, K. Z. & Surprenant, A. Common ionic mechanisms of excitation by substance P and other transmitters in guinea-pig submucosal neurones. J. Physiol. 462, 483–501 (1993). 16. Steinhoff, M. S., Mentzer von, B., Geppetti, P., Pothoulakis, C. & Bunnett, N. W. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease. Physiol. Rev. 94, 265–301 (2014). y 46. Noh, S. et al. The direct modulatory activity of zinc toward ion channels. Integr. Med. Res. 4, 142–146 (2015). 17. Hoover, D. B., Chang, Y., Hancock, J. C. & Zhang, L. Actions of tachykinins within the heart and their relevance to cardiovascular disease. Jpn. J. Pharmacol. 84, 367–373 (2000). 47. Fioranelli, M. et al. Analysis of heart rate variability ﬁve minutes before the onset of paroxysmal atrial ﬁbrillation. Pacing Clin. Electrophysiol. 22, 743–749 (1999). ( ) 18. Mistrova, E., Kruzliak, P. & Dvorakova, M. C. Role of substance P in the cardiovascular system. Neuropeptides 58, 41–51 (2015). 48. Sharifov, O. F. et al. Roles of adrenergic and cholinergic stimulation in spontaneous atrial ﬁbrillation in dogs. J. Am. Coll. Cardiol. 43, 483–490 (2004). 19. Guler, N. et al. Do cardiac neuropeptides play a role in the occurrence of atrial ﬁbrillation after coronary bypass surgery? Ann. Thorac. Surg. 83, 532–537 (2007). 20. Yu, Y., Liu, L., Jiang, J. Y., Qu, X. F. & Yu, G. Parasympathetic and substance P-immunoreactive nerve denervation in atrial ﬁbrillation models. Cardiovasc. Pathol. 21, 39–45 (2012). 49. Wiegerinck, R. F. et al. Transmural dispersion of refractoriness and conduction velocity is associated with heterogeneously reduced connexin43 in a rabbit model of heart failure. Heart Rhythm. 5, 1178–1185 (2008). 21. Enyedi, P. & Czirják, G. Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol. Rev. 90, 559–605 (2010). 50. Krul, S. P. J. et al. Thoracoscopic video-assisted pulmonary vein antrum isolation, ganglionated plexus ablation, and periprocedural conﬁrmation of ablation lesions: ﬁrst results of a hybrid surgical-electrophysiological approach for atrial ﬁbrillation. Circ. Arrhythm. Electrophysiol. 4, 262–270 (2011). 22. Khawaja, A. M. & Rogers, D. F. Tachykinins: receptor to effector. Int. J. Biochem. Cell Biol. 28, 721–773 (2003). 23. Mathie, A. Neuronal two-pore-domain potassium channels and their regulation by G protein-coupled receptors. J. Physiol. 578, 377–385 (2007). y p y 51. de Jong, J. S. S. G. et al. Activated human platelet products induce proarrhythmic effects in ventricular myocytes. J. Mol. Cell Cardiol. 51, 347–356 (2011). References Krul, S. P. J. et al. Treatment of atrial and ventricular arrhythmias through autonomic modulation. JACC Clin. Electrophysiol. 1, 496–508 (2015). 8. Rechardt, L., Aalto-Setälä, K., Purjeranta, M., Pelto-Huikko, M. & Kyösola, K. Peptidergic innervation of human atrial myocardium: an electron 8. Rechardt, L., Aalto-Setälä, K., Purjeranta, M., Pelto-Huikko, M. & Kyösola, K. Peptidergic innervation of human atrial myocardium: an electron 13 Acknowledgements Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ R.C. is supported by the Leducq International network of Excellence RHYTHM. J.R.G. is supported by a personal VIDI grant from the Netherlands Organization for Scientiﬁc Research NWO/ZonMW 016.146.310. C.A.R. is supported by a personal VIDI grant from the Netherlands Organization for Scientiﬁc Research NWO/ZonMW Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. (91714371). B.J.B. is supported by a personal grant from the Dutch Heart Foundation (2016T047). (91714371). B.J.B. is supported by a personal grant from the Dutch Heart Foundation (2016T047). 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ARTICLE Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 °C, dialysed internally with cAMP-free and cAMP-containing solutions. Cell Calcium 25, 321–331 (1999). 31. Gruss, M., Mathie, A., Lieb, W. R. & Franks, N. P. The two-pore-domain K(+) channels TREK-1 and TASK-3 are differentially modulated by copper and zinc. Mol. Pharmacol. 66, 530–537 (2004). 59. Baartscheer, A. et al. Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model. Cardiovasc. Res. 15, 1015–1024 (2003). 32. Wormser, U. et al. Highly selective agonists for substance P receptor subtypes. EMBO J. 5, 2805–2808 (1986). 33. Drapeau, G. et al. Selective agonists for substance P and neurokinin receptors. Neuropeptides 10, 43–54 (1987). 60. Verkerk, A. O. et al. Functional NaV1.8 channels in intracardiac neurons: the link between SCN10A and cardiac electrophysiology. Circ. Res. 111, 333–343 (2012). 34. Regoli, D., Nguyen, Q. T., Juklc, D. & Rouissi, N. Functional characterization of neurokinin receptors with agonists and antagonists. Regul. Pept. 46, 287–289 (1993). 61. Bolger, A. M., . & Lohse, M. & Usadel, B. Trimmomatic: a ﬂexible trimmer for Illumina Sequence Data. Bioinformatics 30, 2114–2120 (2014). 35. Emonds-Alt, X. et al. SR 142801, the ﬁrst potent non-peptide antagonist of the tachykinin NK3 receptor. Life Sci. 56, PL27–PL32 (1995). 62. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2014). 36. Hoover, D. B. & Hancock, J. C. Distribution of substance P binding sites in guinea-pig heart and pharmacological effects of substance P. J. Auton. Nerv. Syst. 23, 189–197 (1988). 63. Anders, S., Pyl, P. T. & Huber, W. HTSeq – a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015). y 37. Walsh, R. J., Weglicki, W. P. & Correa-de-Araujo, R. Distribution of speciﬁc substance P binding sites in the heart and adjacent great vessels of the Wistar white rat. Cell Tissue Res. 284, 495–500 (1996). 64. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). 14 NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunicatio ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06530-5 performed the experiments and the statistical analysis, and edited the manuscript. R.C. ARTICLE conceived the project, designed the experiments, interpreted the data and wrote the manuscript. 65. Ritchie, M. E. et al. limma powers differential expression analyses for RNA- sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015). 66. Law, C. W., Chen, Y., Shi, W. & Smyth, G. K. voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 15, R29 (2014). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06530-5. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06530-5. © The Author(s) 2018 Additional information 67. Ruijter, J. M. et al. Factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology. Retrovirology 3, 2 (2006). Author contributions M.W.V. conceived the project, designed and performed the experiments, analysed and interpreted the data, and wrote the manuscript. G.S.C. performed the experiments, analysed and interpreted the data and wrote the manuscript. A.B. performed the experiments, analysed and interpreted the data and wrote the manuscript. A.O.V. per- formed the experiments, analysed and interpreted the data and wrote the manuscript. C. A.S. performed the experiments and analysed and interpreted the data. G.G.S. performed the experiments and analysed the data. W.R.B. performed the experiments and analysed the data. S.C. performed the experiments and analysed the data. S.C.M.A. performed the experiments and analysed the data. K.T.S. performed the experiments and analysed the data. A.H.G.D. interpreted the data and edited the manuscript. C.N.W.B. performed the experiments. A.C.G.G. interpreted the data and edited the manuscript. J.R.G. interpreted the data and edited the manuscript. J.M.T.B. interpreted the data and edited the manuscript. C.A.R. interpreted the data and wrote the manuscript. B.J.B. 15 NATURE COMMUNICATIONS \| (2018) 9:4357 \| DOI: 10.1038/s41467-018-06530-5 \| www.nature.com/naturecommunications
https://openalex.org/W1979726087	https://escholarship.org/content/qt61m5w598/qt61m5w598.pdf?t=otpano	English	null	Intersections and Conversations	American anthropologist	2,008	cc-by	1,221	UC Irvine UC Irvine Previously Published Works Title Intersections and Conversations Permalink https://escholarship.org/uc/item/61m5w598 Journal American Anthropologist, 110(2) ISSN 0002-7294 Author BOELLSTORFF, TOM Publication Date 2008-06-01 DOI 10.1111/j.1548-1433.2008.00021.x Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Intersections and Conversations Permalink https://escholarship.org/uc/item/61m5w598 Journal American Anthropologist, 110(2) ISSN 0002-7294 Author BOELLSTORFF, TOM Publication Date 2008-06-01 DOI 10.1111/j.1548-1433.2008.00021.x Copyright Information This work is made available under the terms of a Creative C availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Intersections and Conversations Readers will note an unusual number of “From the Editor” pieces in this issue of American Anthropologist. Although I have ﬁnal decision-making authority on all questions re- garding manuscripts submitted to this journal, it bears em- phasizing that managing American Anthropologist depends on the work of many individuals. The somewhat stilted moniker I have inherited, “Editor-in-Chief,” does not tell the whole story. For instance, the AA Editorial Board plays a crucial role in the everyday work of the journal, above all because it is they who help me identify appropriate re- viewers for manuscripts. In addition to this editorial board, I have created ﬁve Associate Editor positions for the journal (these persons also sit on the AA Editorial Board). In my “From the Editor” piece published in the previous issue of American Anthropologist (110.1), I noted that my decision has been to create: “From the Editor” pieces, written not by myself but by the editors of other AAA journals. I have solicited these pieces because I believe that a key aspect of American Anthropolo- gist’s mission is to support other association journals. If I may cite my previous piece once again: American Anthropologist is one of over twenty journals published under the auspices of the American Anthro- pological Association. It is unusual in that unlike every other journal (save Anthropology News), American Anthro- pologist is not published via a section (in the way that, for instance, the Journal of Linguistic Anthropology is pub- lished by the Society for Linguistic Anthropology). As Editor-in-Chief, I am strongly committed to furthering the idea that American Anthropologist’s “section” is not just the membership of the entire AAA, but speciﬁcally these other AAA journals. One immediate action I have taken in support of this idea has been to invite the editors of other AAA journals to have “From the Editor” pieces published in American An- thropologist itself. My hope is that this will acquaint read- ers with AAA journals of which they may be unaware. Fu- ture issues of American Anthropologist will also feature such “From the Editor” pieces written by the editors of other AAA journals. We are lucky as an association to have a stunning range of top-notch journals, all in various ways experimental, innovative, and featuring solid, cutting-edge scholarship. Powered by the California Digital Library University of California eScholarship.org AMERICAN ANTHROPOLOGIST, Vol. 110, Issue 2, pp. 169–170, ISSN 0002-7294 online ISSN 1548-1433. C⃝2008 by the American Anthropological Association. All rights reserved. DOI: 10.1111/j.1548-1433.2008.00021.x Intersections and Conversations I encourage you to take a look at these jour- nals via print or AnthroSource—and if your institution’s library does not subscribe to any of these journals, please urge them to do so. a system in which there are ﬁve Associate Editors for American Anthropologist. For a ﬂagship journal I believe this added layer of editorial oversight will help ensure that manuscripts get the most comprehensive and in- formed reading possible. These Associate Editors repre- sent archaeology, biological anthropology, linguistic an- thropology, and sociocultural anthropology, as well as an Associate Editor for practicing anthropology. One could imagine structures with more Associate Editors (for in- stance, an Associate Editor for practicing anthropology for each subﬁeld, or Associate Editors for ﬁelds like med- ical anthropology, visual anthropology, legal anthropol- ogy, and so on). I believe that the structure of ﬁve As- sociate Editors outlined above strikes the best balance in terms of being maximally broad and minimally cumber- some, and I hope that readers of American Anthropologist will support me in this decision. The research articles featured in this issue of American Anthropologist reﬂect the broad range of subdisciplinary, theoretical, and methodological interests of members of the association. In “Collective Action in Action,” Michael Gurven and Jeffrey Winking investigate questions of altru- ism and prosocial behavior, moving between ethnographic and experimental game methods and also between the In the following pages, you will see welcome messages from all ﬁve of these Associate Editors. I am deeply thankful for their willingness to step forward and participate in the editorial work of American Anthropologist. Following these messages from the Associate Editors, you will ﬁnd four American Anthropologist • Vol. 110, No. 2 • June 2008 170 speciﬁcities of ﬁeldwork in lowland Bolivia and broad ques- tions of methodology in sociocultural research. Barbara L. Voss’s article “Domesticating Imperialism” turns an archae- ological eye toward questions of sexuality and colonial- ism, bringing a fascinating perspective to questions about the place of gender and sex in New World encounters. In “Scaffolding Imitation in Capoeira,” Greg Downey draws together theories of pedagogy and neurological research on imitative learning to craft a novel approach to questions of embodiment and education; we also feature a photo taken by him on this issue’s cover. E. Intersections and Conversations Paul Durrenberger and Dimitra Doukas combine ethnographic, historical, and quantitative methods in “Gospel of Wealth, Gospel of Work” to explore ideologies of labor and consumption in the United States. Bettina Shell-Duncan provides an exten- sive analysis of debates over female genital cutting, self- hood, and justice in “From Health to Human Rights.” Fi- nally, in “The ‘Old West’ in the Middle East,” Stephen W. Silliman examines uses of the “Indian Country” metaphor in the context of current conﬂicts in Iraq and Afghanistan. questions of method and social change. I have not explic- itly grouped the articles around such themes. I might, of course, choose to create such groupings in some future is- sue of American Anthropologist, but for this set of research articles, I think readers will ﬁnd it productive to discover the unexpected and polyvalent manner in which these themes emerge and intersect. As is usual for American Anthropologist, this issue in- cludes a range of fascinating single book reviews and review essays. A more unusual review featured in this issue is by George W. Stocking Jr. and concerns Eskimo Story (Written for My Children), a manuscript written by Franz Boas and cir- culated privately among his family until published in 2007. Stocking both situates this discovery in the broader oeuvre of Boas’s writings during this period and provides helpful speculations concerning the manner in which it was writ- ten (incl. its abrupt ending). I invite you to enjoy this issue of American Anthropol- ogist. Both the messages from the Associate Editors and editors of other AAA journals, on the one hand, and the research articles and reviews, on the other hand, speak to the vitality of the multifaceted conversations characterizing our discipline. It should be clear that a range of crosscutting themes emerge from these articles. These include questions of learn- ing and knowledge, questions of language and politics, and
https://openalex.org/W2919854971	https://hal.archives-ouvertes.fr/hal-02324065/file/applsci-09-00929-v2.pdf	English	null	The Virial Effect—Applications for SF6 and CH4 Thermal Plasmas	Applied sciences	2,019	cc-by	13,656	The Virial Effect-Applications for SF6 and CH4 Thermal Plasmas Andriniaina Harry Solo, Pierre Freton, Jean Jacques Gonzalez To cite this version: Andriniaina Harry Solo, Pierre Freton, Jean Jacques Gonzalez. The Virial Effect-Applications f SF6 and CH4 Thermal Plasmas. Applied Sciences, 2019, 9 (5), pp.929. 10.3390/app9050929. ha 02324076 To cite this version: Andriniaina Harry Solo, Pierre Freton, Jean Jacques Gonzalez. The Virial Effect-Applications for SF6 and CH4 Thermal Plasmas. Applied Sciences, 2019, 9 (5), pp.929. 10.3390/app9050929. hal- 02324076 To cite this version: Andriniaina Harry Solo, Pierre Freton, Jean Jacques Gonzalez. The Virial Effect-Applications for SF6 and CH4 Thermal Plasmas. Applied Sciences, 2019, 9 (5), pp.929. 10.3390/app9050929. hal- 02324076 Distributed under a Creative Commons Attribution 4.0 International License Received: 8 February 2019; Accepted: 1 March 2019; Published: 5 March 2019 Abstract: A tool based on the mass action law was developed to calculate plasma compositions and thermodynamic properties for pure gases and mixtures, assuming a local thermodynamic equilibrium for pressures of up to 300 bar. The collection of the data that was necessary for tool calculation was automated by another tool that was written using Python, and the formats for the model were adapted directly from the NIST and JANAF websites. In order to calculate the plasma compositions for high pressures, virial correction was introduced. The inﬂuences of the parameters that were chosen to calculate the Lennard–Jones (12-6) potential were studied. The results at high pressure show the importance of virial correction for low temperatures and the dependence of the dataset used. Experimental data are necessary to determine a good dataset, and to obtain interaction potential. However, the data available in the literature were not always provided, so they are not well-adapted to a large pressure range. Due to this lack, the formulation provided by L. I. Stiel and G. Thodos (Journal of Chemical and Engineering Data, vol. 7, 1962, p. 234–236) is a good alternative when the considered pressure is not close to the critical point. The results may depend strongly on the system studied: examples using SF6 and CH4 plasma compositions are given at high pressure. Received: 8 February 2019; Accepted: 1 March 2019; Published: 5 March 2019 Keywords: plasma composition; high pressure; virial coefﬁcient; SF6; CH4 HAL Id: hal-02324076 https://hal.science/hal-02324076v1 Submitted on 21 Oct 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License applied sciences Article The Virial Effect—Applications for SF6 and CH4 Thermal Plasmas Andriniaina Harry Solo, Pierre Freton and Jean-Jacques Gonzalez * Laboratoire Plasma et Conversion d’Energie, Université de Toulouse, CNRS, INPT, UPS, 118 Route de Narbonne, F-31062 Toulouse CEDEX 9, France; andriniaina.harry@laplace.univ-tlse.fr (A.H.S.); freton@laplace.univ-tlse.fr (P.F.) * Correspondence: freton@laplace.univ-tlse.fr Received: 8 February 2019; Accepted: 1 March 2019; Published: 5 March 2019 Abstract: A tool based on the mass action law was developed to calculate plasma compositions and thermodynamic properties for pure gases and mixtures, assuming a local thermodynamic equilibrium for pressures of up to 300 bar. The collection of the data that was necessary for tool calculation was automated by another tool that was written using Python, and the formats for the model were adapted directly from the NIST and JANAF websites. In order to calculate the plasma compositions for high pressures, virial correction was introduced. The inﬂuences of the parameters that were chosen to calculate the Lennard–Jones (12-6) potential were studied. The results at high pressure show the importance of virial correction for low temperatures and the dependence of the dataset used. Experimental data are necessary to determine a good dataset, and to obtain interaction potential. However, the data available in the literature were not always provided, so they are not well-adapted to a large pressure range. Due to this lack, the formulation provided by L. I. Stiel and G. Thodos (Journal of Chemical and Engineering Data, vol. 7, 1962, p. 234–236) is a good alternative when the considered pressure is not close to the critical point. The results may depend strongly on the system studied: examples using SF6 and CH4 plasma compositions are given at high pressure. Keywords: plasma composition; high pressure; virial coefﬁcient; SF6; CH4 applied sciences applied sciences Andriniaina Harry Solo, Pierre Freton and Jean-Jacques Gonzalez * Laboratoire Plasma et Conversion d’Energie, Université de Toulouse, CNRS, INPT, UPS, 118 Route de Narbonn F-31062 Toulouse CEDEX 9, France; andriniaina.harry@laplace.univ-tlse.fr (A.H.S.); freton@laplace.univ-tlse.fr (P.F.) C d f l l l f Laboratoire Plasma et Conversion d’Energie, Université de Toulouse, CNRS, INPT, UPS, 118 Route de Narbo F-31062 Toulouse CEDEX 9, France; andriniaina.harry@laplace.univ-tlse.fr (A.H.S.); freton@laplace.univ-tlse.fr (P.F.) Laboratoire Plasma et Conversion d’Energie, Université de Toulouse, CNRS, INPT, UPS, 118 Route de Narbonne, F-31062 Toulouse CEDEX 9, France; andriniaina.harry@laplace.univ-tlse.fr (A.H.S.); freton@laplace.univ-tlse.fr (P.F.) * Correspondence: freton@laplace.univ-tlse.fr 1. Introduction g pp In our research ﬁelds, at high pressures, we are particularly focused on three topics - the plasma behavior in high-voltage circuit breakers (HVCBs) [27–29], where pressures in heating volumes can reach bar values of several tens; - the plasma behavior in high-voltage circuit breakers (HVCBs) [27–29], where pressures in heating volumes can reach bar values of several tens; - hydro–electro formation and hydraulic fracturing using an electric arc in water [30]; - the combustion of methane in thermal gas engines [31]. For each of these topics, the pressure considered and the gases used are different, and they include, within a high-pressure range, SF6 for the ﬁrst application, H2O for the second, and mixtures of air and CH4 for the third. In the case of HVCB, some compositions and thermal properties are available in the literature; nevertheless, a complete databank for describing the current cutoff from the initial pressure to the one obtained during the high-current phase is not given, and authors seem to use the same parameters to calculate the Lennard–Jones potential for the entire pressure range [14,32,33]. This problem is the same for the case of hydro–electro formation and hydraulic fracturing: little data is available for H2O, and only few details on the parameters that are used to calculate the Lennard–Jones potential are given. For engine applications, we did not ﬁnd data related to air–CH4 mixtures in the literature. Therefore, papers from the literature generally consider pure air, and pressure corrections are not taken into account [34–36]. In the ﬁrst part of this paper, the method that was used to calculate the plasma composition is presented. The different equations based on the mass action law, neutrality, conservation of nuclei, and pressure law, used for determining the species densities, are given. The validation of the developed tool is presented with regard to some plasma compositions at atmospheric pressure. In the second part, the importance of the parameters that were used for determining the interaction potential for virial correction is presented. These parameters can be obtained from experimental data, or from a theoretical approach. The two methods are discussed, and they are applied to a mass density calculation. Finally, the plasma compositions of SF6 and CH4 are calculated at high pressures. 1. Introduction The calculation of plasma compositions under the assumption of local thermal equilibrium (LTE) is necessary to determine the thermodynamic properties and the transport coefﬁcients that are needed for magneto-hydrodynamic models of thermal plasma processes and systems as circuit breakers, plasma torches, or thermal engines. These compositions are also necessary as initial equilibrium states for chemical kinetic studies and to determine departures from chemical equilibrium. In the literature, three methods are mainly used to calculate the evolution of species densities with temperature and pressure in thermal plasmas: the Gibbs free enthalpy minimization [1], the mass action law method [2,3], and the collisional radiative model. Based on these methods, numerous papers have reported chemical plasma compositions for pure gases and mixtures, with or without the LTE assumption [2,4–16]. Nevertheless, in most of these papers, even at high pressure (>30 bar), only the ﬁrst-order Debye–Hückel correction is taken into account [2,17]. Although this correction is necessary at high temperatures to consider the Coulombic interactions between the charged particles and others, it is not sufﬁcient for low temperatures (around 300 K), where molecule interactions are important. Under these conditions, it is necessary to correct the perfect gas behavior by considering virial pressure correction. However, in the thermal plasma community, only a few authors have considered this correction at high pressures [4,6,9,14]. Appl. Sci. 2019, 9, 929; doi:10.3390/app9050929 www.mdpi.com/journal/applsci www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 929 2 of 22 The virial pressure correction can be developed over several orders, named virial coefﬁcients. They can be obtained experimentally ([18], for instance) by deducing them from measurements of the mass density, viscosity, or, in some cases, sound velocity [19,20] or by theoretical calculation using intermolecular interaction potentials [18,21,22]. In the literature, we can ﬁnd that, for some gases, the second and sometimes the third coefﬁcients are given as a function of the temperature [19,22–26]. These data are very dependent on the pressure and temperature of the gas, and they are not valid for a large range of temperatures and pressures. The densities of the species, and the mass density of the gases depend very strongly on the applied dataset. This mass density has a direct effect on convection modeling for its applications. 2.1. Partition Functions Whatever the method used to calculate the compositions, partition functions are necessary. Indeed, they are the link between the microscopic and macroscopic properties of the system. For each species, the total partition function Qvol tot can be calculated from Equation (1): Qvol tot (T) = 2.π.m.kB.T h2 3 2 .Qint. e−E0 kB.T (1) (1) where where - m is the mass of the species; - kB is the Boltzmann constant; - T is the temperature; 3 of 22 Appl. Sci. 2019, 9, 929 - h is Planck’s constant; - h is Planck’s constant; - E0 is the energy of formation. The ﬁrst term of this equation corresponds to the translation partition function Qtrans. Qint is the internal partition function, taking into account the vibrational, electronic, and nuclear partition functions. The last term takes into account the chemical energy that is needed for the formation of the particle. This expression differs for each species, due to their speciﬁc masses, from the internal partition function and the energy of formation. This last variable can be obtained from the JANAF table [37], which directly provides the value of the formation enthalpy ∆Hf for each species, which in most cases is estimated at absolute zero temperature. .1.1. The Internal Partition Function of Monoatomic Species For species with only one atom, the internal partition function is given by Equation (2): Qint(T) = imax ∑ i gi.e− Ei kB.T (2) (2) where - gi is the statistical weight of an electronic level i, given by gi = 2.J + 1, where J is the angular momentum; - gi is the statistical weight of an electronic level i, given by gi = 2.J + 1, where J is the angular momentum; - Ei is the energy of the electronic level i; - Ei is the energy of the electronic level i; Ei is the energy of the electronic level i; - imax is the limitation of the summation over the electronic levels, to avoid the divergence of the summation at high temperatures. The limit of the summation is the energy of ionization Eion, which is reduced by the lowering of the ionization potential ∆E (imax ≤Eion −∆E). The lowering of the ionization potential (Equation (3)) is introduced in order to limit the number of electronic energy levels that are considered in the sum (Equation (2)), due to the electric and electromagnetic ﬁeld effects created by the charged particles. This can be estimated from the Debye–Hückel formula (Equation (3)): - imax is the limitation of the summation over the electronic levels, to avoid the divergence of the summation at high temperatures. The limit of the summation is the energy of ionization Eion, which is reduced by the lowering of the ionization potential ∆E (imax ≤Eion −∆E). The lowering of the ionization potential (Equation (3)) is introduced in order to limit the number of electronic energy levels that are considered in the sum (Equation (2)), due to the electric and electromagnetic ﬁeld effects created by the charged particles. This can be estimated from the Debye–Hückel formula (Equation (3)): 2 ∆E = (Z + 1).e2 4.π.ε0.λD (eV) (3) (3) where - Z is the electrical charge of the considered species; - Z is the electrical charge of the considered species; - e is the electrical charge of the electron; - ε0 is the vacuum permittivity; - λD is the Debye length, given by Equation (4): λD = s ε0.kB.T e2. ne + ∑i̸=e Z2 i .ni (4) (4) where ne is the electronic density, and ni is the density of each species. 2.2.1. Choices of Elements and Chemical Species The chemical elements are the basic components. When an ionized gas is described, the electric charge needs to be considered and added to the list of the basic components. The chemical species are constituted of one or several of these basic components. For instance, the oxygen atom is the basic component of several chemical species, such as O, O+, O−, O2, and O2+. Of course, the element and species choices depend on the plasma of interest. The only limit is the availability of the species data in the NIST [38] and JANAF [37] databases for the internal partition functions. In order to properly differentiate the basic components and the chemical species, two parameters, M and N, are deﬁned. M is the number of chemical (or basic) elements, and N is the number of chemical species, with M ≤N. 2.1.2. The Internal Partition Function for Molecules For molecules, the internal partition function can be obtained from Equation (5), by using the spectroscopic data available in the JANAF database [37], and especially the free Gibbs energy and the enthalpy. Qint(T) = Pr kB.T h2 2.π.m.kB.T 3 2 exp " 1 Na.kB [H(0) −H(Tr)]JANAF T − G −H(Tr) T JANAF !# (5) Qint(T) = Pr kB.T h2 2.π.m.kB.T 3 2 exp " 1 Na.kB [H(0) −H(Tr)]JANAF T − G −H(Tr) T JANAF !# (5) where (5) B π B " Na B JANAF !# where where - Tr and Pr are the temperature and pressure, respectively, of the reference in the JANAF database; N i th A d t t - Tr and Pr are the temperature and pressure, respectively, of the reference in the JANAF database; - Na is the Avogadro constant; - Na is the Avogadro constant; g H(0) −H(Tr) represents the enthalpy at absolute zero; [G −H(Tr)]/T is the Gibbs energy, which must be known for each temperature that is considered For the electrons, the internal partition function is constant and equal to 2. The Equation for the Calculation of the LTE Plasma Composition 2.2. The Equation for the Calculation of the LTE Plasma Composition In this work, to calculate the plasma composition, we used the mass action law (Equation (6)) with the method proposed by Godin [2,3]. This method is a generalization of the Saha and Guldberg–Waage laws, and enables a fast solution for the system of equations. N ∏ i (ni)vi = N ∏ i Qvol tot, i vi (6) (6) where ni represents the densities of the species i, νi is the stoichiometric coefﬁcients of the reaction (cf. Section 2.2.4), Qvol tot,i is the total partition function (Equation (1)), and N is the number of species considered in the plasma. The composition calculation can then be divided into several steps, described below. .1.1. The Internal Partition Function of Monoatomic Species The various data on the energy levels, and the angular momentum for the calculation of the statistical weights of the considered levels, were extracted from the NIST database [38]. Appl. Sci. 2019, 9, 929 4 of 22 2.1.2. The Internal Partition Function for Molecules 2.1.2. The Internal Partition Function for Molecules 2.2.3. The Chemical Base 2.2.3. The Chemical Base The chemical base is a subsection of chemical species from which it is possible to express the other species that are not present in the base matrix, but which can be created by chemical processes. This introduces the concept of the base matrix B, and the out-of-base matrix B. Their coefﬁcients can be obtained from the composition matrix by Equation (8): ( Bk,i = Ck,i B∗ j,i = Cj,i with ( i, kϵ{1, M} jϵ{1, N −M} . (8) (8) In order to choose the chemical species of the base, some speciﬁc conditions have to be veriﬁed for each temperature step: In order to choose the chemical species of the base, some speciﬁc conditions have to be veriﬁed for each temperature step: The densities of the species in the base matrix must be the highest densities. - The densities of the species in the base matrix must be the highest densities. - The species of the base matrix must be independent, in order to avoid \|B\| = 0 (the null determinant). - The species that are present at very low densities must not be included in the base matrix. For that, at the beginning of the calculation, the densities of the base species must be evaluated. This estimation depends greatly on initial temperature and on the given pressure. For instance, for the composition matrix proposed in Equation (7), the estimation of the base matrix, starting from T = 30 kK and P = 1 bar, is given in Equation (9): N O charge B =    1 0 1 0 1 1 0 0 −1    N+ O+ e−      Species o f the base . (9) (9) The other species in the composition matrix (Equation (7)) that are not included in matrix B (Equation (9)) are used to constitute the out-of-base matrix B. 2.2.2. The Composition Matrix Once deﬁned as M elements and N species, the composition matrix can be built with elements Cj,i. Its dimension is N × M. This matrix links the chemical species with the basic elements. An example is shown in Equation (7), for plasma composed of dinitrogen and dioxygen. 5 of 22 Appl. Sci. 2019, 9, 929 N O charge C =                 1 0 0 1 0 1 2 0 0 1 1 0 0 1 0 0 1 1 0 2 0 1 2 0 0 0 −1                 N N+ N2 NO O O+ O2 NO2 e−                                Chemical species . (7) N O charge C =                 1 0 0 1 0 1 2 0 0 1 1 0 0 1 0 0 1 1 0 2 0 1 2 0 0 0 −1                 N N+ N2 NO O O+ O2 NO2 e−                                Chemical species . (7) (7) 2.2.4. The Reaction Coefﬁcient The reaction coefﬁcient matrix ν is used to link the species from the base to those that are out of the base. Its dimension is (N −M) × M, and its expression is given in Equation (10): B∗= v.B ⇔B∗ j,i = M ∑ k=1 vj,k.Bk,i (10) (10) where k is the number of species of the base, and j is the number of out-of-base species. where k is the number of species of the base, and j is the number of out-of-base species. From the concept of the reaction matrix coefﬁcient, it is possible to obtain all of the chemical processes, creating out-of-base species from the base ones. 6 of 22 Appl. Sci. 2019, 9, 929 2.2.5. Conservation Equations 2.2.5. Conservation Equations 5. Conservation Equations 2.2.5. Conservation Equations 2.2.5. Conservation Equations For closing the system, several equations of conservation are used. ng the system, several equations of conservation are used. - Conservation of nuclei: - Conservation of nuclei: - Conservation of nuclei: εj N ∑ i=1 ni.Ci,k = εk N ∑ i=1 ni.Ci,jwhere j, k ( ϵ {1, M −1} ̸= charge (11) (11) with the atomic proportion ε. 2.3. Automatization of the Tool The calculation of the composition requires a large amount of information, such as the information required for the determination of the partition functions for each species, the virial coefﬁcient, the considered pressure, and the temperature range or temperature step. 2.2.7. Numerical Methods and System of Equations As the equation system is strongly nonlinear, a linearization algorithm based on the Newton Raphson iterative method is derived, using Equations (18)–(21): M ∑ i=1 Jl,i.δnbi = −Rl ⇔J.δn = −R (18) Rl = −A ◦ l + M ∑ i=1 Al,bi.nbi + N−M ∑ j=1 Al,b∗ j .nb∗ j (19) M ∑ i=1 Jl,i.δnbi = −Rl ⇔J.δn = −R (18) Rl = −A ◦ l + M ∑ i=1 Al,bi.nbi + N−M ∑ j=1 Al,b∗ j .nb∗ j (19) Jl,i = Al,bi + 1 nbi N−M ∑ j=1 Al,b∗ j .nb∗ j .vj,ii and l ϵ {1, M} (20) nbi = nbi + δnbi (21) (18) Rl = −A ◦ l + M ∑ i=1 Al,bi.nbi + N−M ∑ j=1 Al,b∗ j .nb∗ j (19) (19) Jl,i = Al,bi + 1 nbi N−M ∑ j=1 Al,b∗ j .nb∗ j .vj,ii and l ϵ {1, M} (20) (20) (21) nbi = nbi + δnbi nbi = nbi + δnbi (21) where Jl,i is the Jacobian matrix of the system, Rl represents the residuals of the matrix, and δnbi is the correction applied on the densities at each iteration. The convergence criterion is given in Equation (22), with a convergence threshold ε = 10−10 [2]. where Jl,i is the Jacobian matrix of the system, Rl represents the residuals of the matrix, and δnbi is the correction applied on the densities at each iteration. The convergence criterion is given in Equation (22), with a convergence threshold ε = 10−10 [2]. ( \|Rl\| Max Al,ini N i=1 ε )M l=1 . (22) (22) 2.2.6. The Equation for Out-of-Base Species Density The general equation for the out-of-base densities n∗ bj, depending on the base densities nbi, and the total partition functions of the base (Qbi) and the out-of-base (Q∗ bj) species is given in Equation (17): ( n∗ bj = Q∗ bj.exp M ∑ i=1 vj,i. ln nbi −ln Qbi !)N−M j=1 . (17) (17) 2.2.7. Numerical Methods and System of Equations 2.2.7. Numerical Methods and System of Equations with the atomic proportion ε. - Electric neutrality: N ∑ i=1 ni.Zi = 0 (12) (12) where Zi is the electric charge of the species i (which can be directly expressed from Ci, charge). where Zi is the electric charge of the species i (which can be directly expressed from Ci, charge). - Pressure conservation: The plasma pressure P is determined by the Dalton law (the perfect gas law) with Debye–Hückel, and with virial correction for real ﬂuids: P = N ∑ i=1 ni.kb.T + ∆PDebye + ∆Pvirial (13) (13) where where - ∆PDebye is the Debye–Hückel pressure correction: - ∆PDebye is the Debye–Hückel pressure correction: ∆PDebye = − kb.T 24.π.λ3 D ; (14) (14) - ∆Pvirial is the virial pressure correction. For its calculation, several coefﬁcients describing the difference in behavior between real ﬂuids and perfect gases are needed. Taking into account the second- and third-order corrections, the virial pressure correction is given in Equation (15): - ∆Pvirial is the virial pressure correction. For its calculation, several coefﬁcients describing the difference in behavior between real ﬂuids and perfect gases are needed. Taking into account the second- and third-order corrections, the virial pressure correction is given in Equation (15): ∆Pvirial = kB.T.B(T). N ∑ i=1 ni !2 + kB.T.C(T). N ∑ i=1 ni !3 (15) (15) where B(T) and C(T) are the second and third virial coefﬁcients, respectively, and they depend on the temperature. The methods to obtain them are described in Section 3 of this paper (cf. Section 3 « virial coefﬁcient »). More generally, all conservation equations can be written in the form given in Equation (16): ( N ∑ i=1 Al,i.ni = A ◦ l )M l=1 (16) (16) where Al,i and A ◦ l are the coefﬁcients of the conservation equation l for species i. A summary of all of these equations is given in Table 1. where Al,i and A ◦ l are the coefﬁcients of the conservation equation l for species i. A summary of all of these equations is given in Table 1. Table 1. Conservation coefﬁcients. Table 1. Conservation coefﬁcients. Conservation Equation Al,i Ao l Number of Equations Atomic nuclei εjCi,k −εkCi,j 0 M-2 Electric neutrality Ci,charge 0 1 Pressure 1 (P−∆P) kT 1 Appl. Sci. 2019, 9, 929 7 of 22 on for Out-of-Base Species Density 2.2.6. The Equation for Out-of-Base Species Density 2.2.6. The Equation for Out-of-Base Species Density 2.3.2. The Initialization File This ﬁle is very important, as it contains all of the necessary parameters for determining the densities of the species. It generally contains the following: - the number of gases considered in the composition. - the number of gases considered in the composition. - the name(s) of the gas(es) in the composition (for instance, SF6, N2, and O2, Ar, and Cu, e - the molar fraction of each gas in the melt (the sum of all of the fractions must equal unity; for example, it could be 1 SF6, 0.7885 N2 and 0.2115 O2, or 0.8 Ar and 0.2 Cu, etc.); - the number of atoms and species that come from the gas mixture, in order to deﬁne M and N (cf. Section 2.2.1); - the names of the species in the plasma; - the atomic proportions of each atom/nucleus for the considered mixture (for example, 100% SF6: 1 S and 6 F; 78.85% N2 and 21.15% O2: (0.7885 × 2) N and (0.2115 × 2) O); - the atomic proportions of each atom/nucleus for the considered mixture (for example, 100% SF6: 1 S and 6 F; 78.85% N2 and 21.15% O2: (0.7885 × 2) N and (0.2115 × 2) O); - the considered pressure; 1 S and 6 F; 78.85% N2 and 21.15% O2: (0.7885 × 2) N and (0.2115 × 2) O); - the considered pressure; - the considered pressure; - the temperature range and the temperature step of the results. - the temperature range and the temperature step of the results. 2.3.1. Data from the NIST and JANAF Tables To simplify the searching and formatting of the data available in the NIST and JANAF tables, software was developed using Python. This enables all of the required data for the species of the chosen composition to be concatenated and formatted automatically. In the case of the data originating from the NIST [38], the added parameters for each species, complementing the different energy levels and angular momentums, are as follows: - the name of the species, for a link between the initialization ﬁle and the data ﬁle; - the name of the species, for a link between the initialization ﬁle and the data ﬁle; - the energy of formation, needed for the calculation of the total partition function; - the energy of ionization that is used in order to satisfy the limit condition of Equation (2 th it f th l l d di th t t d d t (i −1 i V) - the energy of ionization that is used in order to satisfy the limit condition of Equation (2); - the units for the energy levels, depending on the extracted data (in cm−1 or in eV); th b f l l t id t th i i ti f th id d i gy y q ( ) - the units for the energy levels, depending on the extracted data (in cm−1 or in eV); - the number of energy levels to consider, up to the ionization of the considered species; - the mass of the considered species. For data from the JANAF database [37], two parameters are added: the name of the species and the mass. We have collected 296 chemical species, which can be used for calculating compositions Appl. Sci. 2019, 9, 929 8 of 22 containing carbon (C), hydrogen (H), oxygen (O), nitrogen (N), sulfur (S), ﬂuorine (F), argon (Ar), helium (He), copper (Cu), and iron (Fe). All kinds of particles have been taken into account in this databank: atoms, molecules, positive and negative ions, and molecular ions. 2.4. Results and Discussion In order to validate our tool, we compared our results with two examples found in the literature at atmospheric pressure: In order to validate our tool, we compared our results with two examples found in the literature at atmospheric pressure: - Case 1: pure argon; Case 2: air with 78.084% N2, 20.946% O2, 0.036% CO2, and 0.934% Ar. - Case 2: air with 78.084% N2, 20.946% O2, 0.036% CO2, and 0.934% Ar. - Case 2: air with 78.084% N2, 20.946% O2, 0.036% CO2, and 0.934% Ar In order to properly compare the results with the literature, we considered the same species (Table 2) reported by authors of past studies. Table 2. Chemical species considered for argon and air plasma compositions. Gas Chemical Species Argon Ar, Ar +, Ar +2, Ar +3, e−. Air N, N+, N+2, O, O+, O+2, O−, C, C+, C+2, C−, Ar, Ar+, Ar+2, N2, N2+, NO, NO+, O2, O2+, C2, CO, N2O, NO2, CO2, e−. The composition obtained from our tool in Case 1 is shown with those of three other studies [2,11,16] in Figure 1. For the air plasma, our results were compared with the calculation of André [5], and are plotted in Figures 2 and 3 (Figure 2 shows the atomic and ionic species, and Figure 3 shows the molecular species). In the two cases (Ar and Air), a good agreement could be observed. At low temperatures, the neutral species were dominant. When the temperature increased, we observed different types of processes. In the case of argon, the ionization of the atoms and the simultaneous apparition of electrons occurred. In the case of air, we noted the dissociation of the molecules, the recombination processes, the apparition of electrons, and the ionization of atoms, but also the electronic attachments. Nevertheless, for some species, some of the results displayed differences between the literature and our calculations. These were generally due to the sources used for the internal partition function of each species. In Figure 1, the results from Godin [2] are based on the data available from Drawin & Felenbok [39]. In this database, the internal partition functions are given versus temperature, with values of the lowering of ionization potential. For the results of Rat [16], the source was not given, but the shift in argon atom density observed at high temperature could be due to the fact that the Ar+3 ion 9 of 22 Appl. 2.4. Results and Discussion Sci. 2019, 9, 929 Air was not taken into account by this author. For the air composition, the partition functions calculated by André [5] were determined from data in the literature [37–39]. Furthermore, his calculation method was different from ours, as he used the minimization of Gibbs free energy. The composition obtained from our tool in Case 1 is shown with those of three other studies [2,11,16] in Figure 1. For the air plasma, our results were compared with the calculation of André [5], and are plotted in Figures 2 and 3 (Figure 2 shows the atomic and ionic species, and Figure 3 shows the molecular species). , gy molecular species). Figure 1. Evolution of species: argon; P = 1 bar − local thermal equilibrium (LTE). Comparison of ours results with Colombo et al [11], Godin [2] and Rat et al [16] Figure 1. Evolution of species: argon; P = 1 bar −local thermal equilibrium (LTE). Comparison of ours results with Colombo et al [11], Godin [2] and Rat et al [16]. l. Sci. 2019, 9, 929 9 o Figure 2. Evolution of atomic species: air: P = 1 bar − LTE. Comparison of our results with P. André [5] Figure 2. Evolution of atomic species: air: P = 1 bar −LTE. Comparison of our results with P. André [5]. Although most papers from the literature used tabulated values for the partition functions, ose to calculate them from the available data of the NIST database [38] for monoatomic species, a Figure 1. Evolution of species: argon; P = 1 bar − local thermal equilibrium (LTE). Comparison of ours results with Colombo et al [11], Godin [2] and Rat et al [16] Figure 1. Evolution of species: argon; P = 1 bar −local thermal equilibrium (LTE). Comparison of ours results with Colombo et al [11], Godin [2] and Rat et al [16]. pl. Sci. 2019, 9, 929 9 of Figure 1. Evolution of species: argon; P = 1 bar − local thermal equilibrium (LTE). Comparison of ours results with Colombo et al [11], Godin [2] and Rat et al [16] Figure 1. Evolution of species: argon; P = 1 bar −local thermal equilibrium (LTE). Comparison of ours results with Colombo et al [11], Godin [2] and Rat et al [16]. Sci. 2019, 9, 929 9 Figure 2. Evolution of atomic species: air: P = 1 bar − LTE. 2.4. Results and Discussion Comparison of our results with P. André [5] Figure 2. Evolution of atomic species: air: P = 1 bar −LTE. Comparison of our results with P. André [5]. Although most papers from the literature used tabulated values for the partition functions, we hose to calculate them from the available data of the NIST database [38] for monoatomic species, and om the JANAF database [37] for molecules. Figure 2. Evolution of atomic species: air: P = 1 bar − LTE. C i f lt ith P A d é [5] Figure 2. Evolution of atomic species: air: P = 1 bar −LTE. Comparison of our results with P. André [5]. Comparison of our results with P. André [5] Although most papers from the literature used tabulated values for the partition functions, we chose to calculate them from the available data of the NIST database [38] for monoatomic species, and from the JANAF database [37] for molecules. Comparison of our results with P. André [5] Although most papers from the literature used tabulated values for the partition functions, we chose to calculate them from the available data of the NIST database [38] for monoatomic species, and from the JANAF database [37] for molecules. Appl. Sci. 2019, 9, 929 10 of 22 10 of 22 By using our tool, the LTE compositions of air and argon plasmas were calculated at P = 1 bar. The choices for the temperature range and of the considered species were made with the aim of assisting with the comparison of our results against the works of other authors. The results validate our tool for atmospheric pressure. Figure 2. Evolution of atomic species: air: P = 1 bar − LTE. Comparison of our results with P. André [5] Figure 3. Evolution of molecular species: air; P = 1 bar − LTE. Comparison of our results with P. André [5] Figure 3. Evolution of molecular species: air; P = 1 bar −LTE. Comparison of our results with P. André [5]. Figure 3. Evolution of molecular species: air; P = 1 bar − LTE. Comparison of our results with P. André [5] Figure 3. Evolution of molecular species: air; P = 1 bar −LTE. Comparison of our results with P. André [5]. where where - m is the number of gas for mixing; - m is the number of gas for mixing; - xi, xj, and xk are the respective molar fractions of species i, j, and k; - Bij is the binary interaction parameter; - Cijk is the ternary interaction parameter. 3.1. The Lennard–Jones Potential For calculating the virial coefﬁcients, several kinds of intermolecular potentials can be used, depending on the study [6,18,40–42]. Nevertheless, obtaining the third-order coefﬁcient requires the calculation of a triple integration on the potential. In the literature, the expression of this integral is only given for a hard-sphere potential, a square-shaped potential, and the Lennard–Jones (12-6) potential [18]. This last approach seems to be the best at properly describing inter-particle interactions, and it is used in this paper. In the two cases 3. Virial Coefﬁcients neutral species were dominant. When the temperature increased, we observed different types of processes. In the case of argon, the ionization of the atoms and the simultaneous apparition of electrons occurred. In the case of air, we noted the dissociation of the molecules, the recombination processes, the apparition of electrons, and the ionization of atoms, but also the electronic attachments. Nevertheless, for some species, some of the results displayed differences between the literature and our calculations These were generally due to the sources used for the internal The tool presented in this study is also aimed at determining initial compositions for kinetic studies, in applications where the pressure can reach bar values of several tens. The developments, taking into account the pressure correction, are now presented. Virial coefﬁcients are parameters that are used to describe the real behavior of a ﬂuid. For this description, the second- (B(T)) and third-(C(T)) order coefﬁcients could be used, and they were expressed using intermolecular potential: ies. In Figure 1, the results from Godin [2] are based on the data bok [39]. In this database, the internal partition functions are given B(T) = −2.π.Na. ∞ Z 0 f (r12).r2 12.dr12 (23) data given (23) C(T) = −8.π2.N2 a 3 . ∞ Z Z Z 0 f (r12).f (r13).f (r23).r12.r13.r23.dr1.dr2.dr3 (24) (24) where f rij = exp − u(rij) kB.T −1 is the Mayer function, u rij is the potential for interactions between two molecules, and rij are the coordinates of molecules i, relative to molecule j. = exp − u(rij) kB.T −1 is the Mayer function, u rij is the potential for interactions between two molecules, and rij are the coordinates of molecules i, relative to molecule j. For a mixture, the virial coefﬁcients can be expressed by Equations (25) and (26): B(T)mixture = m ∑ i=1 m ∑ j=1 Bij(T).xi.xj (25) (25) C(T)mixture = m ∑ i=1 m ∑ j=1 m ∑ k=1 Cijk(T).xi.xj.xk (26) (26) 11 of 22 Appl. Sci. 2019, 9, 929 3.1.1. Expression of the Potential The Lennard–Jones (LJ) potential derives from the Mie potential, assuming that n = 12 and m = 6. The number 12 corresponds to the repulsive index at short distances, and 6 to the attractive index, based on Van der Waals interactions. This shape of the potential is very commonly used by other authors [13,18,43,44] for describing particle interactions in pure gas and mixtures. This potential is given by u(r) = 4.ε. σ r 12 − σ r 6 (27) (27) where r is the distance between two atoms. 2019, 9, 929 - bij = 1 8 b 1 3 0i + b 1 3 0j 3 is the interaction parameter of the mixture; 1 - bij = 1 8 b 1 3 0i + b 1 3 0j 3 is the interaction parameter of the mixture; 1 - bij = 1 8 b 1 3 0i + b 1 3 0j 3 is the interaction parameter of the mixture; 1 - T∗ ij = kB.T εij is the reduced temperature of the mixture with εij = εi.εj 1 2 ; - T∗ ij = kB.T εij is the reduced temperature of the mixture with εij = εi.εj 1 2 ; - h Cijk(T) i S.W is the third virial coefﬁcient obtained from the square-shaped potential [18]; - h Cijk(T) i S.W is the third virial coefﬁcient obtained from the square-shaped potential [18]; - A T∗ i , A T∗ j , and A T∗ k are the tabulated values under the reduced temperature [18]. - A T∗ i , A T∗ j , and A T∗ k are the tabulated values under the reduced temperature [18]. When i = j = k, Ciii(T) = [Ciii(T)]S.W. A T∗ i 3 = Ci(T) = b2 0.C∗(T∗). When i = j = k, Ciii(T) = [Ciii(T)]S.W. A T∗ i 3 = Ci(T) = b2 0.C∗(T∗). From these expressions, it appears that the virial coefﬁcients obtained using the LJ potential use parameters of other kinds of potentials (hard-sphere and square-shaped), in order to better describe real ﬂuid behavior at high pressure. 3.1.2. Potential Parameters σ and ε The parameter σ corresponds to the distance at which it can be assumed that u(r) = 0; ε is an energy value, and it represents the depth of the potential. This last parameter is generally given by the expression ε kB . These two parameters play a very important role in the determination of the virial coefﬁcients. Currently, much data concerning these parameters can be found in the literature for several species [18,32,41,43,45,46]. The determination of these parameters was obtained for almost all cases from experimental measurements, based on the transport coefﬁcients (viscosity and diffusion), or the second virial coefﬁcient [18,43,45,46]. Nevertheless, for a given species, the values obtained by several authors [18,22,45–47] present very large differences, depending on the method used for their determination. An example of these disparities is presented for some species in Table 3. The LJ potential (12-6) has been considered in all relevant sources. Table 3. The variation of potential LJ (12-6) parameters for some species. Species σ [Å] ε kB [K] References Ar 2.83–3.48 110.2–124 [18,45,46] N2 3.681–4.21 26.9–95.9 [18,45,46] O2 3.30–3.58 88–118 [18,45,46] SF6 4.8–6.5 155–414.81 [18,22,45,47] CH4 3.22–3.882 137–176.8 [18,45,46] CO2 2.84–4.486 152–594.4 [18,45,46] Table 3. The variation of potential LJ (12-6) parameters for some species. In Table 3, the range for the values of σ is quite narrow, compared to the values of ε kB . In the cases of SF6 or CO2, the ε kB values presented a large range, and it was difﬁcult to make conclusions pertaining to the value to be used. In the literature, several approximation methods have been proposed for determining these parameters. Some of them are based on empirical rules, considering species interactions [18], and others are based on critical properties, such as the critical temperature, pressure, volume, or compressibility factor [45,48–51]. For example, Stiel [51] proposes the determination of the parameters based on Equations (34) and (35): σ = 0.1866 v 1 3c Z −6 5 c (34) ε kB = 65.3 Tc Z 18 5c (35) (34) (35) where vc, Zc, and Tc are critical properties. In order to choose the better dataset, it is necessary to compare the results with the experimental data. Hirschfelder [18] recommends the use of parameters deduced from experimental viscosity for calculations of transport properties, the experimental second virial coefﬁcients for the state equation calculation, and the calculation of thermodynamic properties. where r is the distance between two atoms. where r is the distance between two atoms. With the LJ potential, Hirschfelder [18] proposed the use of reduced amounts of data expressed with hard-sphere potential b0 (Equations (28) and (29)). This approach has been validated by this author for the second and third virial coefﬁcients for several species: B(T) = b0.B∗(T∗) (28) C(T) = b2 0.C∗(T∗) (29) (28) (29) where B(T) and C(T) are reduced values of, respectively, the second and third virial tabulated coefﬁcients versus the reduced temperature T* given in Equation (30): where B(T) and C(T) are reduced values of, respectively, the second and third virial tabulated coefﬁcients versus the reduced temperature T* given in Equation (30): T∗= kB.T ε . (30) (30) Equation (31) expresses the hard-sphere potential: Equation (31) expresses the hard-sphere potential: b0 = 2 3.πσ3. (31) (31) The signiﬁcance of σ and ε is given in the next paragraph. The signiﬁcance of σ and ε is given in the next paragraph. For a gas mixture, the terms Bij(T) and Cijk(T), in Equations (25) and (26), respectively, can be obtained using the LJ potential. Their respective expressions are given in Equations (32) and (33): Bij(T) = b0ij.B∗ T∗ ij (32) Bij(T) = b0ij.B∗ T∗ ij (32) Cijk(T) = h Cijk(T) i S.W. h A(T∗ i ).A T∗ j .A(T∗ k ) i (33) (32) Cijk(T) = h Cijk(T) i S.W. h A(T∗ i ).A T∗ j .A(T∗ k ) i (33) (33) 12 of 22 Appl. Sci. 3.2.1. Choice of Parameters From the values of σ and ε kB obtained with the Stiel formulation, it is possible to calculate the plasma composition and the mass density at high pressure. For validation, experimental gas densities at high pressure are not often available. Nevertheless, experimental mass densities are more widely available. Thus, a validation of the calculations for theoretical composition were performed with this data. As the species densities from our composition tool were previously presented, the mass density of the gas could be calculated according to Equation (36): ρ = N ∑ i=1 ni.mi (36) (36) where N is the total number of species, ni represents the densities of the species i, and mi is the mass of the species. p Table 5 shows a comparison of the calculated mass densities (from our tool and from using the Stiel formulation for the parameters of the Lennard–Jones potential) at different pressures, using the experimental data for SF6. The theoretical data were compared over a temperature range of 300–490 K. In order to study the inﬂuence of the virial coefﬁcients, the calculation was performed without taking them into account. It can be observed in Table 5 that the theoretical results with and without the virial corrections become closer over higher temperatures. This indicates that the virial corrections were predominantly relevant at low temperatures for high pressures. Nevertheless, despite the good agreement between the Stiel formulation-derived values of σ and ε kB and the experimental values (observed in Table 4), these values led to quite large differences in the theoretical mass densities, compared to the experimental values given by Funke [19] and Claus [57] at around the critical point (Pc ≈37.5 bar and Tc ≈318.8 K) (Table 5). Table 6 shows the same comparison for CH4. The mass densities for this gas were less sensitive for the values of σ and ε kB . Indeed, the virial effect was not as preponderant as that observed in Table 6. Even with the consideration of the virial correction, for SF6 (Table 5), large differences could be observed between the calculated and experimental values for some points. In order to better understand this, we tried to calculate the mass density through another method. Indeed, Funke [19] gives the values of the second and third virial coefﬁcients in his paper, deduced from measurements in a homogeneous area of SF6. 3.1.2. Potential Parameters σ and ε Nevertheless, the data in this last case were scarce, and they were only available for some species. where vc, Zc, and Tc are critical properties. In order to choose the better dataset, it is necessary to compare the results with the experimental data. Hirschfelder [18] recommends the use of parameters deduced from experimental viscosity for calculations of transport properties, the experimental second virial coefﬁcients for the state equation calculation, and the calculation of thermodynamic properties. Nevertheless, the data in this last case were scarce, and they were only available for some species. Appl. Sci. 2019, 9, 929 13 of 22 In Table 4, a comparison of the σ and ε kB parameters is given. The values from the literature used for the comparison with Stiel [51] are included in the range given in Table 3. These values were chosen, as they corresponded to the experimental values deduced from the viscosity. Consequently, it appears that the Stiel formulation [51] can be a good option for when data are missing. Table 4. A comparison of the LJ (12-6) potential parameters, based on their use in experimental measurements, as calculated by several authors from the formulation given by Stiel et al. [51]. Table 4. A comparison of the LJ (12-6) potential parameters, based on their use in experimental measurements, as calculated by several authors from the formulation given by Stiel et al. [51]. measurements, as calculated by several authors from the formulation given by Stiel et al. [51]. Species (σcal)[Å] ( ε kB )[K] Reference σlit [Å] ( ε kB )lit [K] Reference Ar 3.451 116.035 [52] 3.465 116 [18] N2 3.694 94.837 [53] 3.681 91.5 [18] O2 3.474 115.008 [54] 3.433 113 [18] SF6 4.993 215.04 [55] 5.199 212 [45] CH4 3.828 143.02 [51] 3.796 144 [18] CO2 3.993 191.143 [56] 3.996 190 [18] 3.2. Results and Discussion 3.2.1. Choice of Parameters Thus, by using the state equation proposed by Funke, it was possible to re-calculate the mass densities on the points given in the article. These values of ρcal are reported in Table 7. We expected that the mass densities calculated from the virial coefﬁcients given by Funke 14 of 22 Appl. Sci. 2019, 9, 929 would lead to values that were close to the experimental ones given in the same paper. Unfortunately, this was not the case, and a perfect agreement was only reached for P = 20 bar. Table 5. SF6 mass densities for several pressure values. SF6 Sources Funke et al. [19] Claus et al. [57] This Work with the Stiel Formulation T [K] Plit [bar] ρlit [kg/m3] Plit [bar] ρlit [kg/m3] ρcal [kg/m3] with Virial % Error without Virial % Error 300 100.1876 1505.417 100.613 1505.939 1673.43 11.2% 585.554 61.1% 40.1390 1382.802 - - 1312.44 5.1% 234.222 83.1% 20.0346 162.945 - - 147.34 9.6% 117.111 28.1% 316 100.2052 1399.646 - - 1444.65 3.2% 555.906 60.3% 40.6363 1152.050 - - 387.055 66.4% 222.362 80.7% 20.0334 142.452 - - 133.724 6.1% 111.181 22.0% 324 100.0588 1340.546 - - 1333.7 0.5% 542.18 59.6% 40.6180 550.994 - - 340.594 38.2% 216.872 60.6% 19.9775 134.527 - - 128.128 4.8% 108.436 19.4% 340 100.0395 1208.301 100.618 1210.5 1124.27 7.0% 516.665 57.2% 41.2991 351.610 - - 288.904 17.8% 206.666 41.2% 19.8607 121.89 - - 118.652 2.7% 103.333 15.2% 360 - - 100.512 1019.111 897.537 11.9% 487.962 52.1% - - 19.9922 111.689 109.6 1.9% 97.592 12.6% 375 - - 100.31 872.05 768.606 11.9% 468.443 46.3% - - 39.9242 241.556 230.316 4.7% 187.377 22.4% - - 20.1877 111.689 113.597 1.7% 93.688 16.1% 410 - - 102.462 644.85 583.902 9.5% 428.454 33.6% - - 40.7729 206.536 196.906 4.7% 171.382 17.0% - - 20.2447 93.8963 91.6877 2.4% 85.690 8.7% 460 - - 100.829 469.477 452.405 3.6% 381.883 18.7% - - 40.5095 170.138 166.066 2.4% 152.753 10.2% - - 20.2695 81.2073 79.7085 1.8% 76.376 5.9% 490 - - 101.215 415.62 404.055 2.8% 358.503 13.7% - - 20.0561 74.4856 74.0648 0.6% 71.700 3.7% Table 6. CH4 mass densities for several pressure values. CH4 Cristancho et al. 3.2.1. Choice of Parameters Five curves are plotted in these ﬁgures: - the experimental mass densities measured by Funke [19]; - the mass densities calculated from the second and third virial coefﬁcients given by Funke [19]; - the mass densities calculated from the values of σ and ε kB by Hirschfelder et al. [18]; - the mass densities calculated from the values of σ and ε kB , deduced by the Stiel formulation [51]; - the mass densities calculated from the values of σ and ε kB , deduced from the optimization process and named “ﬁt”. - the mass densities calculated from the second and third virial coefﬁcients given by Funke [19]; - the mass densities calculated from the values of σ and ε kB by Hirschfelder et al. [18]; - the mass densities calculated from the values of σ and ε kB , deduced by the Stiel formulation [51]; - the mass densities calculated from the values of σ and ε kB , deduced from the optimization process and named “ﬁt”. the mass densities calculated from the second and third virial coefﬁcients given by Funke [1 The mass densities obtained from the optimization process are the closest to the experimental ones. The mass densities obtained from the other sources from the literature are sometimes in good agreement below the critical point, and sometimes agree at values over the critical point, but not over the full range of temperature and pressure. By using the optimized data (ﬁt), the relative errors of the experimental values did not exceed 9%. Thus, when experimental data for the mass densities are available, obtaining σ and ε kB with the optimized software is preferable. This dataset σ = 5.21279 Å and ε kB = 222.27 K gives a better approximation of the calculated mass densities for a pressure range of between 1 and 300 bar. The use of the Stiel [51] formulation is a good option for pressures and temperatures that are not too close to the critical point Pc ≈37.5 bar and Tc ≈318.8 K (cf. Figures 4 and 6), when no other data are available. The curves from Figures 4–6 show discrepancies in the variation of the LJ potential parameters on the mass densities deduced from the composition. 3.2.1. Choice of Parameters [58] This Work with the Stiel Formulation Tlit [K] P [bar] ρlit [kg/m3] ρcal [kg/m3] with virial % Error without Virial % Error 305.236 50.01 34.173 34.011 0.5% 31.636 7.4% 305.231 99.93 72.932 72.321 0.8% 63.216 13.3% 338.049 50 29.983 29.848 0.5% 28.542 4.8% 338.037 69.05 42.093 41.821 0.6% 39.416 6.4% 338.103 99.69 62.054 62.391 0.5% 56.907 8.3% 400.015 100.02 49.746 49.807 0.1% 48.246 3.0% 450.115 344.92 136.647 136.563 0.061% 147.891 7.6% 15 of 22 Appl. Sci. 2019, 9, 929 Table 7. Comparison of the SF6 mass densities between the experimental values [19] and the theoretical value calculated from B and C [19]. Table 7. Comparison of the SF6 mass densities between the experimental values [19] and the theoretical value calculated from B and C [19]. SF6 Sources Funke et al. [19] This Work T [K] B [cm3/mol] C [cm3/mol]2 P [Bar] ρlit [kg/m3] ρcal [kg/m3] from B and C % Error 300 −271.2 ± 0.8 18,160 ± 1250 100.1876 1505.417 1732.788 15.1% 40.1390 1382.802 1532.088 10.8% 20.0346 162.945 162.913 0.01% 316 −241.1 ± 0.8 17,850 ± 1000 100.2052 1399.646 1467.539 4.9% 40.6363 1152.050 1122.203 2.6% 20.0334 142.452 142.439 0.0% 324 −227.4 ± 0.6 16,950 ± 750 100.0588 1340.546 1410.368 5.2% 40.6180 550.994 915.276 66.1% 19.9775 134.527 134.572 0.03% 340 −202.9 ± 0.6 15,860 ± 750 100.0395 1208.301 1235.266 2.2% 41.2991 351.610 355.637 1.1% 19.8607 121.89 121.912 0.01% These large differences close to critical point for SF6 caused us to search for better values for σ and ε kB , by lowering the error between the theoretical and experimental values of mass density. Homemade optimization software was developed for obtaining such values. The software proposes values of σ and ε kB that minimize the difference between the theoretical and experimental mass densities, using all of the experimental data available. This minimization was based on a real genetic algorithm proposed by Ballester [59]. From this model, we obtained two values: σ = 5.21279 Å and ε kB = 222.27 K. Different sources of SF6 mass densities versus temperature are indicated in Figures 4–6, respectively, for pressure P = 20, 40, and 100 bar. These values were compared to the mass densities that were calculated from the parameters deduced from the genetic algorithm. 3.2.1. Choice of Parameters It should be underlined that a dataset for σ and ε kB , obtained from the experimental measurements for the given values of temperature and pressure, cannot be used without care over the whole pressure range. Appl. Sci. 2019, 9, 929 16 of 22 16 of 22 Figure 4. The SF6 mass density at 20 bar. Figure 5. The SF6 mass density at 40 bar. Figure 4. The SF6 mass density at 20 bar. Figure 4. The SF6 mass density at 20 bar. Figure 5. The SF6 mass density at 40 bar. Figure 5. The SF6 mass density at 40 bar. 2.2. SF6 and CH4 Plasma Compositions Using the values for σ and ε kB obtained from the genetic algorithm, the compositions of the nd CH4 plasma were calculated at pressure P = 100 bar. The values proposed by the genetic algorit o minimize the error using the same values of σ and ε kB for the pressure range (1 bar < P < 300 b nd temperature (300 K < T < 60 kK) are given in Table 8. Figure 4. The SF6 mass density at 20 bar. Figure 4. The SF6 mass density at 20 bar. Figure 4. The SF6 mass density at 20 bar. Figure 4. The SF6 mass density at 20 bar. Figure 4. The SF6 mass density at 20 bar. Figure 4. The SF6 mass density at 20 bar. Figure 5. The SF6 mass density at 40 bar. Figure 5. The SF6 mass density at 40 bar. Figure 5. The SF6 mass density at 40 bar. Figure 5. The SF6 mass density at 40 bar. Figure 5. The SF6 mass density at 40 bar. Figure 5. The SF6 mass density at 40 bar. 3.2.2. SF6 and CH4 Plasma Compositions 3.2.2. SF6 and CH4 Plasma Compositions Using the values for σ and ε kB obtained from the genetic algorithm, the compositions of the SF6 and CH4 plasma were calculated at pressure P = 100 bar. The values proposed by the genetic algorithm to minimize the error using the same values of σ and ε kB for the pressure range (1 bar < P < 300 bar) and temperature (300 K < T < 60 kK) are given in Table 8. 3.2.1. Choice of Parameters Using the values for σ and ε kB obtained from the genetic algorithm, the compositions of the SF6 and CH4 plasma were calculated at pressure P = 100 bar. The values proposed by the genetic algorithm to minimize the error using the same values of σ and ε kB for the pressure range (1 bar < P < 300 bar) and temperature (300 K < T < 60 kK) are given in Table 8. 17 of 22 Appl. Sci. 2019, 9, 929 Figure 6. The SF6 mass density at 100 bar. Figure 6. The SF6 mass density at 100 bar. f ε f f Appl. Sci. 2019, 9, 929 17 of 2 Figure 6. The SF6 mass density at 100 bar. Fi 6 Th SF d it t 100 b Appl. Sci. 2019, 9, 929 17 of Figure 6. The SF6 mass density at 100 bar. Figure 6. The SF6 mass density at 100 bar. 2. SF6 and CH4 Plasma Compositions Table 8. Optimized values of σ and ε kB for SF6 and CH4, deduced from the genetic algorithm. Figure 6. The SF6 mass density at 100 bar. ఌ ௞ಳ obtained from the genetic algori d at pressure P = 100 bar. The v using the same values of σ and ఌ ௞ಳ K < T < 60 kK) are given in Table 8 σ [Å] ε kB [K] SF6 5.21279 222.27 CH4 4.083 143.57 ositions ఌ ಳ obtained from the genetic algori d at pressure P = 100 bar. The v using the same values of σ and ఌ The results obtained at P = 1 bar were in agreement with other authors from the literature for SF6 [60–62] and CH4 [2,4] and are not reported here. The plasma compositions at P = 100 bar using the values in Table 8 are presented in Figures 7 and 8 for the case of SF6, and in Figures 9 and 10 for the case of CH4. An increase in pressure induces changes: the higher the pressure, the higher the temperature that is needed for the dissociation of molecules. This leads to the presence of molecules at higher temperatures. The same trend is observed for ionized particles. The results obtained at P = 1 bar were in agreement with other authors from the literature for SF6 [60–62] and CH4 [2,4] and are not reported here. 3.2.1. Choice of Parameters σ [Å] 𝜺𝒌𝑩 ൗ [K] SF6 5.21279 222.27 CH4 4.083 143.57 Figure 9. Atomic species for CH4 plasma densities at P = 100 bar − LTE. Figure 9. Atomic species for CH4 plasma densities at P = 100 bar −LTE. Appl. Sci. 2019, 9, 929 19 of 22 Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar −LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Table 8. Optimized values of σ and ఌ ௞ಳ for SF6 and CH4, deduced from the genetic algorithm. σ [Å] 𝜺𝒌𝑩 ൗ [K] SF6 5.21279 222.27 CH4 4.083 143.57 Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Table 8. Optimized values of σ and ఌ ௞ಳ for SF6 and CH4, deduced from the genetic algorithm. σ [Å] 𝜺𝒌𝑩 ൗ [K] SF6 5.21279 222.27 CH4 4.083 143.57 Figure 9. Atomic species for CH4 plasma densities at P = 100 bar − LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar −LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Table 8. Optimized values of σ and ఌ ௞ಳ for SF6 and CH4, deduced from the genetic algorithm. σ [Å] 𝜺𝒌𝑩 ൗ [K] SF6 5.21279 222.27 CH4 4.083 143.57 Figure 9. Atomic species for CH4 plasma densities at P = 100 bar − LTE. Figure 9. Atomic species for CH4 plasma densities at P = 100 bar −LTE. Appl. Sci. 2019, 9, 929 19 of 2 Figure 10. Molecular species for CH4 plasma densities at P = 100 bar − LTE. 4. Conclusions Figure 10. Molecular species for CH4 plasma densities at P = 100 bar −LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar −LTE. SF6 5.21279 222.27 CH4 4.083 143.57 Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar −LTE. SF6 5.21279 222.27 CH4 4.083 143.57 Table 8. Optimized values of σ and ௞ಳ for SF6 and CH4, deduced from the genetic algorithm. 3.2.1. Choice of Parameters The plasma compositions at P = 100 bar using the values in Table 8 are presented in Figures 7 and 8 for the case of SF6, and in Figures 9 and 10 for the case of CH4. An increase in pressure induces changes: the higher the pressure, the higher the temperature that is needed for the dissociation of molecules. This leads to the presence of molecules at higher temperatures. The same trend is observed for ionized particles. < 300 bar) and temperature (300 K < T < 60 kK) are given in Table 8. The results obtained at P = 1 bar were in agreement with other authors from the literature for SF6 [60–62] and CH4 [2,4] and are not reported here. The plasma compositions at P = 100 bar using the values in Table 8 are presented in Figures 7 and 8 for the case of SF6, and in Figures 9 and 10 for the case of CH4. An increase in pressure induces changes: the higher the pressure, the higher the temperature that is needed for the dissociation of molecules. This leads to the presence of molecules at higher temperatures. The same trend is observed for ionized particles. Figure 7. Atomic species for SF6 plasma densities at P = 100 bar − LTE. Figure 7. Atomic species for SF6 plasma densities at P = 100 bar − LTE. Figure 7. Atomic species for SF6 plasma densities at P = 100 bar −LTE. Figure 7. Atomic species for SF6 plasma densities at P = 100 bar − LTE. Figure 7. Atomic species for SF6 plasma densities at P = 100 bar −LTE. 18 of 22 Appl. Sci. 2019, 9, 929 pp , , Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Table 8. Optimized values of σ and ఌ ௞ಳ for SF6 and CH4, deduced from the genetic algorithm. σ [Å] 𝜺𝒌𝑩 ൗ [K] SF6 5.21279 222.27 CH4 4.083 143.57 Figure 9. Atomic species for CH4 plasma densities at P = 100 bar − LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar −LTE. Figure 8. Molecular species for SF6 plasma densities at P = 100 bar − LTE. Table 8. Optimized values of σ and ఌ ௞ಳ for SF6 and CH4, deduced from the genetic algorithm. 3.2.1. Choice of Parameters σ [Å] 𝜺𝒌𝑩 ൗ [K] SF6 5.21279 222.27 CH4 4.083 143.57 Fi 9 At i i f CH l d iti t P 100 b LTE Figure 9. Atomic species for CH4 plasma densities at P = 100 bar − LTE. Figure 9. Atomic species for CH4 plasma densities at P = 100 bar −LTE. ci. 2019, 9, 929 19 Figure 9. Atomic species for CH4 plasma densities at P = 100 bar − LTE. Figure 9. Atomic species for CH4 plasma densities at P = 100 bar −LTE. ci. 2019, 9, 929 19 Figure 9. Atomic species for CH4 plasma densities at P = 100 bar − LTE. Figure 10. Molecular species for CH4 plasma densities at P = 100 bar − LTE. Conclusions Figure 10. Molecular species for CH4 plasma densities at P = 100 bar −LTE. Appl. Sci. 2019, 9, 929 19 of 22 19 of 22 4. Conclusions and G.J.-J.: conceptualization, methodology, resources, supervision, and original draft preparation. Author Contributions: H.S.A. and G.J.-J.: investigation, software, validation, and original draft preparation; F.P. and G.J.-J.: conceptualization, methodology, resources, supervision, and original draft preparation. Funding: This research received no external funding. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Conclusions Software using the mass action law for obtaining the plasma composition was developed. Under the assumption of LTE, a variation of species densities with temperature can be obtained for atmospheric pressures of up to 300 bar. For such high pressures, the virial corrections that are associated with classical Debye–Hückel corrections are systematically used in ours developments. After a presentation of the theory for calculating the plasma composition, the results of the software were validated with compositions reported from the literature concerning argon and air plasmas at atmospheric pressure. Additional routines were developed using Python to automatically collect the required data from the NIST and JANAF Internet databases. To consider the high pressures, potential interactions that are formally used in virial correction were studied. We focused on the (12-6) Lennard–Jones potential, which is very commonly used in the literature. For this kind of potential, two parameters are necessary: σ, which corresponds to the distance where the potential is zero, and energy ε, corresponding to the depth of the potential. A different set of values can be found in the literature, or determined from measurements of gas mass density or viscosity. Some authors have also directly given values for the second and third virial coefﬁcients. Nevertheless, a wide disparity appears in these two coefﬁcients, leading to quite large differences in composition and hence mass density. Most of the time, the data given in the literature are only valid for a given pressure value, but they are not optimized for other values. We then developed a computing code based on a genetic algorithm to optimize these parameter values from the experimental data over a wide range of pressures. If no measurements are available, we suggest the use of the Stiel formulation, based on the critical values for temperature and pressure. Indeed, for temperatures and pressures that are not too close to the critical values, this formulation leads to a good compromise for values of σ and ε. The software was applied to obtain results for SF6 and CH4 plasma compositions at a pressure P = 100 bar. Plasma Compositions: Even if the software is currently automated, it does not have a user-friendly interface. In the meantime, the authors can be contacted at the given email to obtain the plasma compositions and properties. Author Contributions: H.S.A. and G.J.-J.: investigation, software, validation, and original draft preparation; F.P. References 1. White, W.B.; Johnson, S.M.; Dantzig, G.B. Chemical Equilibrium in Complex Mixtures. J. Chem. Phys. 1958, 28, 751–755. [CrossRef] 1. White, W.B.; Johnson, S.M.; Dantzig, G.B. Chemical Equilibrium in Complex Mixtures. J. Chem. Phys. 1958, 28, 751–755. [CrossRef] 2. Godin, D. Calcul de Compositions Chimiques de Plasmas à l’Equilibre Thermodynamique: Application à la Modélisation de l’Ablation dans les Disjoncteurs. Master’s Thesis, Université de Montréal—Ecole Polytechnique de Montréal, Montreal, QC, Canada, 1998. 2. Godin, D. Calcul de Compositions Chimiques de Plasmas à l’Equilibre Thermodynamique: Application à la Modélisation de l’Ablation dans les Disjoncteurs. Master’s Thesis, Université de Montréal—Ecole Polytechnique de Montréal, Montreal, QC, Canada, 1998. y q 3. Godin, D.; Trépanier, J.Y. A Robust and Efﬁcient Method for the Computation of Equilibrium Composition in Gaseous Mixtures. Plasma Chem. Plasma Process. 2004, 24, 447–473. [CrossRef] 3. Godin, D.; Trépanier, J.Y. A Robust and Efﬁcient Method for the Computation of Equilibrium Composition in Gaseous Mixtures. Plasma Chem. Plasma Process. 2004, 24, 447–473. [CrossRef] 4. Ailas, S. Etude du Spectre Moléculaire Emis par un Plasma Thermique Formé de Mélange CH4-H2. Ph.D. Thesis, Université Abou-Bekr Belkaid, Tlemcen, Algerie, 2017. 4. Ailas, S. Etude du Spectre Moléculaire Emis par un Plasma Thermique Formé de Mélange CH4-H2. Ph.D. Thesis, Université Abou-Bekr Belkaid, Tlemcen, Algerie, 2017. 5. André, P. Etude de la Composition et des Propriétés Thermodynamiques des Plasmas Thermiques à l’Equilibre et Hors d’Equilibre Thermodynamique. Ph.D. Thesis, Université Blaise Pascal, Clermont-Ferrand, France, 1995. 5. André, P. Etude de la Composition et des Propriétés Thermodynamiques des Plasmas Thermiques à l’Equilibre et Hors d’Equilibre Thermodynamique. Ph.D. Thesis, Université Blaise Pascal, Clermont-Ferrand, France, 1995. 6. André, P.; Brunet, L.; Duffour, E.; Lombard, J.M. Composition, Pressure and Thermodynamic Properties Calculated in Plasma Formed in Insulator Vapours of PC and POM at ﬁxed volume. Eur. Phys. J. Appl. Phys. 2002, 17, 53–64. [CrossRef] 7. Aubreton, J. Etude des Propriétés Thermodynamiques et de Transport Dans les Plasmas à l’Equilibre et hors Equilibre Thermodynamique Appliquées aux Plasmas de Mélanges Ar-H2 et Ar-O2. Ph.D. Thesis, Université de Limoges, Limoges, France, 1985. Appl. Sci. 2019, 9, 929 20 of 22 20 of 22 8. Aubreton, A. Modélisation et Etude Expérimentale d’un Plasma Métallique Crée par Ablation Laser. Ph.D. Thesis, Université de Toulouse III, Paul Sabatier, Toulouse, France, 2002. 9. Billoux, T. Elaboration d’une Base de Données Radiatives pour des Plasmas de Type CwHxOyNz et Application au Transfert Radiatif pour des Mélanges Air, CO2 et CO-H2. Ph.D. References Thesis, Université de Toulouse III, Paul Sabatier, Toulouse, 2013. 10. Cabrera, A.M. Modélisation de la Cinétique Chimique d’un Plasma en Extinction dans un Disjoncteur Basse Tension. Ph.D. Thesis, Université de Toulouse III, Paul Sabatier, Toulouse, France, 2003. 11. Colombo, V.; Ghedini, E.; Sanibondi, P. Thermodynamic and Transport Properties in Non-Equilibrium Argon, Oxygen and Nitrogen Thermal Plasmas. Prog. Nuclear Energy 2008, 50, 921–933. [CrossRef] 12. Colombo, V.; Ghedini, E.; Sanibondi, P. Two Temperature Thermodynamic and Transport Properties of Argon-Hydrogen and Nitrogen-Hydrogen plasma. J. Phys. D Appl. Phys. 2009, 42, 12. [CrossRef] 13. Cressault, Y. Propriétés des Plasmas Thermiques dans des Mélanges Argon-Hydrogène-Cuivre. Ph.D. Thesis, Université de Toulouse III, Paul Sabatier, Toulouse, France, 2001. 14. Cressault, Y.; Connord, V.; Hingana, H.; Teulet, P.; Gleizes, A. Transport properties of CF3I thermal plasmas mixed with CO2, air or N2 as an alternative to SF6 plasmas in high-voltage circuit breakers. J. Phys. D Appl. Phys. 2011, 44, 495202. [CrossRef] 15. Hingana, H. Contribution à l’Etude des Propriétés des Plasmas à Deux Températures: Application A Et Air. Ph.D. Thesis, Université Toulouse III, Paul Sabatier, Toulouse, France, 2010. 16. Rat, V.; André, P.; Aubreton, J.; Elchinger, M.F.; Fauchais, P.; Vacher, D. Transport coefﬁcients including diffusion in a two-temperature argon plasma. J. Phys. D Appl. Phys. 2002, 35, 981–991. [CrossRef] 17. Rochette, D.; Bussière, W.; André, P. Composition, Enthalpy, and Vaporization temperature, calculation of Ag-SiO2 plasmas with Air in the temperature range from 1000 to 6000 K and for pressure included between 1 and 50 bars. Plasma Chem. Plasma Process. 2004, 24, 475–492. [CrossRef] 18. Hirschfelder, J.O.; Curtiss, C.F.; Bird, R.B. Molecular Theory of Gases and Liquids; J. Wiley and Sons Chapman and Hal, Inc.: New York, NY, USA, 1954. 19. Funke, M.; Kleinrahm, R.; Wagner, W. Measurement and correlation of the (p, ρ, T) relation of sulphur hexaﬂuoride. I. The homogeneous gas and liquid region in the temperature range from 225 K to 340 K at pressures up to 12 MPa. J. Chem. Thermodyn. 2002, 34, 717–734. [CrossRef] 20. Gilgen, R.; Kleinrahm, R.; Wagner, W. Supplementary measurements of the (pressure, density, temperature) relation of carbon dioxide in the homogeneous region at temperatures from 220 K to 360 K and pressures up to 13 MPa. J. Chem. Thermodyn. 1992, 24, 1243–1250. [CrossRef] y 21. Hurly, J.J.; Deﬁbaugh, D.R.; Moldover, M.R. Thermodynamic properties of sulfur hexaﬂuoride. Int. J. Thermophys. 2000, 21, 739–765. [CrossRef] 22. Zarkova, L.; Hohm, U. References pVT—Second virial coefﬁcients B(T), viscosity η(T), and self-diffusion ρD(T) of the gases: BF3, CF4, SiF4, CCl4, SiCl4, SF6, MoF6, WF6, UF6, C(CH3)4, and SI(CH3)4, determined by means of an isotopic temperature-dependent potential. J. Phys. Chem. Ref. Data 2002, 31, 183–216. [CrossRef] 23. Sengers, J.M.H.L.; Klein, M.; Gallagher, J.S. Pressure-Volume-Temperature relationships of gases virial coefﬁcients. In American Institute of Physics Handbook, 3rd ed.; McGraw-Hill: New York, NY, USA, 1971. W W S h d A f f S d d h d l 24. Wagner, W.; Ewers, J.; Schmidt, R. An equation of state for oxygen vapour—Second and third virial coefﬁcients. Cryogenics 1984, 24, 37–43. [CrossRef] 25. Zarkova, L.; Hohm, U.; Damyanova, M. Viscosity and pVT-Second virial coefﬁcient of binary noble-globular gas and globular-globular gas mixtures, calculated by means of an isotropic temperature-dependent potential. J. Phys. Chem. Ref. Data 2003, 32, 1591–1705. [CrossRef] 26. Zarkova, L.; Hohm, U.; Damyanova, M. Viscosity and pVT-Second virial coefﬁcient, and diffusion of pure and mixed small alkanes CH4, C2H6, C3H8, n-C4H10, i-C4H10, n-C5H12, i-C5H12, and C(CH3)4, calculated by means of an isotopic temperature-dependent potential. I. Pure alkanes. J. Phys. Chem. Ref. Data 2006, 35, 1331–1364. [CrossRef] 27. Petchanka, A.; Reichert, F.; Gonzalez, J.J.; Freton, P. Modelling of deformation of PTFE nozzles in high voltage circuit breaker due to multiple interruptions. J. Phys. D Appl. Phys. 2016, 49, 135201. [CrossRef] 28. Reichert, F.; Petchanka, A.; Freton, P.; Gonzalez, J.J. Studies on the Thermal Re-ignition in SF6 High-Voltage Circuit-Breakers by means of Coupled Simulation. Plasma Phys. Technol. J. 2015, 2, 2336–2626. 29. Gonzalez, J.J.; Freton, P.; Reichert, P.; Pechanka, A. PTFE vapor contribution to pressure changes in high voltage circuit breakers. IEEE Trans. Plasma Sci. 2015, 43, 2703–2714. [CrossRef] 21 of 22 21 of 22 Appl. Sci. 2019, 9, 929 30. Laforest, Z.; Gonzalez, J.J.; Freton, P. Experimental study of a plasma bubble created by a wire explosion in water. Int. J. Res. Rev. Appl. Sci. 2018, 34, 93–105. 31. Benmouffok, M.; Freton, P.; Gonzalez, J.J. Theoretical plasma characterization during current pulse. Int. J. Res. Rev. Appl. Sci. 2018, 34, 3. 32. André, P.; Brunet, L.; Bussière, W.; Caillard, J.; Lombard, J.M.; Picard, J.P. Transport coefﬁcients of plasmas consisting of insulator vapours: Application to PE, POM, PMMA, PA66 and PC. Eur. Phys. J. Appl. Phys. 2004, 25, 169–182. [CrossRef] 33. Chervy, B.; Gleizes, A.; Razaﬁnimanana, M. References Thermodynamic properties and transport coefﬁcients in SF6-Cu mixtures at temperatures of 300 K–30 kK and pressures of 0.1–1 MPa. J. Phys. D Appl. Phys. 1994, 27, 1193–1206. [CrossRef] 34. Kravchik, T.; Sher, E.; Heywood, J.B. From spark ignition to ﬂame initiation. Combust. Sci. Technol. 1995, 108, 1–30. [CrossRef] 35. Maly, R.; Vogel, M. Initiation and Propagation of Flame Fronts in Lean Methane/Air Mixtures by the Three Modes of the Ignition Spark. In Symposium (International) on Combustion; Elsevier: New York, NY, USA, 1979; Volume 17, pp. 821–831. 36. Zaepffel, C. Etude Expérimentale et Numérique d’une Décharge Electrique Appliquée à L’allumage d’un Milieu Réactif, Ph.D. Thesis, Université d’Orléans, Orléans, France, 2008. 37. Chase, M.W.; Davies, C.A.; Downey, J.R.; Frurip, D.J.; McDonald, R.A.; Syverud, A.N. NIST-JANAF Thermochemical Tables (ver. 1.0), Online, Standard Reference Data Program; National Institute of Standards and Technology: Gaithersburg, MD, USA, 1985. Available online: https://janaf.nist.gov/ (accessed on 7 November 2018). [CrossRef] 38. Kramida, A.; Ralchenko, Y.; Reader, J.; NIST ASD Team. NIST Atomic Spectra Database (ver. 5.5.6); Online; National Institute of Standards and Technology: Gaithersburg, MD, USA, 2018. Available online: https: //physics.nist.gov/PhysRefData/ASD/levels_form.html (accessed on 7 November 2018). 39. Drawin, H.W.; Felenbok, P. Data of Plasmas in Local Thermodynamic Equilibrum; Editions Gauthier: Paris, France, 1965. 40. Hryniewicki, M.K. Accurate and Efﬁcient Evaluation of the Second Virial Coefﬁcient Using Practical Intermolecular Potentials for Gases. Ph.D. Thesis, University of Toronto, Toronto, ON, Canada, 2011. 41. Mamedov, B.A.; Somuncu, E. Analytical evaluation of third virial coefﬁcient with Lennard-Jones (12-6) potential and its applications. In AIP Conference Proceedings; AIP Publishing: Melville, NY, USA, 2016; pp. 020121-1–020121-4. [CrossRef] 42. Rice, W.E.; Hirschfelder, J.O. Second virial coefﬁcients of gases obeying a modiﬁed Buckingham (exp-6) potential. J. Chem. Phys. 1954, 22, 187–192. [CrossRef] 43. Edalat, M.; Lan, S.S.; Pang, F.; Mansoori, G.A. Optimized parameters and exponents of Mie (n, m) intermolecular potential energy function based on the shape of molecules. Int. J. Thermophys. 1980, 1, 177–184. [CrossRef] 44. Mahfouf, A. Calcul des Coefﬁcients de Transport Dans des Plasmas Hors de L’équilibre. Ph.D. Thesis, Université Blaise Pascal, Clermont-Ferrand, France, 2016. 45. Mouritz, F.M.; Rummens, F.H.A. A critical evaluation of Lennard-Jones and Stockmayer potential parameters and of some correlation methods. Can. J. Chem. 1977, 55, 3007–3020. [CrossRef] 6. Youssef, A.; Hanna, M.M.; Saad, S.M. Determination of the Lennard-Jones force constants from diffu measurements. Z. Phys. Chem. 1969, 241, 81–100. [CrossRef] 47. Aziz, R.A.; Slaman, M.J. References An improved intermolecular potential for sulfur hexaﬂuoride. J. Chem. Phys. 1991, 94, 2. [CrossRef] 48. Bird, R.B.; Stewart, W.E.; Lightfoot, E.N. Transport Phenomena, 2nd ed.; J. Wiley and Sons, Inc.: New York, NY, USA, 2002; ISBN 0-471-41077-5. 49. Flynn, L.W.; Thodos, G. Lennard-Jones force constants from viscosity data: Their relationship to critical properties. AIChE J. 1962, 8, 362–365. [CrossRef] 50. Hirschfelder, J.O.; Bird, R.B.; Spotz, E.L. The transport properties of gases and gaseous mixtures II. Chem. Rev. 1949, 44, 1205–1231. [CrossRef] 51. Stiel, L.I.; Thodos, G. Lennard-Jones force constants predicted from critical properties. J. Chem. Eng. Data 1962, 7, 234–236. [CrossRef] Appl. Sci. 2019, 9, 929 22 of 22 52. Angus, S.; Armstrong, B.; Gosman, A.L.; McCarty, R.D.; Hust, J.G.; Vasserman, A.A.; Rabinovich, V.A. International Thermodynamic Tables of the Fluid State—1 Argon; Butterworths: London, UK, 1972. 53. Angus, S.; de Reuck, K.M.; Armstrong, B.; Jacobsen, R.T.; Stewart, R.B. International Thermodynamic Tables of the Fluid State—6 Nitrogen; Pergamon Press: Pergamon, NY, USA, 1979. 54. NIST Chemistry WebBook, NIST Standard Reference Database 69. Available online: https://webbook.nist. gov/chemistry/ (accessed on 7 November 2018). 55. Makarevich, L.A.; Sokolova, E.S.; Sorina, G.A. Critical parameters of Sulfur Hexaﬂuoride. Zh Fiz. Khim. 1968, 42, 3–22. 56. Suehiro, Y.; Nakajima, M.; Yamada, K.; Uematsu, M. Critical parameters of {xCO2 + (1 −x)CHF3} for x = (1.0000, 0.7496, 0.5013, and 0.2522). J. Chem. Therm. 1996, 28, 1153–1164. [CrossRef] 57. Claus, P.; Kleinrahm, R.; Wagner, W. Measurements of the (p, ρ, T) Relation of Ethylene, Ethane, and Sulphur Hexaﬂuoride in the Temperature Range From 235 K to 520 K at Pressures Up to 30 MPa using an Accurate Single-Sinker Densimeter. J. Chem. Thermodyn. 2003, 35, 159–175. [CrossRef] 58. Cristancho, D.E.; Mantilla, I.D.; Ejaz, S.; Hall, K.R. Accurate PρT data for methane from 300 to 450 K up to 180 MPa. J. Chem. Eng. 2010, 55, 826–829. 59. Balester, P.J.; Carter, J.N. Real-parameter Genetic Algorithms for ﬁnding Multiple Optimal Solutions in Multi-modal Optimization. In Proceedings of the Genetic and Evolutionary Computation Conference GECCO 2003, Chicago, IL, USA, 12–16 July 2003; Lecture Notes in Computer Science. Springer: Berlin/Heidelberg, Germany, 2003; Volume 2723. 60. Chervy, B. Calcul des Propriétés de Transport et Etude du Pouvoir de Coupure des Mélanges Hexaﬂuorure de Soufre (SF6)-Fluorure de Carbone (CF4 ou C2F6) et Hexaﬂuorure de Soufre—Vapeurs de Cuivre. Ph.D. Thesis, Université Toulouse III, Paul Sabatier, Toulouse, France, 1995. 61. Belhaouari, J.B. References Modélisation de l’Extinction d’un Arc de SF6 Hors Equilibre Thermodynamique Local. Ph.D. Thesis, Université de Toulouse III, Paul Sabatier, Toulouse, France, 1997. 62. Brand, K.P.; Kopainsky, J. Particle densities in a decaying SF6 plasma. Appl. Phys. 1978, 6, 425–432. [CrossRef] © 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/). © 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/W4214879059	https://link.springer.com/content/pdf/10.1007/s43938-022-00009-y.pdf	English	null	Combining experimental and theoretical insights for reduction of CO2 to multi-carbon compounds	Discover chemical engineering	2,022	cc-by	20,187	* Abdesslem Jedidi, ajedidi@kau.edu.sa; * Shahid Rasul, Shahid.Rasul@Northumbria.ac.uk; Ian Brewis, Ian3.Brewis@Northumbria.ac.uk; Rana‑Faisal Shahzad, Rana-Faisal.Shahzad@Northumbria.ac.uk; Robert W. Field, Robert.Field@Northumbria.ac.uk \| 1Faculty of Engineering and Environment, Northumbria University, Ellison Pl, Newcastle Upon Tyne NE1 8ST, UK. 2Department of Chemistry, King AbdulAziz University, Abdullah Sulayman, Jeddah 21589, Saudi Arabia. Abstract The electrochemical reduction of carbon dioxide is a promising method for both recycling of atmospheric CO2 and storing renewably produced electrical energy in stable chemical bonds. In this paper, we review the current challenges within this promising area of research. Here we provide an overview of key findings from the perspective of improving the selectivity of reduction products, to serve as a contextual foundation from which a firmer understanding of the field can be built. Additionally, we discuss recent innovations in the development of catalytic materials selective toward C3 and liquid products. Through this, we form a basis from which key mechanisms into C3 products may be further examined. Carbon–carbon (C–C) bond formation provides a key step in the reduction of CO2 to energy dense and high value fuels. Here we demonstrate how variations in catalytic surface morphology and reaction kinetics influence the formation of multi-carbon products through their impact on the formation of C–C bonds. Finally, we discuss recent developments in the techniques used to characterise and model novel electrocatalysts. Through these insights, we hope to provide the reader with a perspective of both the rapid progress of the field of electrocatalysis, as well as offering a concise overview of the challenges faced by researchers within this rapidly developing field of research. Keywords Electrochemistry · Catalysis · CO2 reduction · Energy storage Keywords Electrochemistry · Catalysis · CO2 reduction · Energy storage Combining experimental and theoretical insights for reduction of CO2 to multi‑carbon compounds Ian Brewis1 · Rana‑Faisal Shahzad1 · Robert W. Field1 · Abdesslem Jedidi2 · Shahid Rasul1 Received: 24 December 2021 / Accepted: 8 February 2022 © The Author(s) 2022 OPEN Discover Chemical Engineering Discover Chemical Engineering 1 Introduction By implementing carbon capture technology to siphon waste CO2 from the atmosphere, as well as natural and industrial processes. CO2 reduction aims to mimic the natural process of photosynthesis (i.e. “artificial photosyn- thesis”) [3, 6], effectively managing atmospheric levels of CO2 whilst providing a carbon neutral source of fuels. An added bonus of recycling CO2 via electrochemical reduction is that the fuels produced are highly compatible with existing technology which would otherwise be powered by fossil fuels, with most only requiring minor modifica- tions [7, 15, 16]. It is for these reasons that the electrochemical reduction of CO2 has begun to see notable interest within the field of electrochemistry [15, 17–20]. The electrochemical reduction of CO2 however is still an emerging technology. There are significant challenges to current electrocatalyst performance, including: issues regarding energy efficiency, reaction selectivity both between CO2 reduction (CO2RR) products and with the competing hydrogen evolution reaction (HER), as well as overall conversion rate. Before carbonaceous fuels produced via the reduction of CO2 can be considered as a viable method of energy storage, each of these challenges must first be addressed [20]. A 2008 review by Hori [21] examined many elemental metal catalysts in terms of their overall catalytic performance and product selectivity. Since then, considerable work has been conducted into improving the activity and selectivity of reduction products. A overview of some of the most active electrocatalysts selective towards specific products can be observed in Table 1. Despite the stability of the CO2 molecule [22], it can be reduced to simple products requir- ing only a 2-electron transfer process such as CO and HCOOH with low overpotential and high Faradaic efficiency. For larger products requiring higher numbers of electron transfers however, such as ethylene or alcohols, markedly higher overpotentials and lower selectivities among CO2RR products are observed [20]. The aim of this review is to examine and discuss some of the history of electrochemical CO2 reduction, as well as its more recent advances, to provide the reader with an overview of the field itself. Through this, this review aims to provide a sense of where the field has come from, as well as how it maybe further develop in the future [17, 18, 17–18]. 1 Introduction Fossil fuels form the cornerstone of industrial and economic growth globally, accounting for over 87% of the world’s total energy consumption [1, 2]. Dwindling fuel reserves and mounting environmental damage as a result of burning fossil fuels demonstrate the need to move beyond our global dependence on non-renewable sources of energy. As of 2015, the remaining oil and natural gas reserves were estimated at 1697.6 billion barrels and 186.9 trillion cubic meters respectively. Based on global consumption levels as of 2015 and the remaining supply of fossil fuels at that time, it was estimated that around 50 years of oil and gas remain [2]. The burning of such large quantities of fossil fuels, particularly in the transport sector, has resulted in the uncon- trolled release of CO2 into the atmosphere, significantly contributing toward climate change [3–5]. This realisation has led to considerable efforts being made to transition from fossil fuels toward low carbon and renewable alterna- tives such as biofuels, hydroelectric power, solar, wind, geothermal, and nuclear energy [1]. Whilst some low carbon \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 Discover Chemical Engineering (2022) 2:2 1 3 ngineering (2022) 2:2 Discover Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y Review Review energy sources such as nuclear and hydroelectric energy are being implemented on a large scale [1], most alternatives to fossil fuels, such as solar and wind, have been unable to achieve this due to intermittent operating times and are incapable of consistently meeting global energy demands in their current state [6]. A key method in tackling this inherent intermittancy is through the conversion of otherwise surplus energy into a medium which is both portable and storable so that it may be implemented wherever and whenever it is needed, much like fossil fuels are used today [3, 7–11]. CO2 reduction poses an attractive method of converting said energy. By using the abundant greenhouse gas CO2 to produce carbonaceous fuels compatible with existing infrastructure it effectively forms a carbon neutral cycle for the production of hydrocarbons [12–14]. An example of such a cycle as this can be observed in Fig. 1. cycle for the production of hydrocarbons [12–14]. An example of such a cycle as this can be observed in Fig. 1. cycle for the production of hydrocarbons [12–14]. An example of such a cycle as this can be observed in Fig. 1. Fig. 1 The “carbon neutral cycle”. Here atmospheric CO2 is captured and reduced to form synthetic hydrocarbons in the order of C1 to C3 chains (adapted from [3]) 2.1 Methods of CO2 reduction Broadly speaking, the various methods of CO2 reduction can be classified in the following four categories: thermochemi- cal, biochemical, photochemical and electrochemical reduction. Thermochemical CO2 reduction methods have been in use for a number of years, an example of which is the formation of methanol using CO2 and H2 chemical feed-stocks under a catalyst typically composed of Cu/ZnO/AlO3 . This process, similar to the current industrial method for the formation of methanol from syngas [3, 41–44], requires high temperatures and pressures of around 220 to 330 ◦C and 50 to 100 atm respectively [43, 44]. Similarly, at lower pressures, CO2 and H2 can be used as reactants to form hydrocarbons using Fischer–Tropsch style catalysts, albeit at still elevated temperatures [45, 46]. Consequently, due to the high temperatures and pressures required, thermochemical reduction methods are highly energy intensive, and are therefore most com- monly driven by fossil fuels. Biochemical methods commonly employ the use of enzymes or autotrophic organisms in order to capture and convert CO2 into complex products [47, 48]. Photochemical methods, by contrast, use photo- and electrocatalysts in order to mimic natural photosynthesis by both splitting water and reducing CO2 using sunlight to drive the reaction process [49–51]. By convention, research in both biochemical and photochemical reduction methods aim to use sunlight as a near unlimited source of energy to convert CO2 to energy dense fuels under ambient conditions. A limitation of this however is that such methods neglect the use of other renewable energy sources. Electrochemical reduction methods, unlike biochemical and photochemical methods, are not limited to a single renewable energy source, and with the use of a well suited electrocatalyst, are capable of effectively reducing CO2 under ambient conditions [15, 52]. Due to this, electrochemical methods can be easily adapted to be used in conjunction with almost any renewable energy source, particularly as most renewables are used to generate electrical energy. Additionally, high energy efficiencies observed at mild reaction conditions make electrochemical reduction methods an attractive avenue for the reduction of CO2 to fuels [53, 54]. Yao et al. for example, reduced CO2 to hydrocarbons with an overall selectivity of 47.8% across all carbonaceous products, predominantly in the range of C8 to C11 [55]. Such results were produced based on a novel Fe–Mn–K tri-metallic electrocatalyst, producing mainly fuels in a range compatible with most modern aircraft. Another such example is that of Sumit et al. 1 Introduction Among the topics discussed within this paper are: the CO2 reaction processes; including initial activation, catalyst design; both past and present, and finally the use of computational methods to improve our fundamental understanding of the CO2 reduction reaction (CO2RR). Vol:.(1234567890) 1 3 Fig. 1 The “carbon neutral cycle”. Here atmospheric CO2 is captured and reduced to form synthetic hydrocarbons in the order of C1 to C3 chains (adapted from [3]) Vol:.1 3 :.(123456789 3 \| https://doi.org/10.1007/s43938-022-00009-y (2022) 2:2 Discover Chemical Engineering Review Review Table 1 Highly active electrocatalysts selective toward specific CO2RR products [20] CO2RR product Electrocatalyst Faradaic effi- ciency (%) Overpotential (V) jtotal (mA cm−2 Electrolyte (CO2 saturated) Ref. HCOOH Pb 97.4 − 1.19 V 5.0 0.1 M KHCO3 [25] Sn 88.4 − 1.04 V 5.0 0.1 M KHCO3 [25] Pd nanoparticles/C 99 − 0.15 V 2.4–7.0 2.8 M KHCO3 [26] Pd70Pt30 nanoparticles/C 90 − 0.36 V 4.0–7.5 0.2M PO3− 4 buﬀer [27] CO Au 87.1 − 0.64 V 5 0.1 M KHCO3 [25] Au nanoparticles 97 − 0.58 V 3.49 ± 0.61 0.1 M KHCO3 [28] OD-Au nanoparticles > 96 − 0.25 V 2–4 0.5 M NaHCO3 [29] Ag 94 − 0.99 V ∼5 0.1 M KHCO3 [30] CH4 Cu poly 40.4 − 1.34 V ∼7 0.1 M KHCO3 [31] Cu(210) 64 − 1.29 V 5 0.1 M KHCO3 [32] C2H4 Cu poly 26 − 1.13 V 1–2 0.1 M KHCO3 [31] O2 plasma-treated Cu 60 − 0.98 V ∼15 0.1 M KHCO3 [33] Cu-halide 60.5–79.5 − 2.11 V 46.1–39.2 3 M KBr [34] Graphite/carbon NPs/Cu/PTEE 70 − 0.63 V 75–100 7 M KOH [35] CH3OH PtxZn nano-alloys/C 81.4 − 0.90 V ∼3 0.1 M NaHCO3 [36] Co nanoparticles 71.4 − 0.90 V 4 0.1 M NaHCO3 [37] C2H5OH Cu poly 9.8 − 1.14 V ∼0.6 0.1M KHCO3 [31] Cu2O 9-16 − 1.08 V 30–35 0.1 M KHCO3 [38] CuO nanoparticles 36.1 N/A ∼11.7 0.2 M KI [39] Cu/CNS 63 − 1.29 V 2 0.1 M KHCO3 [40] ocatalysts selective toward specific CO2RR products [20] 2.2 The activation of CO2 When considering the activation of CO2, the following 4 equations govern the reactions taking place (1) ∗+CO2 + H+ + e−→∗COOH (2) ∗+CO2 + H+ + e−→∗OCHO (3) ∗+CO2 + e−→∗CO− 2 (4) ∗+H+ + 2e−→∗H− (1) (3) (4) where * denotes a binding site on the catalyst surface. Here COOH is considered to likely be the be intermediate for CO formation and OCHO the likely intermediate in the formation of formic acid for transition and post-transition metals [77]. where * denotes a binding site on the catalyst surface. Here COOH is considered to likely be the be intermediate for CO formation and OCHO the likely intermediate in the formation of formic acid for transition and post-transition metals [77]. A 2017 work by Adrien et al. [78], postulates that the reaction takes place in agreement with previous literature, how- ever with subtle changes. Rather than following only the concerted proton–electron transfer (CPET) reactions as shown in Eqs. (1) and (2), the CO2RR could instead follow a combination of both a CPET reaction and sequential proton–electron transfer (SPET) For the SPET reaction the single reaction step depicted in Eq (1) instead takes place as a 2 step process A 2017 work by Adrien et al. [78], postulates that the reaction takes place in agreement with previous literature, how- ever with subtle changes. Rather than following only the concerted proton–electron transfer (CPET) reactions as shown in Eqs. (1) and (2), the CO2RR could instead follow a combination of both a CPET reaction and sequential proton–electron transfer (SPET). For the SPET reaction, the single reaction step depicted in Eq. (1) instead takes place as a 2-step process consisting of the initial activation of CO2 as shown in Eq. (3), followed by (5) ∗CO− 2 + H+ →∗COOH. ∗CO− 2 + H+ →∗COOH. (5) It was suggested in the same work by Adrien et al. that the conditions affecting the ratio of CPET to SPET reactions were tied to the local pH in the vicinity of the catalyst surface. In the vicinity of pH 3 for example, it was found that the SPET pathway became competitive with CPET, in agreement with experimental results, which saw a dramatic increase in the faradaic efficiency toward CO production. 2.2 The activation of CO2 Formed through the combustion of organic matter, CO2 is a particularly stable molecule [22, 58]. Due to this, it is kineti- cally challenging to convert CO2 to products due to the high activation energy required for the reduction process [12, 59]. The single electron reduction of CO2 to CO− 2 has been shown to have a high thermodynamic potential of Eo = −1.90V versus the standard hydrogen electrode (SHE) in aqueous media (pH = 7) [29, 60–62]. This initial activation step is criti- cal as it forms the rate limiting step of the CO2RR [18]. The coordination of this intermediate additionally determines the whether the CO2 molecule will be reduced to CO or formate following the transfer of 2 electrons [63]. For example, post-transition metals selective toward the formation of formic acid, such as Pb [64], Sn [65], Bi [66, 67] and In [68] prefer to bind CO2 via oxygen. Transition metals such as Cu [69] and Ag [70] however prefer to bind via carbon, resulting in the production of CO. Bagger et al. [71] however, suggests an alternative. For Cu surfaces they demonstrate how oxygen bonded HCOO* is stabilised more effectively than carbon bonded COOH, suggesting a possible oxygen-bound pathway. In addition to this, they suggest the formation of formic acid to be related to the relative standing of the binding energies of H and COOH. This was supported by the lack of experimental evidence showing a lack of HCOO* surface saturation prior to the onset of HER, despite adsorption energy values suggesting an earlier onset of HCOO* production [72–75]. Selectivity of the reaction pathway towards products such as this can be further improved using the appropriate catalyst [25, 27]. It should be noted however, that the choice of material alone does not solely dictate product selectivity. One clear example of this was shown in a recent work by Scholten et al. [76] which demonstrated how highly ordered single-crystal Cu surfaces produced mainly hydrogen. By contrast, it was found that only through the introduction of surface defects such as with etching and plasma pre-treatment methods that significant amounts of hydrocarbons were produced. 2.1 Methods of CO2 reduction who reduced CO2 to CO in an alkaline flow electrolyzer with :(01231 0123456789) 1 3 (01234 1 3 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 Discover Chemical Engineering Review an energy efficiency of 64% using an Au electrode [56]. Such values of efficiency rival that of the hydrogen evolution reaction (HER) using a Pt catalyst in alkaline electrolyte [57]. an energy efficiency of 64% using an Au electrode [56]. Such values of efficiency rival that of the hydrogen evolution reaction (HER) using a Pt catalyst in alkaline electrolyte [57]. 2.2 The activation of CO2 This pH dependence of CO2 reduction pathways, and possibly differing pH dependence compared to the competing hydrogen evolution reaction (HER) was used to explain the strong pH depend- ence of product selectivity for graphite-immobilized CO-protoporphyrin, where H2 was observed as the main reduction product at pH 1, and CO as the main product at pH 3 [79, 80]. This pH dependence of reaction products is further agreed upon by Koper [81], who suggested that due to the nature of this pH dependence, an optimal pH for desired reaction products must be attainable, with a suitable catalyst. The pH dependent, decoupled proton–electron reaction pathways however, Koper explained, are more prevalent if the interaction between the catalyst and intermediates is weak, as CPET Vol:1 :.(123456789 3 Discover Chemical Engineering \| https://doi.org/10.1007/s43938-022-00009-y (2022) 2:2 Review reactions dominate where catalyst-intermediate interactions are strong, resulting in a low pH dependence of reaction products. Not only have reaction conditions been shown to be sensitive toward electrolyte pH, but also to the nature of the electrolyte used. Monteiro et al. [82] for example, demonstrated how a lack of metal ions prevents the formation of CO on Cu surfaces. Their work highlighted the crucial importance of short-range electrostatic interactions between the electrolyte and catalytic surface in promoting CO2RR. reactions dominate where catalyst-intermediate interactions are strong, resulting in a low pH dependence of reaction products. Not only have reaction conditions been shown to be sensitive toward electrolyte pH, but also to the nature of the electrolyte used. Monteiro et al. [82] for example, demonstrated how a lack of metal ions prevents the formation of CO on Cu surfaces. Their work highlighted the crucial importance of short-range electrostatic interactions between the electrolyte and catalytic surface in promoting CO2RR. 2 For the formation of formate and formic acid, a similar situation is observed, particularly for molecular catalysts such as Rh, In and Sn metalloprotoporphyrins which were reported to produce formic acid selectively in aqueous electrolyte [83]. It should be noted however that the formation of formate using an Rh protoporphyrin is only capable at elevated pressures [84], with Rh ordinarily producing H2 as the main product under ambient conditions [85]. Density functional theory (DFT) studies into molecular catalysts such as those mentioned previously showed a strong pH dependence on the formation of formic acid [86]. 2.2 The activation of CO2 Here it was found that within a strongly acidic electrolyte the HER is favourable, following the same reaction path as outlined in Eq. (4). This result was in strong agreement with the literature [87, 88]. From the production of an anionic hydride as shown in Eq. (4), the anion binds to the carbon atom of CO2 like so (6) CO2 + H−→HCOO−. CO2 + H−→HCOO−. (6) The subsequent stability of the HCOO− intermediate can then dictate the formation of either CO or HCOOH. Discussion of this hydride dependent reaction pathway has been further examined for a range of catalysts observed to produce formic acid among their reaction products. Such catalysts include copper-hydride nanostructures [89], palladium elec- trodes [26], and in solution [90]. The subsequent stability of the HCOO− intermediate can then dictate the formation of either CO or HCOOH. Discussion of this hydride dependent reaction pathway has been further examined for a range of catalysts observed to produce formic acid among their reaction products. Such catalysts include copper-hydride nanostructures [89], palladium elec- trodes [26], and in solution [90]. When considering Eqs. (3) and (4), as opposed to Eqs. (1) and (2), the pH sensitivities resulting from the charge of key intermediates suggests the another challenge when designing and implementing an electrochemical cell. For CO2 reduc- tion, this challenge is ensuring an optimal local and system-wide pH for the production of desired products. This is true in particular for reduction products formed following a 2-electron transfer such as CO and HCOOH. The formation of larger and more energy dense products from the CO2RR is however another desired result within the field of electrochemistry. As such, the following section shall cover some works published in this regard. 2.3 Formation of multi‑carbon compounds In order to form larger and more energy rich products such as hydrocarbons and alcohols, CO2 reaction intermediates must undergo additional reaction steps requiring the transfer of multiple electrons. Due to the added complexity of the reaction, a clear drop in selectivity can be observed for C2 and greater products when compared with the production of simpler products such as CO and formate which require fewer reaction steps. As well as this, notably higher overpoten- tials can also be observed [17]. Figure 2 illustrates some of the proposed reaction pathways to larger and more complex products following the formation of CO. It should be noted that following the initial activation of the CO2 molecule, the standard reduction potentials for sequential reaction steps under ambient conditions are relatively low. Even for the formation of large molecules such as ethanol and propanol, reduction potentials are of the order of ≈−0.5V [93, 94]. Whilst this would suggest the formation of hydrocarbons and alcohols would not be much more energetically demanding than the formation of formate or CO, the formation of larger hydrocarbons have been shown to be kinetically challenging [94, 95]. This is due to the reaction kinetics involved in transferring large numbers of electrons, as well as the large structures of multiple carbon products involved in the reaction. The formation of C2+ and greater products such as propane and butane from the CO2RR is of particular interest due to the greater energy density provided by these products. Copper, and copper-based catalysts have been shown as being almost exclusively capable of providing the necessary conditions to form Carbon–carbon bonds at significant current densities [21, 69]. Copper demonstrates an intermediate adsorption energy for CO, providing more ideal conditions for the formation of C–C bonds. Metals with higher binding energies, such as Pt, Ni, Fe and Ti, for example, show a greater selectivity toward hydrogen due to tighter CO binding on the catalyst surface [21, 96, 97]. By contrast, metals such as Au, Ag and Zn, which exhibit a lower adsorption energy, produce mainly CO as the intermediate desorbs from the catalyst surface before further reactions can take place [25, 98–100]. A more complete picture of the binding energies for Cu with various reaction intermediates can be observed in Fig. 3. 2.3 Formation of multi‑carbon compounds This pH sensitivity has been attributed to the rate-determining step in the formation of C–C bonds involving a decoupled proton–electron transfer. This hypothesis is based on a combina- tion of both DFT calculations and experimental data [110]. A recent report by Perez-Gallent et al. [122] observed the formation of a hydrogenated CO-dimer (OCCOH) intermedi- ate during CO reduction using Fourier transform infrared spectroscopy at low overpotentials in LiOH solutions. All results for this work were supported with DFT calculations. In agreement with previous literature, the formation of CO dimers was structurally sensitive; only being observed on the fcc(100) facet. Thus it was concluded, in agreement with prior works, that the formation of the dimer was favoured both thermodynamically and kinetically on Cu(100) compared to Cu(111) [111, 117].f Whilst the effect of adding certain organic layers to copper catalysts such as pyridium [106] or triazole [107] is yet to be fully understood, studies have suggested the modification of copper with such layers results in an enhanced selectivity toward the production of C2 products. Currently however, this effect is believed to possibly be due to local pH effects caused by the presence of such organic materials. One recent study by Hoang et al. [107] for example, reported faradaic efficiencies for the production of C2H4 and C2H5OH reaching approximately 60 and 25% respectively for a cathode potential of − 0.7 V vs. RHE. The same study also reported a total current density of ∼−300 mA∕cm2 , one of the highest catalytic activities at low potential reported to date. Despite the volume of research published into possible reaction pathways towards the formation of C–C bonds however, current faradaic efficiencies for the formation of C3 products such as propane and propanol remain relatively low [64, 123]. Previous studies have suggested that by promoting a high selectivity toward the formation of C2, the right conditions to produce C3 or larger products could be achieved. The current understanding of the reaction mechanisms towards the formation of propane and larger hydrocarbons however, is still unclear. Due to the large number of electron transfers involved, developing a clear picture of a reaction pathway would be a time-consuming and difficult process. An alternative method of examining C3 however, as shown by Bagger et al. [124] could use descriptors for product distribu- tion on specific facets to categorise reaction products by comparing coordination number distribution and the binding energies of intermediates. 2.3 Formation of multi‑carbon compounds Liquid fuels provide both a higher energy density and greater economic value, as well as a simpler method of storage [109]. Due to this, the study of the reaction pathway from CO2 to the most typically produced liquid fuels from the CO2RR such as eth- ylene and ethanol have been subject to a plethora of theoretical [110–114] and experimental studies [32, 52, 115–118].i ylene and ethanol have been subject to a plethora of theoretical [110 114] and experimental studies [32, 52, 115 118]. From prior experimental studies, two pathways for ethylene production have been observed. The first takes place mainly on Cu(111) at high overpotential using a shared intermediate with methane, and the second on Cu(100) at lower overpotential which does not yield methane as a product [117]. The second method also differs from the first in that it is pH independent. It forms Carbon–carbon bonds through the formation of a CO dimer from adsorbed surface materials, as opposed to individual transfers of protons and electrons [69, 84, 91, 119]. As a consequence of this, the production of ethylene is favoured on the face-centred cubic (fcc) (100) facet as local pH can be optimised toward CO formation over the HER with no impact on the selectivity of C2 products. The formation of ethylene using the fcc(100) facet is in stark contrast with the formation of methane from copper electrodes which has been observed to be most actively produced on the fcc(211) facet [25, 120]. Numerous experimental [52, 115] and theoretical works [110–112] however confirm the different reaction pathways taken when producing either ethylene or methane. Such works demonstrate drastic dif- ferences in the reaction conditions required to form larger hydrocarbons over smaller products. Based on results from DFT calculations, it is believed that at more negative potentials the dimerisation of CO is replaced by a more favourable reaction based on the coupling of CO and CHO. The reaction between these intermediates is believed to be due to the lower activation energy of the latter reaction [112, 121]. DFT calculations additionally confirm the tendency for CO dimerisation to be favoured on Cu(100) facets. Furthermore, DFT calculations provide additional insights into C–C bond formation in this case, such that the CO dimer is energetically favoured in the presence of a water layer, a local electric field and in the presence of alkali cations [111, 113]. 2.3 Formation of multi‑carbon compounds A small number of other catalyst designs have been proposed including NiGa, PdAu, NiP and N-doped carbon catalysts which have been capable of producing C2 and greater products, however none have been able to do this as efficiently (0121 3456789) 3 3456789) 3 Discover Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering Review Review Fig. 2 Molecular reaction pathway diagram of the electrochemical reduction of CO2 to larger products (adapted from [91, 92]) r reaction pathway diagram of the electrochemical reduction of CO2 to larger products (adapted from [91, 92]) Fig. 2 Molecular reaction pathway diagram of the electrochemical reduction of CO2 to larger products (adapte Fig. 3 Adsorption energy scaling. A proposed pathway for the reduction of CO2 to CH4 for a metal surface is shown at the top of the image and the adsorption energies of bound intermediates on the fcc (face-centred cubic)(211) facet are shown on the two lower figures. (More tightly bound adsorbates correspond to more negative binding energies.) The adsorption energies of adsorbates bound to the surface by carbon (pictured left) and by oxygen (pictured right) are correlated and plotted against the binding energy of key intermediates CO and OH respectively (adapted from [101]) Fig. 3 Adsorption energy scaling. A proposed pathway for the reduction of CO2 to CH4 for a metal surface is shown at the top of the image and the adsorption energies of bound intermediates on the fcc (face-centred cubic)(211) facet are shown on the two lower figures. (More tightly bound adsorbates correspond to more negative binding energies.) The adsorption energies of adsorbates bound to the surface by carbon (pictured left) and by oxygen (pictured right) are correlated and plotted against the binding energy of key intermediates CO and OH respectively (adapted from [101]) Vol:1 :.(12345678 3 .(12345678 3 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 Review as copper as of yet [102–105]. Recently, notable developments in improving the selectivity of copper catalysts toward C2 products have been observed in a number of ways. Examples of this include altering the catalyst surface structure, such as through high-surface-area oxide-derived (OD) copper, as well as by altering the electrolyte composition and by employing organic films on copper [35, 106–108]. Another point of interest in the formation of C2 and greater products is the possibility of liquid products. 2.3 Formation of multi‑carbon compounds Currently however, researchers propose either the further use of CO as a reaction intermediate, or the formation of C–C bonds between C1 and C2 intermediates [31, 125, 126]. Current research also seems to suggest a likely intermediate toward C3 being formed through the coupling of CO and C2H4 precursors [127, 128]. A 2019 study by Wang et al. [123] reported the highest Faradaic efficiency to date for the formation of propanol of 33 ± 1% with a cathodic energy conversion efficiency of 21% for metal-doped copper catalysts. Due to the increased energy density and (01231 3 3456789) 3 Discover Chemical Engineering (2022) 2:2 Discover Chemical Engineering \| https://doi.org/10.1007/s43938-022-00009-y Review ease of storage of liquid fuels, a greater understanding of the reaction mechanisms required to form C–C bonds in order to produce larger hydrocarbon and alcohol products could prove revolutionary in improving the commercial viability of the electrochemical reduction of CO2. ease of storage of liquid fuels, a greater understanding of the reaction mechanisms required to form C–C bonds in order to produce larger hydrocarbon and alcohol products could prove revolutionary in improving the commercial viability of the electrochemical reduction of CO2. 3 Optimising catalyst structure for the formation of fuels Following Hori’s review in 2008 [84], the development of catalysts has seen drastic improvement. From his results it was found, as previously stated, that copper most effectively produces fuels from the electrochemical reduction of CO2 (see Table 2). Among the metals tested, it was found consistently that the formation of CO from the CO2RR occurred on face cen- tred cubic (fcc) transition metal lattices [84, 129, 130], suggesting a strong dependence on the surface morphology on overall product selectivity. Due to this, considerable work has been conducted aimed toward optimising catalyst surface structure to produce an electrocatalyst highly selective toward a given CO2RR product, whilst minimising the effect of the competing HER. Studies have included the alloying of surface materials [24, 131–134], production of nanoparticles [12, 65, 135] as well as an Ag “nano-coral” structure prepared by an oxide-reduction method in the presence of chloride anions in an aqueous medium [136]. 3.1 Bimetallic alloy catalysts With regards to alloy catalysts, the introduction of heteroatoms to form bimetallic alloys allows for both the structural and electronic properties of the catalyst to be altered due to the interaction between the two elements involved. For example, one study using Ru–Pd catalysts saw in increase in the selectivity of formic acid production from 70% at −0.7V for pure Pd catalysts to 90% at −1.1V vs. NHE [137]. Similarly, a 2015 work by Rasul et al. [132] saw a dramatic improve- ment in the selectivity of CO production through the introduction of In hetero-atoms into an oxide-derived copper catalyst structure. Here a total Faradaic efficiency of 95% at −0.7V vs. RHE was observed, higher than values observed for either Cu or In separately. It was suggested in this study that the enhanced Faradaic efficiency toward the production of CO was due to the suppression of the HER through structure changes. These changes were believed to have been caused by the preferential binding sites of In within the Cu lattice, based on the high overpotentials observed for HER for In in comparison with copper. Figure 4 demonstrates the preferential binding sites of In within the Cu lattice based on calculations in a follow-up paper by Jedidi et al. [131] demonstrating the preference for In atoms to replace Cu atoms in positions of low coordination such as defects and the fcc(211) facet step-site. 3.3 Oxide‑derived electrocatalysts Another method of surface morphology engineering has been observed through the oxidation and subsequent reduc- tion of bulk metal catalysts in order to improve catalytic activity [29, 73, 120, 144–146]. Studies on oxide-derived metal catalysts such as Cu, Au and Sn have demonstrated an enhanced density of active sites through the modification of surface structures, leading to improved overpotentials observed for CO2RR [29, 73, 144]. In a 2012 work by Li et al. [120] conducted on oxide-derived copper ( Cu2O ) electrodes observed a coarse surface structure consisting of nanowires, with the reduction activity being strongly dependent on the initial thickness of the Cu2O layer. The primary products observed from the examined Cu2O catalysts were formate and CO, produced at both enhanced selectivity and activity. Based on the observed Tafel slope for Cu2O catalysts, a similar reaction mechanism could be suggested as bulk Cu for the forma- tion of products based on the formation of an activated CO2 molecule, as observed in Eq. (3), as the rate-determining step. Due to the study focusing on the comparison of geometrical surface area, it would be difficult to conclude as to whether or not the activity of individual catalytic sites was improved without further characterisation with regards to the electrochemically active surface area. Conversely, for oxide-derived Au, it was found that, as opposed to the nanowire morphology observed for Cu, oxide- derived Au formed a thick layer ( ∼1 μm ) of nanoparticles [29]. Similarly to oxide-derived Cu, an enhanced faradaic effi- ciency was, at lower potential, observed for oxide-derived Au when compared with the performance of the bulk metal. This increase in both selectivity and activity was attributed to a higher stability of the reaction intermediate (see Fig. 5). For Au nanoparticles, the enhanced activity has been directly correlated to the density of grain boundaries resulting from the oxidation-reduction step [147, 148]. Oxygen plasma activation has been found to be another form of pre-treatment which results in drastically enhanced CO2RR activity [33, 149]. Following plasma treatment, the surfaces of oxide-derived metal catalysts were suggested to be defect rich. For plasma treated Cu, both the enhanced reactivity and selectivity have been attributed to the presence of Cu+ ions and subsurface oxygen. A 2016 work by Eilert et al. [150] suggested that the involvement of subsurface oxygen lead to increased CO binding energies, primarily in the vicinity of grain boundaries. 3.2 Nanoparticles An alternative approach to optimising catalyst selectivity through morphological changes is the fabrication of nano- structures such as nanoparticle and nano-coral structures. The size of metal nanoparticles has been reported to have a strong influence on both the activity and selectivity of CO2 reduction catalysts [138–140]. For example, a 2014 study by Reske et al. [140] on Cu nanoparticles 2–15 nm in diameter found that for nanoparticles below 5nm the formation of hydrocarbons such as methane and ethylene were suppressed, resulting in an enhanced selectivity toward CO and H2 formation. Such changes in selectivity were attributed to higher concentrations of low-coordinated surface sites, resulting in stronger chemisorption, as well as boosted catalytic activity. A similar study on the size dependency of Au Vol:.(1234567890) 1 3 Table 2 Faradaic yields in CO2 reduction on fcc metal electrodes for experiments at 5 mA cm−2 current density in a 0.1 M KHCO3 buffer at 18.5◦C . Adapted from Peterson et al. [101] Electrode V vs. RHE Hydrocarbons/ organics CO HCOOH H2 Total Ni − 1.09 2.1 0.0 1.4 88.9 92.4 Cu − 1.05 72.3 1.3 9.4 20.5 103.5 Pd − 0.81 2.9 28.3 2.8 26.2 60.2 Ag − 0.98 0.0 81.5 0.8 12.4 94.6 Pt − 0.68 0.0 0.0 0.1 95.7 95.8 Au − 0.75 0.0 87.1 0.7 10.2 98.0 Vol:1 Discover Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering Review nanoparticles on catalytic activity in the range ∼ 1–8 nm observed a similar trend [141]. On the other hand, for Ag, Pd and Sn nanoparticles, a volcano effect can be observed [139, 142, 143]. For Ag nanoparticles for example, CO2 reduction current first increased as particle size decreased to 5 nm, below which the reduction current then began to decrease [142]. This volcano effect was suggested to have been related to the binding energy of key intermediates in relation to particle size, however convolution effects may have been at play due to the experiments taking place in ionic liquid electrolyte as opposed to aqueous media. Fig. 4 Side views of a Cu(100), b C (111) and c Cu(211) with a single In atom replacing the first layer (1ML) and second layer (2ML). The energies relative to the pure Cu lattice (pictured left) are presented. d Top view of bulk Cu11In9 (adapted from [131]) nanoparticles on catalytic activity in the range ∼ 1–8 nm observed a similar trend [141]. 3.2 Nanoparticles On the other hand, for Ag, Pd and Sn nanoparticles, a volcano effect can be observed [139, 142, 143]. For Ag nanoparticles for example, CO2 reduction current first increased as particle size decreased to 5 nm, below which the reduction current then began to decrease [142]. This volcano effect was suggested to have been related to the binding energy of key intermediates in relation to particle size, however convolution effects may have been at play due to the experiments taking place in ionic liquid electrolyte as opposed to aqueous media. nanoparticles on catalytic activity in the range ∼ 1–8 nm observed a similar trend [141]. On the other hand, for Ag, Pd and Sn nanoparticles, a volcano effect can be observed [139, 142, 143]. For Ag nanoparticles for example, CO2 reduction current first increased as particle size decreased to 5 nm, below which the reduction current then began to decrease [142]. This volcano effect was suggested to have been related to the binding energy of key intermediates in relation to particle size, however convolution effects may have been at play due to the experiments taking place in ionic liquid electrolyte as opposed to aqueous media. Fig. 4 Side views of a Cu(100), b C (111) and c Cu(211) with a single In atom replacing the first layer (1ML) and second layer (2ML). The energies relative to the pure Cu lattice (pictured left) are presented. d Top view of bulk Cu11In9 (adapted from [131]) 3.3 Oxide‑derived electrocatalysts further supports this hypothesis [153], demonstrating stable sub-surface oxygen present on Cu surfaces for up to 1 h at 1.15 V vs. Reversible hydrogen electrode (RHE). It was further suggested that the presence of subsurface oxygen withdrew charge from the copper sp- and d-bands, selectively enhancing the binding energy of CO. Recent DFT-based work however disputes this, suggesting it unlikely for subsurface oxygen to remain stable at the negative potentials at which CO2RR takes place due to their low diffusion barriers [154]. A 2018 work by Lum and Ager [155] conducted using 18O labelling similarly concluded that subsurface oxides should indeed be unstable under CO2RR conditions. Addition- ally, theoretical work has shown that subsurface oxygen is not a prerequisite for CO2 adsorption [156] and coordinatively unsaturated Cu atoms promote C–C bond formation [54, 113, 157, 158]. Furthermore, a 2018 work by Fields et al. [154] demonstrated how, from thermodynamic and kinetic perspective, subsurface oxygen should have a negligible effect on the activity of crystalline Cu under reducing potentials. et al. further supports this hypothesis [153], demonstrating stable sub-surface oxygen present on Cu surfaces for up to 1 h at 1.15 V vs. Reversible hydrogen electrode (RHE). It was further suggested that the presence of subsurface oxygen withdrew charge from the copper sp- and d-bands, selectively enhancing the binding energy of CO. Recent DFT-based work however disputes this, suggesting it unlikely for subsurface oxygen to remain stable at the negative potentials at which CO2RR takes place due to their low diffusion barriers [154]. A 2018 work by Lum and Ager [155] conducted using 18O labelling similarly concluded that subsurface oxides should indeed be unstable under CO2RR conditions. Addition- ally, theoretical work has shown that subsurface oxygen is not a prerequisite for CO2 adsorption [156] and coordinatively unsaturated Cu atoms promote C–C bond formation [54, 113, 157, 158]. Furthermore, a 2018 work by Fields et al. [154] demonstrated how, from thermodynamic and kinetic perspective, subsurface oxygen should have a negligible effect on the activity of crystalline Cu under reducing potentials. In short, the role of subsurface oxygen within OD electrocatalysts remains a controversial topic with, as yet, no single agreed solution. Reliable methods capable of measuring, with a certain degree of accuracy, the electrochemically active surface area of metal electrodes [159, 160] will be of paramount importance in once and for all, resolving this ongoing debate of effective surface area effects. 4 Computational methods Whilst the implementation of Density functional theory (DFT) calculations on the electrode surface can be a complicated affair, a considerable amount of effort has been put into improving models. A recent comprehensive review of such works was produced by Rendòn-Calle et al. [161]. Here we provide a brief review of some of the more fundamental approaches and issues regarding current computational models, in addition to advances in calculating the kinetics of electrochemical steps, structure-sensitive screening, ion effects, and machine learning. Vol:.(123456789 1 3 3.3 Oxide‑derived electrocatalysts It was believed this was due to its influence on the electronic structure of the catalyst, reducing 휎-repulsion. An alternative explanation is that the presence of subsurface oxygen enhances CO2RR activity by facilitating neutral and charged Cu sites, resulting in in chemisorbed CO2 in the presence of water [151]. Studies using DFT calculations agree that interstitial oxygen is stable on the Cu fcc(111) surface, unlike the fcc(211) facet, and are capable of improving surface binding of CO2 [152]. Cavalca (01234 1 3 0123456789) 1 3 (012345678 1 3 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 \| h Discover Chemical Engineering Review et al. further supports this hypothesis [153], demonstrating stable sub-surface oxygen present on Cu surfaces for up to 1 h at 1.15 V vs. Reversible hydrogen electrode (RHE). It was further suggested that the presence of subsurface oxygen withdrew charge from the copper sp- and d-bands, selectively enhancing the binding energy of CO. Recent DFT-based work however disputes this, suggesting it unlikely for subsurface oxygen to remain stable at the negative potentials at which CO2RR takes place due to their low diffusion barriers [154]. A 2018 work by Lum and Ager [155] conducted using 18O labelling similarly concluded that subsurface oxides should indeed be unstable under CO2RR conditions. Addition- ally, theoretical work has shown that subsurface oxygen is not a prerequisite for CO2 adsorption [156] and coordinatively unsaturated Cu atoms promote C–C bond formation [54, 113, 157, 158]. Furthermore, a 2018 work by Fields et al. [154] demonstrated how, from thermodynamic and kinetic perspective, subsurface oxygen should have a negligible effect on the activity of crystalline Cu under reducing potentials. I h t th l f b f ithi OD l t t l t i t i l t i ith t i l Fig. 5 FEs for CO and HCO− 2 production on oxide-derived Au and polycrystalline Au electrodes at various potentials between − 0.2 and − 0.5 V in 0.5 M NaHCO3 , pH 7.2. The dashed line indicates the CO equilibrium potential (adapted from [29]) Fig. 5 FEs for CO and HCO− 2 production on oxide-derived Au and polycrystalline Au electrodes at various potentials between − 0.2 and − 0.5 V in 0.5 M NaHCO3 , pH 7.2. The dashed line indicates the CO equilibrium potential (adapted from [29]) et al. 4.1 Kinetic and thermodynamic models Estimations of catalytic activities based on theoretical calculations have, in recent years, provided fast and increasingly accurate results [24]. By employing DFT and the computational hydrogen electrode model (CHE), calculations can be performed on an atomic scale. This opens up the possibility of screening many novel electrochemical materials and active sites from a thermochemical perspective [162]. The CHE model can be seen discussed in greater detail in Sect. 4.2 however the model has seen widespread use since its creation [163–165]. Through the use of such methods, it has been made feasible to examine possible lowest-overpotential pathways from CO to various C1 and C2 products including ethylene, acetaldehyde and ethanol. For any given reaction pathway, the limiting potential UL is the electrode potential at which all steps are exergonic, i.e. the change in Gibbs energy ΔG is always negative. UL can be found as the inverse of the largest reaction energy (ΔGL) [101]. Vol:.(123456789 1 3 Vol:.(123456789 1 3 Discover Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering Review (7) UL = −ΔGL e UL = −ΔGL e (7) The first DFT-based studies detailing mechanisms to C1 species were proposed by Nørskov et al. [166]. By analysing adsorption energies for Cu(100) surfaces, they concluded the lowest energy pathway to be CO2 ←∗COOH ←∗CO ←∗CHO ←∗CH2O ←∗CH3O ←CH4 + ∗O ←∗OH ←H2O . Using this mechanism, Durand et al. [167] calculated the Gibbs free energies of each reaction intermediate on various Cu facets. Their work found that adsorbates were more easily stabilised on the (211) facet, followed by (100) and (111), in line with simple coordination rules [113, 168–170]. Further studies have since expanded upon this work by examining further Cu facets [171], as well as other transition metals and alloys [172–174]. CO2RR screening techniques based on scaling relations assuming a single mechanism for all facets and materials however can be further improved [161, 175, 176]. Further information on this can be found in Sect. 4.3. Nie et al. [114, 177], for example, examined the activation barriers for every possible transition state from CO2 to methane. Based upon their findings, it was discovered that for the Cu(111) facet, CO is more favourably hydrogenated to COH rather than CHO, supporting work performed by Hussain et al. [178]. The pathway continues from here as ∗COH →∗CH →∗CH2 →∗CH3 →CH4 . A later work by Luo et al. 4.1 Kinetic and thermodynamic models [179] further supports this, demonstrating how unlike the (111) facet, Cu(100) kinetically favours the CHO pathway over COH, suggesting a structure-sensitive mechanism dictated by elementary-step kinetics. Beyond methane formation, thermodynamic studies into the formation of C2 products such as ethylene ( C2H4 ) and ethanol ( C2H6O ) have been explored thermodynamically by Calle-Vallejo and Koper [110]. In their work, the electro- chemical reduction of CO (CORR) was studied on Cu(100), considering CO dimerization as the believed first step toward ethylene, acetaldehyde and ethanol [180]. The electroreduction of CO is contained within CO2RR, forming a rate limiting step in most CO2RR reduction processes. The square sites of the Cu(100) facet exhibit strong stabilisation of CO dimers, helping to explain the Cu(100) facet’s preference for ethylene production, whereas step sites tune the product selectivity towards ethanol production [180]. Cheng et al. [157], supports this hypothesis, suggesting in their work that Cu structures with stepped square sites show enhanced selectivity and catalytic activity toward C2 reaction products. Garza et al. [121] further expanded upon these studies, proposing reaction pathways for all reported C2 products from CORR (ethylene, ethanol, acetaldehyde, ethylene glycol, glycolaldehyde, glyoxal, and acetate) on both Cu(100) and Cu(111) facets. The activation energies for C–C bond formation on Cu in vacuum were determined by Montoya et al. [181]. From their work it was found that kinetic barriers depend on the degree of hydrogenation of adsorbates. It was concluded that CO dimerization is kinetically unfavourable in a vacuum. An initial protonation step of CO followed by the dimerization of CHO molecules however, was agreed to be more favourable by comparison. In subsequent work however demonstrated that water-solvated cations stabilize CO dimers, thus making ∗CO ←∗CO coupling feasible under CORR conditions, particularly on Cu(100) facets [111]. y A key challenge currently facing researchers within catalysis is the formation of multiple C–C bonds, forming C3 and greater products such as n-propanol. Due to the increasing kinetic barriers faced due to the increased complexity of the n-propanol molecule, faradaic efficiencies for C3 products currently remain at consistently low values around 10–13% [127, 182] for n-propanol. Small quantities of other C3 products such as allyl alcohol, acetone, propylene and propane have additionally been observed at low Faradaic efficiencies of < 3% [31, 182]. Rui et al. 4.2 The computational hydrogen electrode (CHE) model The CHE model is commonly used in the simulation of electrocatalytic systems [162]. It involves the posteriori correc- tion of standard constant charge, allowing for the electrode potential to be taken into account [193]. When utilised in combination with DFT calculations, CHE can provide a picture of the possible reaction pathways and the potentials at which the redox reactions take place [101, 110, 194]. By approximating the equilibrium between protons and electrons with hydrogen, CHE avoids the explicit treatment of the solvated particles like so (8) 1 2H2(g) ↔H+ + e−. (8) 1 2H2(g) ↔H+ + e−. 1 2H2(g) ↔H+ + e−. (8) The chemical potential of a proton–electron pair can therefore be written on pair can therefore be written The chemical potential of a proton–electron pair can therefore be written The chemical potential of a proton–electron pair can therefore be written (9) 1 2휇H2 = 휇(H++e−) (9) 1 2휇H2 = 휇(H++e−) (9) 1 2휇H2 = 휇(H++e−) (9) where 휇x is the chemical potential of species x. For a reductive process, the Gibbs free energy G can be related to the electrode potential U as where 휇x is the chemical potential of species x. For a reductive process, the Gibbs free energy G can be related to the electrode potential U as (10) ΔG = −eU (10) ΔG = −eU thus providing a consistent evaluation of Gibbs energy for all species involved. It should be noted here that e is the positive (+) electron charge. The hydrogenation of CO, ∗CO + H+ + e−→∗CHO for example, can, for a given potential U, be written (11) ΔG∗CO−∗CHO = 휇∗CHO −휇CO −1 2휇H2 + eU. (11) Additionally, through the combination of CHE and DFT calculations, one can gain insight into the coverage of surface species at specific pH using a Pourbaix diagram [195]. Works using a combination of both Pourbaix diagrams and CHE can also be found throughout electrochemistry literature [196, 197]. Additionally, through the combination of CHE and DFT calculations, one can gain insight into the coverage of surface species at specific pH using a Pourbaix diagram [195]. Works using a combination of both Pourbaix diagrams and CHE can also be found throughout electrochemistry literature [196, 197]. Typically, CHE methods incorporate only CPET steps, however a recent study by Göttle and Koper [78] introduced a method, based on first-principles calculations of acid-base equilibrium constants, which incorporates decoupled pro- ton–electron transfer pathways. 4.1 Kinetic and thermodynamic models suggests that the coupling mechanism for multi-carbon products follows a “polymerisation” scheme of adsorbed CO that obeys the Flory–Schulz distribution [183]. Initial works by Hori performed under strictly galvanostatic conditions ( −5 mA cm−2 , E = −1.1 V vs. NHE) gave a faradaic efficiency of 4.2% [72, 184]. Under such extreme conditions, Rahaman et al. explains how poor selectivity is a direct result of high overpotentials lowering the required kinetic activation barriers for a variety of competing hydro- genation and C–C coupling processes, particularly those involving CO [72, 182]. Since then, many strategies aiming to improve product selectivity of CO2RR have often followed the path of reducing required overpotentials. In his 2017 work Rahaman demonstrates how the addition of a thin oxide “skin” on catalytic surfaces which can be later reduced under CO2RR conditions [171, 185] can reduce the required overpotentials for C–C bond formation [182]. Such reductions are achieved through the formation of low-coordinated sites from the precursor species, resulting in improved stabilisation of chemisorbed * CO.− 2 radical anions, as well as CO [23]. Such methods were applied to form Cu meshes using anneal- ing and electrodeposition methods to produce catalysts capable of producing n-propanol at 13.1% FE at − 1.0V vs. RHE. y Ebaid et al. [186] supports this hypothesis, suggesting surface roughness to be a strong indicator of catalyst perfor- mance, as higher surface roughness was attributed to high population of under-coordinated sites. Among the various (01231 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 Discover Chemical Engineering (2022) 2:2 Review methods used to roughen catalyst surfaces, the reduction of Cu2O [54, 187, 188] and Cu3N [189] have been shown to be the most effective methods of achieving high selectivity toward C2+ products. C3 product selectivity however, remains a issue in virtually all Cu-based catalysts [186, 189–192]. Whilst surface rough- ening methods have shown a drastic improvement in C3 production, the lack of a controlled or regimented method of surface roughening limits the extent to which structure and activity relationships can be investigated for CO2RR performance. 4.2 The computational hydrogen electrode (CHE) model Through this, the sequential proton–electron transfer (SPET) pathways can be estimated and provide a computational understanding of pH effects observed experimentally for molecular, metal and oxide- derived catalyst2s [23, 198, 199]. This method has seen use, for example, in rationalising the experimentally determined pH dependence of CO reduction on immobilized cobalt protoporphyrins [79]. 4.4 Effect of structure and pH pH, structural sensitivity and ion effects are all interconnected during CO2RR, the interplay of their effects altering reaction mechanisms. Observed onset potentials, faradaic efficiencies and product distributions have been shown to be not only dependant on the chosen catalyst material [23, 209, 210], but also on its surface structure [32, 117]. Typically however, studies based on scaling relation screenings only consider a single reaction mechanism [101, 174, 206]. A work by Calle-Vallejo and Koper [176] however showed that metal- and structure-sensitive bifurcating pathways can be incorporated into screening routines using scaling relations. It should be noted however that breaking scaling relations between CO and CHO may lead to enhanced CO2RR electrocatalysis [101, 206]. 2 In addition to this, it has been observed that both electrolyte pH and local pH effects play an important role in the CO2RR mechanism. When modelling a system, successive proton-coupled electron transfers are often assumed to take place at every step of the reaction process, so as to enable the use of the CHE model [194]. It should be noted however that the CHE model cannot capture pH effects as the adsorption energies of all intermediates shift proportionally. This problem becomes most apparent when analyzing the CORR due to the strong pH and structure dependent features of the CO intermediate. For example, for pristine Cu, whilst ethene is produced most commonly on the fcc(100) terrace site, CO and methane are more preferentially produced on the fcc(111) facet, both with a strong dependence on pH [209, 211]. A report by Hori [91] suggests the disparity between pH responses indicate that the rate-limiting step of CORR to CH4 involves proton-coupled electron transfers, making the study of CO2RR and CORR to CH4 suitable for CHE models. The formation of C2H4 however does not include such reactions, and should be analyzed cautiously. In a 1991 work by Murata et al. [212], it was shown that the production of multi-carbon species could be enhanced through the use of alkaline cations. The selectivity toward C2H4 , for example, can be increased by the inclusion of larger cations as their smaller hydration spheres better favour adsorption on cathodic surfaces, yielding more positive potential values. Singh et al. [213] explained cation effects based on the pka values for their hydrolysis. 4.3 Scaling relations Scaling relations, i.e. linear correlations between adsorption energies of adsorbates [200], are effective in simplify- ing DFT-based catalytic models. Through their implementation, scaling relations may impose additional constraints toward further optimising current electrocatalysts. Due to this, scaling relations have been studied extensively [201–203]. Recent works have examined the possibility of breaking scaling relations [113, 204, 205], however breaking such relations has proven to be difficult to achieve experimentally. In order to effectively break scaling relations, one must first stabilise one intermediate with respect to the others. For example, Li et al. [113] explained in their 2014 work how the preference for the producing C2 products over methane on Cu(100) facets was due to the breaking of scaling relations. They suggest the scaling relations between bound CO and C2O2 on the facet are broken due strong ensemble effects observed during the dimerization step. For CO2RR, a number of methods l:.(123456789 3 r Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 Discover Chemical Engineering Review have been discussed within the literature with regards to breaking scaling relations. Alloying, addition of promot- ers, tethering, the introduction of p-block dopants and creation of low-coordination sites are among some of the strategies which have been suggested throughout the literature [101, 206]. As well as this, some other methods of note are; the alteration of adsorbate solvation via modification of the electrolyte composition/dielectric constant [101, 206], strain effects [207], the study of transition-metal-free catalysts [197] and the anchoring of active ligands on active sites [208]. have been discussed within the literature with regards to breaking scaling relations. Alloying, addition of promot- ers, tethering, the introduction of p-block dopants and creation of low-coordination sites are among some of the strategies which have been suggested throughout the literature [101, 206]. As well as this, some other methods of note are; the alteration of adsorbate solvation via modification of the electrolyte composition/dielectric constant [101, 206], strain effects [207], the study of transition-metal-free catalysts [197] and the anchoring of active ligands on active sites [208]. Conversely, recent studies have shown, based on scaling-relation-based volcano plots, that whilst a single mecha- nism is operative on all materials and facets [101, 206], the CO2RR itself is highly sensitive to both structure and material, as previously discussed. 4.4 Effect of structure and pH The larger CO2 concentrations near the cathode and lower local pH observed with increasing cation size both being satisfactorily explaining cation effects for the CO2RR but not those observed for the CORR. 2 When factoring-in cation effects into computational models, cation effects may be included implicitly or explicitly. For example, one study by Chen et al. [214] used an implicit method of modelling cation effects, applying a general electric field as opposed to individual point charges. The electric field then interacts with adsorbates, modifying the adsorption energies depending on the on the dipole moment of a given species [215]. Conversely, Akhade et al. [216], used an explicit model of cation effects in combination with DFT calculations to account for the impact of adsorbed and co-adsorbed I− and K+ ion, on CO protonation. Through this work it was found that K∗ improves CO and CHO binding over COH, improving the selectivity toward the CHO pathway. By contrast, I∗ weakens the binding energy of CO, COH and CHO, resulting in an improved selectivity toward the formation CO as the primary reduction product. In later works, it was suggested that specific halide adsorption on Cu may occur at negative electrode potentials [179]. As a consequence of this, it was found that the specific adsorption of Cl− , Br− , and I− affect the CO2RR. A 2016 work by Vaela et al. [217] supports these results. Their experimental observations showing a 3.5 times higher selectivity toward CO production using Br− as opposed to Cl− , as well as I− favouring the reduction of CO to methane over CO desorption. Perez-Gallent et al. [122] presented a joint computational-experimental study on the impact of cation effects on CORR. Using an explicit model, it was observed that hydrogenation of monomers was particularly more difficult than that of dimers. This was attributed to the fact that cations stabilize C2 adsorbates but not C1 intermedi- ates. This discovery could explain why C2H4 exhibits earlier onset potentials than CH4 [122]. (01231 3456789) 3 3456789) 3 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering (2022) 2:2 Discover Chemical Engineering Review 4.5 Machine learning Despite the tremendous potential of machine-learning algorithms, their predictive capabilities depend largely on the size and quality of the training dataset, as well as the descriptor of choice. For example, assuming a single mechanism for all materials, datasets made of unrelaxed calculations and similar simplifications can lead to incorrect predictions [219].i To summarise, significant progress has been made in the development of computational models, however a number of challenges must be overcome in order to develop more accurate screening routines for the design of CO2 and CO reduction materials [175]. 4.5 Machine learning Computational works fully based upon DFT calculations for CO2RR providing a comprehensive amount of detail for the catalytic activity of materials. such works however are often computationally expensive. In addition to the study of scal- ing relations, machine learning algorithms offer an alternative approach toward electrocatalyst selection and design. By using an extensive library of preexisting data from previous research, trends within previously documented results are detected and examined during an initial “training phase”. Following this, provided suitable descriptors have been selected, a process of statistical analyses can be used to predict, within a degree of accuracy, new materials selective toward a desired product without performing new DFT calculations [218, 219]. One such machine learning framework, for example, was presented by Ulissi et al. [220]. Through this framework, they examined numerous configurations and active sites on intermetallic compounds. The results of their method explained the activity of certain NiGa sites and suggested the need for a composition-, configuration- and structure-sensitive meth- ods to speed up the discovery of new catalytic materials. Research conducted by Jiang et al. used genetic algorithms to find stable solid-liquid interfaces on Cu to account for solvation effects when modelling CORR [221]. Similarly, Xin et al. used a neural network-based chemisorption model in tandem with scaling relations to predict CO2RR to C2 reac- tion products on (100−) terminated catalysts [222]. One such work by Zhong et al. [223], developed a machine learning accelerated, high-throughput DFT framework allowing for new materials to be screened ab-initio. Through this particular work, Zhong et al. discovered a promising bi-metallic CuAl compound with near optimal CO adsorption energy ( −0.67 eV) with an observed faradaic efficiency of ∼80% with a partial current density of 600 mA cm−2. Despite the tremendous potential of machine-learning algorithms, their predictive capabilities depend largely on the size and quality of the training dataset, as well as the descriptor of choice. For example, assuming a single mechanism for all materials, datasets made of unrelaxed calculations and similar simplifications can lead to incorrect predictions [219]. To summarise, significant progress has been made in the development of computational models, however a number of challenges must be overcome in order to develop more accurate screening routines for the design of CO2 and CO reduction materials [175]. Authors’ contributions SR conceived the project. IB constructed the framework of the manuscript and summarized the literature. All the authors were involved in writing the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the Engineering and Physical Sciences Research Council [Grant Number EP/S023836/1]. Data availability All data analysed during this review paper is within the article. Authors’ contributions SR conceived the project. IB constructed the framework of the manuscript and summarized the literature. All the authors were involved in writing the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Competing interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adapta- tion, 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/. References 1. Ritchie H. Energy. Our world in data; 2014. https://ourworldindata.org/energy. y y 2. Ritchie H. Fossil fuels. Our world in data; 2017. https://ourworldindata.org/fossil-fuels. 2. Ritchie H. Fossil fuels. Our world in data; 2017. https://ourworldindata.org/fossil-fuels. 3. Olah GA, Goeppert A, Surya Prakash GK. Chemical recycling of carbon dioxide to methanol and dimethyl e to renewable, environmentally carbon neutral fuels and synthetic hydrocarbons. J Org Chem. 2009;74(2):48 y y y 4. Carlson KM, Gerber JS, Mueller ND, Herrero M, MacDonald GK, Brauman KA, Havlik P, O’Co Greenhouse gas emissions intensity of global croplands. Nat Clim Change. 2017;7(1):63–8. 4. Carlson KM, Gerber JS, Mueller ND, Herrero M, MacDonald GK, Brauman KA, Havlik P, O’Connell CS, Johnson JA, Saatchi S, West PC. Greenhouse gas emissions intensity of global croplands. Nat Clim Change. 2017;7(1):63–8. y g p g Mansoori GA. A unique view on carbon dioxide emissions around the world. Glob J Earth Sci Eng. 2017;4(1):8–17. Mohammed SJ, Mansoori GA. A unique view on carbon dioxide emissions around the world. Glob J Earth Sci En , q g ; ( ) 6. Lewis NS, Nocera DG. Powering the planet: chemical challenges in solar energy utilization. Proc Natl Acad Sci. 2006;103(43):15729–35. 6. Lewis NS, Nocera DG. Powering the planet: chemical challenges in solar energy utilization. Proc Natl Aca 7. Olah GA. Beyond oil and gas: the methanol economy. Angew Chem Int Ed. 2005;44(18):2 y g y g 8. Fuqiang W, Ziming C, Jianyu T, Yuan Y, Yong S, Linhua L. Progress in concentrated solar power technology with parabolic trough collector system: a comprehensive review. Renew Sustain Energy Rev. 2017;79(C):1314–28. 8. Fuqiang W, Ziming C, Jianyu T, Yuan Y, Yong S, Linhua L. Progress in concentrated so system: a comprehensive review. Renew Sustain Energy Rev. 2017;79(C):1314–28 9. Korkas C, Baldi S, Michailidis I, Kosmatopoulos E. Occupancy-based demand response and thermal comfort optimization in microgrids with renewable energy sources and energy storage. Appl Energy. 2016;163:93–104, 02.fi 10. Muhich CL, Ehrhart BD, Al-Shankiti I, Ward BJ, Musgrave CB, Weimer AW. A review and perspective of efficient hydrogen generation via solar thermal water splitting. WIREs Energy Environ. 2016;5(3):261–87. y Wang A, Zhang T, Tang J. Photoelectrochemical devices for solar water splitting—materials and challenges. Chem 4645–60. 11. Jiang C, Moniz SJA, Wang A, Zhang T, Tang J. Photoelectrochemical devices for solar water splitting—mater Soc Rev. 2017;46(15):4645–60. 12. Kumar B, Brian JP, Atla V, Kumari S, Bertram KA, White RT, Spurgeon JM. References New trends in the development of heterogeneous catalysts for electrochemical CO2 reduction. Catal Today. 2016;270:19–30. 2 y 13. Theaker N, Strain JM, Bijandra Kumar J, Brian P, Kumari S, Spurgeon JM. Heterogeneously catalyzed two-step cascade electrochemical reduction of CO2 to ethanol. Electrochim Acta. 2018;274:1–8. 2 14. White JL, Herb JT, Kaczur JJ, Majsztrik PW, Bocarsly AB. Photons to formate: efficient electrochemical solar energy conversion via reduc- tion of carbon dioxide. J CO2 Util. 2014;7:1–5. 2 PJA. Prospects of CO2 utilization via direct heterogeneous electrochemical reduction. J Phys Chem Lett 2 15. Whipple DT, Kenis PJA. Prospects of CO2 utilization via direct heterogeneous electrochemical redu 2010;1(24):3451–8. 16. Centi G, Perathoner S. Opportunities and prospects in the chemical recycling of carbon dioxide to fuels. Catal Today. 2009;148:191–205, 11. 17. Durst J, Rudnev A, Dutta A, Yongchun F, Herranz J, Kaliginedi V, Kuzume A, Permyakova AA, Paratcha Y, Broekmann P, Schmidt TJ. Elec- trochemical CO2 reduction—a critical view on fundamentals, materials and applications. Chimia. 2015;69(12):769–76. 2 18. Jones JP, Surya Prakash GK, Olah GA. Electrochemical CO2 reduction: recent advances and current trends. Israel J Chem. 2014;54(10):1451–66. 19. Zuman P. Modern aspects of electro-chemistry. Adv Coll Interface Sci. 1981;14(4):282–3. 20. Birdja YY, Pérez-Gallent E, Figueiredo MC, Göttle AJ, Calle-Vallejo F, Koper MTM. Advances and challenges in understanding the electro- catalytic conversion of carbon dioxide to fuels. Nat Energy. 2019;4(9):732–45. y gy 21. Hori Y. Electrochemical CO2 reduction on metal electrodes. In: Modern aspects of electrochemistry. New York: Springer; 2008. p. 101. 22. Ibram G. Conversion of carbon dioxide into methanol—a potential liquid fuel: fundamental challenges and opportunities (a review). Renew Sustain Energy Rev. 2014;31:221–57. 2 p y p g ; p 22. Ibram G. Conversion of carbon dioxide into methanol—a potential liquid fuel: fundamental challenges and opportunities (a review). Renew Sustain Energy Rev. 2014;31:221–57. gy 23. Kortlever R, Shen J, Klaas JP, Schouten FC-V, Koper MTM. Catalysts and reaction pathways for the electrochemical reduction of carbon dioxide. J Phys Chem Lett. 2015;6(20):4073–82.f y 24. Seh ZW, Kibsgaard J, Dickens CF, Chorkendorff IB, Nørskov JK, Jaramillo TF. Combining theory and experiment into materials design. Science. 2017;355(6321):eaad4998. Dickens CF, Chorkendorff IB, Nørskov JK, Jaramillo TF. Combining theory and experiment in electrocatalysis: insights . Science. 2017;355(6321):eaad4998. g ; ( ) 25. Hori Y, Wakebe HHI, Tsukamoto T, Koga O. Electrocatalytic process of CO selectivity in electrochemical reduction of CO2 at metal elec- trodes in aqueous media. Code availability Not applicable. Ethics approval and consent to participate Not applicable. 5 Conclusion The reduction of CO2 to multi-carbon products such as hydrocarbons consists of multiple complex reaction steps involv- ing many shared intermediates depending on the desired reaction product. In recent decades, a significant amount of progress has been made regarding the design of highly active or selective electrocatalysts, particularly for the formation of CO and formate. For the formation of larger products involving multiple carbon atoms such as ethylene however, a greater understanding of the reaction mechanisms is required. In particular, improving understanding of key areas such as the effect of the electrode surface morphology on product selectivity, sub-surface structure as well as the effect of local pH. Now, due to a lack of effective probes able to examine quantities outside macroscopic equilibria, a combina- tion of both experimental and computational methods could provide the key to developing a greater understanding of the reaction phenomena taking place. In terms of computational modelling of electrocatalysts, one possible method of improving the understanding of a system could be through combining numerous theoretical models to develop beyond the specific cases which certain models are tuned toward. This would require both a more standardised approach toward future experimental setups, as well as a conscious effort to develop future computational models beyond merely the surface characteristics of potential catalyst designs. Even after 35 years of research, there is still much work to be done, particularly in the development of catalysts more selective toward the production of liquid fuels. The “one-pot” synthesis of chemicals from waste CO2 however, particularly fuels, represents a useful and potentially game-changing method of moving toward a carbon neutral future with minimal impact on existing infrastructure. Authors’ contributions SR conceived the project. IB constructed the framework of the manuscript and summarized the literature. All the authors were involved in writing the manuscript. All authors read and approved the final manuscript. Data availability All data analysed during this review paper is within the article. Vol:1 :.(123456789 3 Discover Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y Discover Chemical Engineering Review Code availability Not applicable. References Selective electrochemical reduction of CO2 to ethylene at a three-phase interface on copper(I) halide-confined Cu-mesh electrodes in acidic solutions of potassium halides. J Electroanal Chem. 2004;565(2):287–93.i i 35. Dinh CT, Burdyny T, Kibria MG, Seifitokaldani A, Gabardo CM, García de Arquer FP, Kiani A, Edwards JP, De Luna P, Bushuyev OS, Zou C. CO2 electroreduction to ethylene via hydroxide-mediated copper catalysis at an abrupt interface. Science. 2018;360(6390):783–7. 36. Payra S, Shenoy S, Chakraborty C, Tarafder K, Roy S. Structure-sensitive electrocatalytic reduction of CO2 to methanol over carbon- supported intermetallic PtZn nano-alloys. ACS Appl Mater Interfaces. 2020;12(17):19402–14. 37. Huang J, Guo X, Yue G, Qiong H, Wang L. Boosting CH3 OH production in electrocatalytic CO2 reduction ov cobalt nanoparticles dispersed on single-layer nitrogen-doped graphene. ACS Appl Mater Interfaces. 2018; 37. Huang J, Guo X, Yue G, Qiong H, Wang L. Boosting CH3 OH production in electrocatalytic CO2 reduction over partially oxidized 5 nm cobalt nanoparticles dispersed on single-layer nitrogen-doped graphene. ACS Appl Mater Interfaces. 2018;10(51):44403–14. 38 Ren D DengY Handoko AD Chen CS Malkhandi S Yeo BS Selective electrochemical reduction of carbon dioxide to ethylene and ethanol 38. Ren D, Deng Y, Handoko AD, Chen CS, Malkhandi S, Yeo BS. Selective electrochemical reduction of carbon dioxi on copper(I) oxide catalysts. ACS Catal. 2015;5(5):2814–21. ko AD, Chen CS, Malkhandi S, Yeo BS. Selective electrochemical reduction of carbon dioxide to ethylene and ethano talysts. ACS Catal. 2015;5(5):2814–21. pp y 39. Chi D, Yang H, Yanfang D, Lv T, Sui G, Wang H, Jiaxing L. Morphology-controlled CuO nanoparticles for electroreduction of CO2 to ethanol. RSC Adv. 2014;4(70):37329–32. 40. Song Y, Peng R, Hensley DK, Bonnesen PV, Liang L, Zili W, Meyer HM, Chi M, Ma C, Sumpter BG, Rondinone AJ. High-selectivity electro- chemical conversion of CO2 to ethanol using a copper nanoparticle/N-doped graphene electrode. ChemistrySelect. 2016;1(19):6055–61. 40. Song Y, Peng R, Hensley DK, Bonnesen PV, Liang L, Zili W, Meyer HM, Chi M, Ma C, Sumpter BG, Rondinone AJ. High-selectivity electro- chemical conversion of CO2 to ethanol using a copper nanoparticle/N-doped graphene electrode. ChemistrySelect. 2016;1(19):6055–61. 41. Saito M. R&D activities in Japan on methanol synthesis from CO2 and H2. Catal Surv Jpn. 1998;2(2):175–84. p y 2 2 p 42. Saito M, Takeuchi M, Watanabe T, Toyir J, Luo S, Wu J. Methanol synthesis from CO2 and H2 over a CuZnO-based multicomponent catalyst. Energy Convers Manag. 1997;38:S403–8. 43. Bartholomew CH, Farrauto RJ. References Electrochim Acta. 1994;39(11–12):1833–9. q 26. Min X, Kanan MW. Pd-catalyzed electrohydrogenation of carbon dioxide to formate: high mass activity at low overpotential and iden- tification of the deactivation pathway. J Am Chem Soc. 2015;137(14):4701–8. 26. Min X, Kanan MW. Pd-catalyzed electrohydrogenation of carbon dioxide to fo tification of the deactivation pathway. J Am Chem Soc. 2015;137(14):4701–8. (01231 3456789) 3 Discover Chemical Engineering \| https://doi.org/10.1007/s43938-022-00009-y Review (2022) 2:2 27. Kortlever R, Peters I, Koper S, Koper MTM. Electrochemical CO2 reduction to formic acid at low overpotential and with high faradaic efficiency on carbon-supported bimetallic Pd–Pt nanoparticles. ACS Catal. 2015;5(7):3916–23. fi y pp p 28. Cave ER, Montoya JH, Kuhl KP, Abram DN, Hatsukade T, Shi C, Hahn C, Nørskov JK, Jaramillo TF. Electrochemical CO2 reduction on Au surfaces: mechanistic aspects regarding the formation of major and minor products. Phys Chem Chem Phys. 2017;19(24):15856–63. 29. Chen Y, Li CW, Kanan MW. Aqueous CO2 reduction at very low overpotential on oxide-derived au nanoparticles. J Am Chem Soc. 2012;134(49):19969–72. fi y pp p 28. Cave ER, Montoya JH, Kuhl KP, Abram DN, Hatsukade T, Shi C, Hahn C, Nørskov JK, Jaramillo TF. Electrochemical CO2 reduction on Au surfaces: mechanistic aspects regarding the formation of major and minor products. Phys Chem Chem Phys. 2017;19(24):15856–63. 29 Ch Y Li CW K MW A CO d ti t l t ti l id d i d ti l J A Ch S 29. Chen Y, Li CW, Kanan MW. Aqueous CO2 reduction at very low overpotential on oxide-derived au nano 2012;134(49):19969–72. 30. Hatsukade T, Kuhl KP, Cave ER, Abram DN, Jaramillo TF. Insights into the electrocatalytic reduction of CO2 on metallic silver surfaces. Phys Chem Chem Phys. 2014;16(27):13814–9. y 31. Kuhl KP, Cave ER, Abram DN, Jaramillo TF. New insights into the electrochemical reduction of carbon dioxide on metallic copper surfaces. Energy Environ Sci. 2012;5(5):7050–9. gy 32. Hori Y, Takahashi I, Koga O, Hoshi N. Electrochemical reduction of carbon dioxide at various series of copper single crystal electrodes. J Mol Catal A Chem. 2003;199(1–2):39–47. 33. Mistry H, Varela AS, Bonifacio CS, Zegkinoglou I, Sinev I, Choi YW, Kisslinger K, Stach EA, Yang JC, Strasser P, C plasma-activated copper catalysts for carbon dioxide reduction to ethylene. Nat Commun. 2016;7:1–9. 34. Yano H, Tanaka T, Nakayama M, Ogura K. References Hydrogen production and synthesis gas reactions. In: Fundamentals of industrial catalytic processes. Hoboken: Wiley; 2006. p. 339–486. y p 44. Behrens M, Studt F, Kasatkin I, Kühl S, Hävecker M, Abild-Pedersen F, Zander S, Girgsdies F, Kurr P, Kniep B methanol synthesis over Cu/ZnO/Al2O3 industrial catalysts. Science. 2012;336(6083):893–7. p Kasatkin I, Kühl S, Hävecker M, Abild-Pedersen F, Zander S, Girgsdies F, Kurr P, Kniep B-L, et al. The active site o ver Cu/ZnO/Al2O3 industrial catalysts. Science. 2012;336(6083):893–7. y 2 3 y 45. Russell WW, Miller GH. Catalytic hydrogenation of carbon dioxide to higher hydrocarbons. J Am Chem Soc. 1950;72(6):2446–54. Russell WW, Miller GH. Catalytic hydrogenation of carbon dioxide to higher hydrocarbons. J Am Chem Soc. 1950 , y y g g y ; ( ) 46. Dorner RW, Hardy DR, Williams FW, Willauer HD. Heterogeneous catalytic CO2 conversion to value-added hydrocarbons. Energy Environ Sci. 2010;3(7):884–90. ang B, Li Y, Nan W, Lan CQ. CO2 bio-mitigation using microalgae. Appl Microbiol Biotechnol. 2008;79(5):707–18. 48. Yaashikaa PR, Senthil Kumar P, Varjani Sunita J, Saravanan A. A review on photochemical, biochemical and electrochemical transforma- tion of CO2 into value-added products. J CO2 Util. 2019;33(1):131–47. p 2 Froehlich J, Dang T, Sathrum A, Kubiak CP. Photochemical and photoelectrochemical reduction of CO2. Annu Rev 1):541–69. 2 2 49. Kumar B, Llorente M, Froehlich J, Dang T, Sathrum A, Kubiak CP. Photochemical and photoelectrochemical r Phys Chem. 2012;63(1):541–69. y 50. Morris AJ, Meyer GJ, Fujita E. Molecular approaches to the photocatalytic reduction of carbon dioxide for solar fuels, accounts of chemi- cal research. Acc Chem Res. 2009;42(12):1983–94. 51. Li K, An X, Park KH, Khraisheh M, Tang J. A critical review of CO2 photoconversion: catalysts and reactors. Catal Today. 2014;224:3–12. 52 U d M Niit Y H ik T M t d S O M El t h i l d ti f CO t h d b t t bl l t i l 51. Li K, An X, Park KH, Khraisheh M, Tang J. A critical review of CO2 photoconversion: catalysts and reactors. Catal Today. 2014;224:3–12. 52. Umeda M, Niitsuma Y, Horikawa T, Matsuda S, Osawa M. Electrochemical reduction of CO2 to hydrocarbons to store renewable electrical energy and upgrade biogas. Energy Convers Manag. 2007;48(4):1255–65. 51. Li K, An X, Park KH, Khraisheh M, Tang J. A critical review of CO2 photoconversion: catalysts and reactors. Cat 52. References On the photoelectrocatalytic reduction of carbon dioxide. Mater Chem Phys. 1989;22(3–4):249–80. 62. Bockris JOM, Wass JC. On the photoelectrocatalytic reduction of carbon dioxide. Mater Chem Phys. 1989;22(3–4):249–80. 63 Delacourt C Ridgway PL Newman J Mathematical modeling of CO reduction to CO in aqueous electrolytes J Electrochem Soc 64. Lee CH, Kanan MW. Controlling H+ vs CO2 reduction 64. Lee CH, Kanan MW. Controlling H+ vs CO2 reduction selectivity on Pb electrodes. ACS Catal. 2015;5(1):465–9.f 2 65. Del Castillo A, Alvarez-Guerra M, Solla-Gullón J, Sáez A, Montiel V, Irabien A. Sn nanoparticles on gas diffu characterization and use for continuous CO2 electroreduction to formate. J CO2 Util. 2017;18:222–8. 66. Zhang X, Lei T, Liu Y, Qiao J. Enhancing CO2 electrolysis to formate on facilely synthesized Bi catalysts at low overpotential. Appl Catal B. 2017;218:46–50.l ; 67. Kim S, Dong WJ, Gim S, Sohn W, Park JY, Yoo CJ, Jang HW, Lee JL. Shape-controlled bismuth nanoflakes as highly selective catalysts for electrochemical carbon dioxide reduction to formate. Nano Energy. 2017;39(May):44–52. gy y 68. Rabiee A, Nematollahi D. Electrochemical reduction of CO2 to formate ion using nanocubic mesoporous In(OH)3/carbon black system. Mater Chem Phys. 2017;193:109–16. N, Co A. A review of the aqueous electrochemical reduction of CO2 to hydrocarbons at copper. J Electroanal Ch . y 69. Gattrell M, Gupta N, Co A. A review of the aqueous electrochemical reduction of CO2 to hydrocarbons at co 2006;594(1):1–19. 70. Singh MR, Goodpaster JD, Weber AZ, Head-Gordon M, Bell AT. Mechanistic insights into electrochemical reduction of CO2 over Ag using density functional theory and transport models. Proc Natl Acad Sci USA. 2017;114(42):E8812–21. y y p ; ( ) 71. Bagger A, Ju W, Varela AS, Strasser P, Rossmeisl J. Electrochemical CO2 reduction: a classification problem. ChemPhysChem. 2017;18(22):3266–73. ; ( ) 72. Hori Y, Murata A, Takahashi R. Formation of hydrocarbons in the electrochemical reduction of carbon dioxide at a copper electrode in aqueous solution. J Chem Soc Faraday Trans 1. 1989;85:2309–26. y n oxide dependence of the CO2 reduction efficiency on tin electrodes and enhanced activity for tin/tin oxide thin- hem Soc. 2012;134(4):1986–9. y 73. Chen Y, Kanan MW. Tin oxide dependence of the CO2 reduction efficiency on tin electrodes and enhanced ac film catalysts. J Am Chem Soc. 2012;134(4):1986–9. i y 74. Luc W, Collins C, Wang S, Xin H, He K, Kang Y, Jiao F. References Solis BH, Maher AG, Dogutan DK, Nocera DG, Hammes-Schiffer S. Nickel phlorin intermediate formed by proto in hydrogen evolution mechanism. Proc Natl Acad Sci USA. 2016;113(3):485–92. y g 87. Göttle AJ, Koper MTM. Determinant role of electrogenerated reactive nucleophilic species on selectivity during reduction of CO2 cata- lyzed by metalloporphyrins. J Am Chem Soc. 2018;140(14):4826–34. 87. Göttle AJ, Koper MTM. Determinant role of electrogenerated reactive nucleophilic species on selectivity during reduction of CO2 cata- lyzed by metalloporphyrins. J Am Chem Soc. 2018;140(14):4826–34. y y p p y 88. Loewen ND, Neelakantan TV, Berben LA. Renewable formate from C–H bond formation with CO2: using iron carbonyl clusters as elec- trocatalysts. Acc Chem Res. 2017;50(9):2362–70. y y p p y 88. Loewen ND, Neelakantan TV, Berben LA. Renewable formate from C–H bond formation with CO2: using iron carbonyl clusters as elec- trocatalysts. Acc Chem Res. 2017;50(9):2362–70. y 89. Tang Q, Lee Y, Li DY, Choi W, Liu CW, Lee D, Jiang DE. Lattice-hydride mechanism in electrocatalytic CO2 reduction by structurally precise copper-hydride nanoclusters. J Am Chem Soc. 2017;139(28):9728–36. y 89. Tang Q, Lee Y, Li DY, Choi W, Liu CW, Lee D, Jiang DE. Lattice-hydride mechanism in electrocatalytic CO2 reduction by structurally precise copper-hydride nanoclusters. J Am Chem Soc. 2017;139(28):9728–36. y 90. Groenenboom MC, Keith JA. Quantum chemical analyses of BH4 − and BH3OH− hydride transfers to CO2 in aqu tials of mean force. ChemPhysChem. 2017;18(22):3148–52. y 91. Hori Y, Takahashi R, Yoshinami Y, Murata A. Electrochemical reduction of CO at a copper electrode. J Phys Chem B. 1997;101(36):7075–81. 91. Hori Y, Takahashi R, Yoshinami Y, Murata A. Electrochemical reduction of CO at a copper electrode. J Phys Chem B. 1997;101(36):7075–81. 92. Varela AS. The importance of pH in controlling the selectivity of the electrochemical CO2 reduction. Curr Opin Green Sustain Chem. 2020;26:11. 91. Hori Y, Takahashi R, Yoshinami Y, Murata A. Electrochemical reduction of CO at a copper electrode. J Phys Chem B. 1997;101(36):7075–81. 92. Varela AS. The importance of pH in controlling the selectivity of the electrochemical CO2 reduction. Curr Opin Green Sustain Chem. 2020;26:11. pp y 92. Varela AS. The importance of pH in controlling the selectivity of the electrochemical CO2 reduction. Curr Opin Green Sustain Chem. 2020;26:11. f 93. Haag MR. Handbook of chemistry. Agron J. 1950;42(2):111–2. g y g 94. Lim RJ, Xie M, Sk MA, Lee JM, Fisher A, Wang X, Lim KH. References Ag-–Sn bimetallic catalyst with a core-shell structure for CO2 reduction. J Am Chem Soc. 2017;139(5):1885–93. 75. Baruch MF, Pander III JE, White JL, Bocarsly AB. Mechanistic insights into the reduction of CO2 on tin electrodes using in situ ATR-IR spectroscopy. ACS Catal. 2015;5(5):3148–56. 76. Scholten F, Nguyen K-LC, Bruce JP, Heyde M, Cuenya BR. Identifying structure-selectivity correlations in the electrochemical reduction of CO2: a comparison of well-ordered atomically clean and chemically etched copper single-crystal surfaces. Angew Chem Int Ed. 2021;60(35):19169–75. ; ( ) 77. Yoo JS, Christensen R, Vegge T, Nørskov JK, Studt F. Theoretical insight into the trends that guide the electrochemical reduction of carbon dioxide to formic acid. ChemSusChem. 2016;9(4):358–63. 78. Göttle AJ, Koper MTM. Proton-coupled electron transfer in the electrocatalysis of CO2 reduction: prediction of sequential vs. concerted pathways using DFT. Chem Sci. 2016;8(1):458–65. as R, Birdja YY, Diaz-Morales O, Kwon Y, Ledezma-Yanez I, Schouten KJ, Mul G, Koper M. Electrocatalytic reduction carbon monoxide and methane at an immobilized cobalt protoporphyrin. Nat Commun. 2015;6(1):1–8. p y g 79. Shen J, Kortlever R, Kas R, Birdja YY, Diaz-Morales O, Kwon Y, Ledezma-Yanez I, Schouten KJ, Mul G, Koper M. of carbon dioxide to carbon monoxide and methane at an immobilized cobalt protoporphyrin. Nat Commu 80. Shen J, Kolb MJ, Göttle AJ, Koper MTM. DFT study on the mechanism of the electrochemical reduction of CO2 catalyzed by cobalt por- phyrins. J Phys Chem C. 2016;120(29):15714–21. p y y 81. Koper MTM. Theory of the transition from sequential to concerted electrochemical proton–electron transfer. Phys Chem Chem Phys. 2013;15(5):1399–407. 82. Monteiro MCO, Dattila F, Hagedoorn B, García-Muelas R, López N, Koper M. Absence of CO2 electroreduction on copper, gold and silver electrodes without metal cations in solution. Nat Catal. 2021;4(8):654–62.l 83. Birdja YY, Shen J, Koper MTM. Influence of the metal center of metalloprotoporphyrins on the electrocat acid. Catal Today. 2017;288:37–47. y 84. Hori Y. Electrochemical CO2 reduction on metal electrodes. New York: Springer; 2008. p. 89–189. y 84. Hori Y. Electrochemical CO2 reduction on metal electrodes. New York: Springer; 2008. p. 89–189. 2 p g p 85. Azuma M. Electrochemical reduction of carbon dioxide on various metal electrodes in low-temperature aqueous KHCO3 media. J Elec- trochem Soc. 1990;137(6):1772.f 85. Azuma M. Electrochemical reduction of carbon dioxide on various metal electrodes in low-temperature aqueous KHCO3 media. J Elec- trochem Soc. 1990;137(6):1772.f 86. References Umeda M, Niitsuma Y, Horikawa T, Matsuda S, Osawa M. Electrochemical reduction of CO2 to hydrocarbons to energy and upgrade biogas. Energy Convers Manag. 2007;48(4):1255–65. gy pg g gy g ; ( ) 53. Zhong S, Cao Z, Yang X, Kozlov SM, Huang KW, Tung V, Cavallo L, Li LJ, Han Y. Electrochemical conversion of CO2 to 2-bromoethanol in a membraneless cell. ACS Energy Lett. 2019;4(2):600–5. gy 54. Jiang K, Sandberg RB, Akey AJ, Liu X, Bell DC, Nørskov JK, Chan K, Wang H. Metal ion cycling of Cu foil for selective C–C coupling in electrochemical CO2 reduction. Nat Catal. 2018;1(2):111–9. 2 55. Yao B, Xiao T, Makgae OA, Jie X, Gonzalez-Cortes S, Guan S, Kirkland AI, Dilworth JR, Al-Megren HA, Alshihri SM, Dobson PJ. Transforming carbon dioxide into jet fuel using an organic combustion-synthesized Fe–Mn–K catalyst. Nat Commun. 2020;11(1):1–12. y y 56. Verma S, Hamasaki Y, Kim C, Huang W, Lu S, Jhong HR, Gewirth AA, Fujigaya T, Nakashima N, Kenis PJ. Insights electroreduction of CO2 to CO on a supported gold catalyst in an alkaline flow electrolyzer. ACS Energy Lettf electroreduction of CO2 to CO on a supported gold catalyst in an alkaline flow electrolyzer. ACS Energy Lett. 2018;3(1):193–8. 57. De Luna P, Hahn C, Higgins D, Jaffer SA, Jaramillo TF, Sargent EH. What would it take for renewably powered electrosynthesis to displace petrochemical processes? Science. 2020;350(3):eaav3506. p p ; ( ) 58. Tascedda P, Weidmann M, Dinjus E, Duach E. Recent developments in electrochemical and photoelectrochemical CO2 reduction: involve- ment of the ( CO2)2⋅− dimer radical anion. Appl Organomet Chem. 2001;15(2):113–20. p p 58. Tascedda P, Weidmann M, Dinjus E, Duach E. Recent developments in electrochemical and photoelect ment of the ( CO2)2⋅− dimer radical anion. Appl Organomet Chem. 2001;15(2):113–20. 2 pp g 59. Rosen BA, Salehi-Khojin A, Thorson MR, Zhu W, Whipple DT, Kenis PJ, Masel RI. Ionic liquid-mediated selective conversion of CO2 to co at low overpotentials. Science. 2011;334(6056):643–4. 61. Chandrasekaran K, Bockris LM. In-situ spectroscopic investigation of adsorbed intermediate radicals in electrochemical reactions: CO2 − on platinum. Surf Sci. 1987;185(3):495–514. 61. Chandrasekaran K, Bockris LM. In-situ spectroscopic investigation of adsorbed intermediate radicals in electrochemical reactions: CO2 − on platinum. Surf Sci. 1987;185(3):495–514. Vol:1 :.(12345678 3 Discover Chemical Engineering \| https://doi.org/10.1007/s43938-022-00009-y (2022) 2:2 Review ckris JOM, Wass JC. On the photoelectrocatalytic reduction of carbon dioxide. Mater Chem Phys. 1989;22(3–4):24 62. Bockris JOM, Wass JC. References Structure sensitivity of the electrochemical reduction of carbon monoxide on copper single 013;3(6):1292–5. 116. Schouten KJ, Pérez Gallent E, Koper MT. Structure sensitivity of the electrochemical reduction of carbo crystals. ACS Catal. 2013;3(6):1292–5. y 117. Schouten KJ, Qin Z, Pérez Gallent E, Koper MT. Two pathways for the formation of ethylene in CO reduction on single-crystal copper electrodes. J Am Chem Soc. 2012;134(24):9864–7. 118. Wuttig A, Liu C, Peng Q, Yaguchi M, Hendon CH, Motobayashi K, Ye S, Osawa M, Surendranath Y. Tracking a common surface-bound intermediate during CO2-to-fuels catalysis. ACS Cent Sci. 2016;2(8):522–8. 118. Wuttig A, Liu C, Peng Q, Yaguchi M, Hendon CH, Motobayashi K, Ye S, Osawa M, Surendranath Y. Tr intermediate during CO2-to-fuels catalysis. ACS Cent Sci. 2016;2(8):522–8. 2 119. Hori Y, Murata A, Takahashi R, Suzuki S. Electroreduction of CO to CH4 and C2H4 at a copper electrode in aqueous solutions at ambient temperature and pressure. J Am Chem Soc. 1987;109(16):5022–3.i 120. Li CW, Kanan MW. CO2 reduction at low overpotential on Cu electrodes resulting from the reduction of th 2012;134(17):7231–4. an MW. CO2 reduction at low overpotential on Cu electrodes resulting from the reduction of thick Cu2O films. J Am 17):7231–4. 121. Garza AJ, Bell AT, Head-Gordon M. Mechanism of CO2 reduction at copper surfaces: pathways to C2 products. ACS Catal. 2018;8(2):1490–9. 121. Garza AJ, Bell AT, Head-Gordon M. Mechanism of CO 121. Garza AJ, Bell AT, Head-Gordon M. Mechanism of CO2 reduction at copper surfaces: pathways to C2 products. ACS Catal. 2018;8(2):1490–9. 122. Pérez-Gallent E, Figueiredo MC, Calle-Vallejo F, Koper MT. Spectroscopic observation of a hydrogenated CO dimer intermediate during 121. Garza AJ, Bell AT, Head-Gordon M. Mechanism of CO2 reduction at copper surfaces: pathways to C2 products. ACS Catal. 2018;8(2):1490–9. 122. Pérez-Gallent E, Figueiredo MC, Calle-Vallejo F, Koper MT. Spectroscopic observation of a hydrogenated CO dimer intermediate during CO reduction on Cu(100) electrodes. Angew Chem Int Ed. 2017;56(13):3621–4. , , 2 pp p y p 22. Pérez-Gallent E, Figueiredo MC, Calle-Vallejo F, Koper MT. Spectroscopic observation of a hydrogenated CO CO reduction on Cu(100) electrodes. Angew Chem Int Ed. 2017;56(13):3621–4. 123. References A review on the electrochemical reduction of CO2 in fuel cells, metal electrodes and molecular catalysts. Catal Today. 2014;233:169–180. y y 95. Prieto G. Carbon dioxide hydrogenation into higher hydrocarbons and oxygenates: thermodynamic and kin with heterogeneous and homogeneous catalysis. Chemsuschem. 2017;10(6):1056–70. 96. Hori Y, Murata A, Yoshinami Y. Adsorption of CO, intermediately formed in electrochemical reduction of CO2, at a copper electrode. J Chem Soc Faraday Trans. 1991;87(1):125–8. (0121 3456789) 3 Review Discover Chemical Engineering (2022) 2:2 \| https://doi.org/10.1007/s43938-022-00009-y 97. Hori Y, Murata A, Tsukamoto T, Wakebe H, Koga O, Yamazaki H. Adsoprtion of carbon monoxide at a copper electrode accompanied by electron transfer observed by voltammetry and IR spectroscopy. Electrochim Acta. 1994;39(17):2495–500. 97. Hori Y, Murata A, Tsukamoto T, Wakebe H, Koga O, Yamazaki H. Adsoprtion of carbon monoxide at a copper l f b d b l d l h 97. Hori Y, Murata A, Tsukamoto T, Wakebe H, Koga O, Yamazaki H. Adsoprtion of carbon monoxide at a copper electron transfer observed by voltammetry and IR spectroscopy. Electrochim Acta. 1994;39(17):2495–500. y y y 98. Production at, metal of, electrochemical reduction, of CO, Chiba Hori, Synthetic Katsuhei Kikuchi, Faculty Chiba Shin, and Suzuki Engi- neering. The chemicals. Laboratory Techniques in Biochemistry and Molecular Biology. 1990;20(C):5–51. 99. Vielstich W, Lamm A, Gasteiger HA. Materials and manufacturing processes. In: Reitz W, editor. Handbook o technology, and applications (Volume 2). Hoboken: Wiley; 2007. p. 789. 100. Hara K, Kudo A, Sakata T. Electrochemical reduction of high pressure carbon dioxide on Fe electrodes at large current density. J Electroanal Chem. 1995;386(1–2):257–60. 101. Peterson AA, Nørskov JK. Activity descriptors for CO2 electroreduction to methane on transition-metal catalysts. J Phys Chem Lett. 2012;3(2):251–8. 102. Torelli DA, Francis SA, Crompton JC, Javier A, Thompson JR, Brunschwig BS, Soriaga MP, Lewis NS. Nickel-gallium-catalyzed electrochemi- cal reduction of CO2 to highly reduced products at low overpotentials. ACS Catal. 2016;6(3):2100–4. 2 g y p p 03. Kortlever R, Peters I, Balemans C, Kas R, Kwon Y, Mul G, Koper MTM. Palladium-gold catalyst for the electroch C1–C5 hydrocarbons. Chem Commun. 2016;52(67):10229–32.f y 104. Calvinho KU, Alherz AW, Yap KM, Laursen AB, Hwang S, Bare ZJ, Clifford Z, Musgrave CB, Dismukes GC. Selective CO2 reduction to C3 and C4 oxyhydrocarbons on nickel phosphides at overpotentials as low as 10 mV. Energy Environ Sci. 2018;11(9):2550–9. y y p p p gy 105. W. Transition metal (Mo, Fe Co, and Ni)-based catalysts for electrochemical CO2 reduction. Cuihua Xuebao/C ):1157–66. References Fan Q, Zhang M, Jia M, Liu S, Qiu J, Sun Z. Electrochemical CO2 reduction to C2+ species: heterogeneous electrocatalysts, reaction path- ways, and optimization strategies. Mater Today Energy. 2018;10:280–301. y g y gy 106. Han Z, Kortlever R, Chen HY, Peters JC, Agapie T. CO2 reduction selective for C ≥2 products on polycrystalline copper with N-substituted pyridinium additives. ACS Cent Sci. 2017;3(8):853–9. y 07. Hoang TT, Verma S, Ma S, Fister TT, Timoshenko J, Frenkel AI, Kenis PJ, Gewirth AA. Nanoporous copper–silver a electrodeposition for the selective electroreduction of CO2 to ethylene and ethanol. J Am Chem Soc. 2018;1 electrodeposition for the selective electroreduction of CO2 to ethylene and ethanol. J Am Chem Soc. 2018;140(17):5791 7. 108. Gao D, McCrum IT, Deo S, Choi YW, Scholten F, Wan W, Chen JG, Janik MJ, Roldan Cuenya B. Activity and selectivity control in CO2 elec- troreduction to multicarbon products over CuOx catalysts via electrolyte design. ACS Catal. 2018;8(11):10012–20. 08. Gao D, McCrum IT, Deo S, Choi YW, Scholten F, Wan W, Chen JG, Janik MJ, Roldan Cuenya B. Activity and sele troreduction to multicarbon products over CuOx catalysts via electrolyte design. ACS Catal. 2018;8(11):1001 109. Bushuyev OS, De Luna P, Dinh CT, Tao L, Saur G, van de Lagemaat J, Kelley SO, Sargent EH. What should we make with CO2 and how can we make it? Joule. 2018;2(5):825–32. 110. Calle-Vallejo F, Koper MT. Theoretical considerations on the electroreduction of CO to C2 species on Cu(100) electrodes. Angew Chem Int Ed. 2013;52(28):7282–5. 11. Montoya JH, Shi C, Chan K, Nørskov JK. Theoretical insights into a CO dimerization mechanism in CO2 elec Lett. 2015;6(11):2032–7.i 12. Goodpaster JD, Bell AT, Head-Gordon M. Identification of possible pathways for C–C bond formation during of CO2: new theoretical insights from an improved electrochemical model. J Phys Chem Lett. 2016;7(8):1471 2 113. Li H, Li Y, Koper MT, Calle-Vallejo F. Bond-making and breaking between carbon, nitrogen, and oxygen in electrocatalysis. J Am Chem Soc. 2014;136(44):15694–701. 114. Nie X, Esopi MR, Janik MJ, Asthagiri A. Selectivity of CO2 reduction on copper electrodes: the role of the kinetics of elementary steps. Angew Chem Int Ed. 2013;52(9):2459–62. 115. Schouten KJP, Kwon Y, Van Der Ham CJM, Qin Z, Koper MTM. A new mechanism for the selectivity to C1 and C2 species in the electro- chemical reduction of carbon dioxide on copper electrodes. Chem Sci. 2011;2(10):1902–9. allent E, Koper MT. nsition metal (Mo, Fe Co, and Ni)-based catalysts for electrochemical CO2 reduction. Cuihua Xuebao/Chin J C 66 129. Hao J, Shi W. Transition metal (Mo, Fe Co, and Ni)-based catalysts for electrochemical CO2 reduction. Cuihua Xuebao/Chin J Catal. 2018;39(7):1157–66. References Subsurface oxide plays a critical role in CO2 acti form chemisorbed CO2, the first step in reduction of CO2. Proc Natl Acad Sci USA. 2017;114(26):6706– y y y Favaro M, Xiao H, Cheng T, Goddard WA, Crumlin EJ. Subsurface oxide plays a critical role in CO2 activation form chemisorbed CO the first step in reduction of CO Proc Natl Acad Sci USA 2017;114(26):6706 11 152. Xiao J, Kuc A, Frauenheim T, Heine T. CO2 reduction at low overpotential on Cu electrodes in the presence of impurities at the sub- surface. J Mater Chem A. 2014;2(14):4885–9. ; ( ) 153. Cavalca F, Ferragut R, Aghion S, Eilert A, Diaz-Morales O, Liu C, Koh AL, Hansen TW, Pettersson LG, Nilsson A. Nature and distribution of stable subsurface oxygen in copper electrodes during electrochemical CO2 reduction. J Phys Chem C. 2017;121(45):25003–9. 153. Cavalca F, Ferragut R, Aghion S, Eilert A, Diaz-Morales O, Liu C, Koh AL, Hansen TW, Pettersson LG, Nilsson A. Nature and distribution of stable subsurface oxygen in copper electrodes during electrochemical CO2 reduction. J Phys Chem C. 2017;121(45):25003–9. 154. Fields M, Hong X, Nørskov JK, Chan K. Role of subsurface oxygen on Cu surfaces for CO2 electrochemical reduction. J Phys Chem C. 153. Cavalca F, Ferragut R, Aghion S, Eilert A, Diaz Morales O, Liu C, Koh AL, Hansen TW, Pettersson LG, Nilsson A. Nature and distribution of stable subsurface oxygen in copper electrodes during electrochemical CO2 reduction. J Phys Chem C. 2017;121(45):25003–9. 154. Fields M, Hong X, Nørskov JK, Chan K. Role of subsurface oxygen on Cu surfaces for CO2 electrochemical reduction. J Phys Chem C. 2018;122(28):16209–15. 154. Fields M, Hong X, Nørskov JK, Chan K. Role of subsurface oxygen on Cu surfaces for CO2 electrochemical reduction. J Phys Chem C. 2018;122(28):16209–15. 55. Lum Y, Ager JW. Stability of residual oxides in oxide-derived copper catalysts for electrochemical CO2 re 18o labeling. Angew Chem Int Ed. 2018;57(2):551–4. 18o labeling. Angew Chem Int Ed. 2018;57(2):551–4. 156. Garza AJ, Bell AT, Head-Gordon M. Is subsurface oxygen necessary for the electrochemical reduction of CO2 on copper? J Phys Chem Lett. 2018;9(3):601–6. g g 156. Garza AJ, Bell AT, Head-Gordon M. Is subsurface oxygen necessary for the electrochemical reduction of CO2 on copper? J Phys Chem Lett. 2018;9(3):601–6. ; ( ) 157. Cheng T, Xiao H, Goddard WA. Nature of the active sites for CO reduction on copper nanoparticles; suggestions for optimizing performance. References A highly selective copper–indium bim electrochemical reduction of aqueous CO2 to CO. Angew Chem Int Ed. 2015;54(7):2146–50. q 2 g 133. Li D, Wu J, Liu T, Liu J, Yan Z, Zhen L, Feng Y. Tuning the pore structure of porous tin foam electrodes for enhanced electrochemical reduc- tion of carbon dioxide to formate. Chem Eng J. 2019;375:122024. g ; 134. Sarfraz S, Garcia-Esparza AT, Jedidi A, Cavallo L, Takanabe K. Cu–Sn bimetallic catalyst for selective aqueous electroreduction of CO2 to CO. ACS Catal. 2016;6(5):2842–51. 135. Cheng J, Xuan X, Yang X, Zhou J, Cen K. Selective reduction of CO2 to alcohol products on octahedral catalyst of carbonized Cu(BTC) doped with Pd nanoparticles in a photoelectrochemical cell. Chem Eng J. 2019;358:860–8. p p p g 36. Hsieh YC, Senanayake SD, Zhang Y, Xu W, Polyansky DE. Effect of chloride anions on the synthesis and e of silver nanocoral electrodes for CO2 electroreduction. ACS Catal. 2015;5(9):5349–56. p p g e SD, Zhang Y, Xu W, Polyansky DE. Effect of chloride anions on the synthesis and enhanced catalytic activity ectrodes for CO2 electroreduction. ACS Catal. 2015;5(9):5349–56. 2 137. Furuya N, Yamazaki T, Shibata M. High performance Ru–Pd catalysts for CO2 reduction at gas-diffusion electrodes. J Electroanal Chem. 1997;431(1):39–41. 138. Back S, Yeom MS, Jung Y. Active sites of Au and Ag nanoparticle catalysts for CO2 electroreduction to CO. ACS Catal. 2015;5(9):5089–96. 139 D f G H Zh JW Mi S Y F W G W J B X Si d d t l t t l ti d ti f CO Pd 38. Back S, Yeom MS, Jung Y. Active sites of Au and Ag nanoparticle catalysts for CO2 electroreduction to CO. AC 138. Back S, Yeom MS, Jung Y. Active sites of Au and Ag nanoparticle catalysts for CO2 electroreduction to CO. ACS Catal. 2015;5(9):5089–96. 139. Dunfeng Gao H, Zhou JW, Miao S, Yang F, Wang G, Wang J, Bao X. Size-dependent electrocatalytic reduction of CO2 over Pd nano- particles. J Am Chem Soc. 2015;137(13):4288–91. 2 139. Dunfeng Gao H, Zhou JW, Miao S, Yang F, Wang G, Wang J, Bao X. Size-dependent electrocatalytic re particles. J Am Chem Soc. 2015;137(13):4288–91. p 140. Reske R, Mistry H, Behafarid F, Roldan Cuenya B, Strasser P. Particle size effects in the catalytic electroreduction of CO2 on Cu nano- particles. J Am Chem Soc. 2014;136(19):6978–86. p ; ( ) 141. References Mistry H, Reske R, Zeng Z, Zhao ZJ, Greeley J, Strasser P, Cuenya BR. Exceptional size-dependent activity enhancement in the elec- troreduction of CO2 over Au nanoparticles. J Am Chem Soc. 2014;136(47):16473–6. 2 142. Salehi-Khojin A, Jhong HR, Rosen BA, Zhu W, Ma S, Kenis PJ, Masel RI. Nanoparticle silver catalysts that show enhanced activity for carbon dioxide electrolysis. J Phys Chem C. 2013;117(4):1627–32. y y yer TJ. Nanostructured tin catalysts for selective electrochemical reduction of carbon dioxide to formate. J Am 5):1734–7. 143. Zhang S, Kang P, Meyer TJ. Nanostructured tin catalysts for selective electrochemical reduction of car Chem Soc. 2014;136(5):1734–7. 144. Xie J, Huang Y, Hanqing Yu. Tuning the catalytic selectivity in electrochemical CO2 reduction on copper oxide-derived nanomaterials. Front Environ Sci Eng. 2015;9(5):861–6. g 145. Dutta A, Rahaman M, Luedi NC, Mohos M, Broekmann P. Morphology matters: tuning the product distribution of CO2 electroreduc- tion on oxide-derived Cu foam catalysts. ACS Catal. 2016;6(6):3804–14. y 146. Ma M, Djanashvili K, Smith WA. Selective electrochemical reduction of CO2 to CO on CuO-derived Cu nanowires. Phys Chem Chem Phys. 2015;17(32):20861–7. elvey K, White HS, Kanan MW. Selective increase in CO2 electroreduction activity at grain-boundary surface te 2017;358(6367):1187–92. y 47. Mariano RG, McKelvey K, White HS, Kanan MW. Selective increase in CO2 electroreduction activity at grain nations. Science. 2017;358(6367):1187–92. 148. Feng X, Jiang K, Fan S, Kanan MW. Grain-boundary-dependent CO2 electroreduction activity. J Am Chem Soc. 2015;137(14):4606–9. 148. Feng X, Jiang K, Fan S, Kanan MW. Grain-boundary-dependent CO2 electroreduction activity. J Am Chem Soc. 2015;137(14):4606–9. 149. Mistry H, Choi YW, Bagger A, Scholten F, Bonifacio CS, Sinev I, Divins NJ, Zegkinoglou I, Jeon HS, Kisslinger K, Stach EA, et al. 148. Feng X, Jiang K, Fan S, Kanan MW. Grain-boundary-dependent CO2 electroreduction activity. J Am Chem Soc. 2015;137(14):4606–9. 149. Mistry H, Choi YW, Bagger A, Scholten F, Bonifacio CS, Sinev I, Divins NJ, Zegkinoglou I, Jeon HS, Kisslinger K, Stach EA, et al. Enhanced carbon dioxide electroreduction to carbon monoxide over defect-rich plasma-activated silver catalysts. Angew Chem Int Ed. 2017;56(38):11394–8. ; ( ) 150. Eilert A, Cavalca F, Roberts FS, Osterwalder J, Liu C, Favaro M, Crumlin EJ, Ogasawara H, Friebel D, Pettersson LG, Nilsson A. Subsurface oxygen in oxide-derived copper electrocatalysts for carbon dioxide reduction. J Phys Chem Lett. 2017;8(1):285–90. yg pp y y 151. Favaro M, Xiao H, Cheng T, Goddard WA, Crumlin EJ. References Wang X, Wang Z, Zhuang T-T, Dinh C-T, Li J, Nam D-H, Li F, Huang C-W, Tan C-S, Chen Z, Chi M, Gabardo CM, Seifitokaldani A, Todorović P, Proppe A, Pang Y, Kirmani AR, Wang Y, Ip AH, Richter LJ, Scheffel B, Aoni X, Lo S-C, Kelley SO, Sinton D, Sargent EH. Efficient upgrading of CO to C3 fuel using asymmetric C–C coupling active sites. Nat Commun. 2019;10(1):5186. g y p g arela AS, Strasser P, Rossmeisl J. Electrochemical CO2 reduction: classifying cu facets. ACS Catal. 2019;9(9):7894–9 Goddard WA Atomistic mechanisms underlying selectivities in C1 and C2 products from electrochemical reductio y 24. Bagger A, Ju W, Varela AS, Strasser P, Rossmeisl J. Electrochemical CO2 reduction: classifying cu facets. ACS C 125. Xiao H, Cheng T, Goddard WA. Atomistic mechanisms underlying selectivities in C1 and C2 products from electrochemical reduction of CO on Cu(111). J Am Chem Soc. 2017;139(1):130–6.i 126. Zhuang TT, Pang Y, Liang ZQ, Wang Z, Li Y, Tan CS, Li J, Dinh CT, De Luna P, Hsieh PL, Burdyny T, et al. Copper nanocavities confine inter- mediates for efficient electrosynthesis of C3 alcohol fuels from carbon monoxide. Nat Catal. 2018;1(12):946–51. fi 27. Ren D, Wong NT, Handoko AD, Huang Y, Yeo BS. Mechanistic insights into the enhanced activity and sta nanocrystals for the electrochemical reduction of carbon dioxide to n-propanol. J Phys Chem Lett. 2016;7(1 y y 128. Tang MT, Peng HJ, Stenlid JH, Abild-Pedersen F. Exploring trends on coupling mechanisms toward C3 product formation in CO(2)R. J Phys Chem C. 2021;125(48):26437–47. 128. Tang MT, Peng HJ, Stenlid JH, Abild-Pedersen F. Exploring trends on coupling mechanisms toward C3 product formation in CO(2)R. J Phys Chem C. 2021;125(48):26437–47. 129. Hao J, Shi W. Transition metal (Mo, Fe Co, and Ni)-based catalysts for electrochemical CO2 reduction. Cuihua Xuebao/Chin J Catal. 2018;39(7):1157–66. Vol:1 :.(123456789 3 Discover Chemical Engineering \| https://doi.org/10.1007/s43938-022-00009-y (2022) 2:2 Review 130. Grote JP, Zeradjanin AR, Cherevko S, Savan A, Breitbach B, Ludwig A, Mayrhofer KJ. Screening of material libraries for electrochemical CO2 reduction catalysts—improving selectivity of Cu by mixing with Co. J Catal. 2016;343:248–56. 2 y p g y y g 131. Jedidi A, Rasul S, Masih D, Cavallo L, Takanabe K. Generation of Cu–In alloy surfaces from CuInO2 as selective catalytic sites for CO2 electroreduction. J Mater Chem A. 2015;3(37):19085–92. ; ( ) 132. Rasul S, Anjum DH, Jedidi A, Minenkov Y, Cavallo L, Takanabe K. References Methanol synthesis on Cu(100) from a binary gas mixture of CO2 and H2. Catal Lett. 1994;26(3):373–81. y g 2 ; ( ) 167. Durand WJ, Peterson AA, Studt F, Abild-Pedersen F, Nørskov JK. Structure effects on the energetics of the electrochemical reduction of CO2 by copper surfaces. Surf Sci. 2011;605(15):1354–9.f 2 y pp 168. Calle-Vallejo F, Loffreda D, Koper M, Sautet P. Introducing structural sensitivity into adsorption-energy scaling relations by means of coordination numbers. Nat Chem. 2015;7(5):403–10. lejo F, Schuhmann W, Bandarenka AS. Making the hydrogen evolution reaction in polymer electrolyte membrane ter. Nat Commun. 2016;7(1):10990.f 69. Tymoczko J, Calle-Vallejo F, Schuhmann W, Bandarenka AS. Making the hydrogen evolution reaction in poly electrolysers even faster. Nat Commun. 2016;7(1):10990.f y 170. Calle-Vallejo F, Martínez JI, García-Lastra JM, Sautet P, Loffreda D. Fast prediction of adsorption properties for platinum nanocatalysts with generalized coordination numbers. Angew Chem Int Ed. 2014;53(32):8316–9.f g g 171. Tang W, Peterson AA, Varela AS, Jovanov ZP, Bech L, Durand WJ, Dahl S, Nørskov JK, Chorkendorff I. The importance of surface morphol- ogy in controlling the selectivity of polycrystalline copper for CO2 electroreduction. Phys Chem Chem Phys. 2012;14(1):76–81. 172 Hi it P El t d ti f b di id t th il d ld t l t DFT t d J Ph Ch 171. Tang W, Peterson AA, Varela AS, Jovanov ZP, Bech L, Durand WJ, Dahl S, Nørskov JK, Chorkendorff I. The importance of surface morphol- ogy in controlling the selectivity of polycrystalline copper for CO2 electroreduction. Phys Chem Chem Phys. 2012;14(1):76–81. 172. Hirunsit P. Electroreduction of carbon dioxide to methane on copper, copper–silver, and copper–gold catalysts: a DFT study. J Phys Chem C. 2013;117(16):8262–8. ogy in controlling the selectivity of polycrystalline copper for CO2 electroreduction. Phys Chem Chem Phys. 2012;14(1):76–81. 172. Hirunsit P. Electroreduction of carbon dioxide to methane on copper, copper–silver, and copper–gold catalysts: a DFT study. J Phys Chem C. 2013;117(16):8262–8. ng W, Limtrakul J. CO2 electrochemical reduction to methane and methanol on copper-based alloys: theoretica C. 2015;119(15):8238–49. 73. Hirunsit P, Soodsawang W, Limtrakul J. CO2 electrochemical reduction to methane and methanol on coppe insight. J Phys Chem C. 2015;119(15):8238–49. g y 174. Hansen HA, Shi C, Lausche AC, Peterson AA, Nørskov JK. Bifunctional alloys for the electroreduction of CO2 and CO. Phys Chem Chem Phys. 2016;18(13):9194–201.f 175. Jovanov ZP, Hansen HA, Varela AS, Malacrida P, Peterson AA, Nørskov JK, Stephens IE, Chorkendorff I. References J Am Chem Soc. 2017;139(34):11642–5. 158. Xiao H, Goddard WA, Cheng T, Liu Y. Cu metal embedded in oxidized matrix catalyst to promote CO2 activation and CO dimerization for electrochemical reduction of CO2. Proc Natl Acad Sci USA. 2017;114(26):6685–8. 2 159. Fisica C, Milano U. And applied chemistry commission on electrochemistry * real surface area measurements prepared for publica- tion by. Pure Appl Chem. 1991;63(5):711–34. 160 McCrory CC Jung S Peters JC Jaramillo TF Benchmarking heterogeneous electrocatalysts for the oxygen evolution reaction J Am 2 59. Fisica C, Milano U. And applied chemistry commission on electrochemistry * real surface area measureme tion by. Pure Appl Chem. 1991;63(5):711–34. y 60. McCrory CC, Jung S, Peters JC, Jaramillo TF. Benchmarking heterogeneous electrocatalysts for the oxygen Chem Soc. 2013;135(45):16977–87. ; ( ) 161. Rendón-Calle A, Builes S, Calle-Vallejo F. A brief review of the computational modeling of CO2 electroreduction on Cu electrodes. Curr Opin Electrochem. 2018;9:158–65. 162. Nørskov JK, Rossmeisl J, Logadottir A, Lindqvist L, Kitchin JR, Bligaard T, Jónsson H. Origin of the overpotential for oxygen reduction at a fuel-cell cathode. J Phys Chem B. 2004;108(46):17886–92. (0123456789) 1 3 3456789) 3 (01231 \| https://doi.org/10.1007/s43938-022-00009-y (2022) 2:2 Discover Chemical Engineering Review 163. Chen R, Chen D, Xiao Y. Theoretical scanning of bimetallic alloy for designing efficient N2 electroreduction catalyst. Mater Today Energy. 2021;20:100684. 163. Chen R, Chen D, Xiao Y. Theoretical scanning of bimetallic alloy for designing efficient N2 electroreduction catalyst. Mater Today Energy. 2021;20:100684. 163. Chen R, Chen D, Xiao Y. Theoretical scanning of bimetallic alloy for designing efficient N2 electrored Energy. 2021;20:100684. 163. Chen R, Chen D, Xiao Y. Theoretical scanning of bimetallic alloy for designing efficient N2 electroreduction catalyst. Mater Today Energy. 2021;20:100684. gy 164. Xiao Y, Tang L, Zhang W, Shen C. Theoretical insights into the selective and activity of CuAu catalyst for O2 and CO2 electroreduction. Comput Mater Sci. 2021;192:110402. derian M, Mathew K, Hennig RG, Grob A. Density functional theory study of the electrochemical interface bet and an aqueous electrolyte using an implicit solvent method. J Chem Phys. 2015;142(23):234107. p 65. Sakong S, Naderian M, Mathew K, Hennig RG, Grob A. Density functional theory study of the electrochem Pt electrode and an aqueous electrolyte using an implicit solvent method. J Chem Phys. 2015;142(23):23 166. Rasmussen PB, Holmblad PM, Askgaard T, Ovesen CV, Stoltze P, Nørskov JK, Chorkendorff I. References Enhanced formation of ethylene and alcohols at ambient temperature and pressure d f b d d l d Ch S Ch C y p chemical reduction of carbon dioxide at a copper electrode. J Chem Soc Chem Commun. 1988;1:17–9. pp . Frese KW. Electrochemical reduction of CO2 at intentio y pp 186. Ebaid M, Jiang K, Zhang Z, Drisdell WS, Bell AT, Cooper JK. Production of C2/C3 oxygenates from planar copper nitride-derived mesoporous copper via electrochemical reduction of CO2. Chem Mater. 2020;32(7):3304–11. 187. Hori Y, Takahashi I, Koga O, Hoshi N. Selective formation of C2 compounds from electrochemical reduction of CO2 at a series of copper single crystal electrodes. J Phys Chem B. 2002;106(1):15–7. g y y 88. Kas R, Kortlever R, Milbrat A, Koper MTM, Mul G, Baltrusaitis J. Electrochemical CO2 reduction on Cu2O-deri controlling the catalytic selectivity of hydrocarbons. Phys Chem Chem Phys. 2014;16(24):12194–201. controlling the catalytic selectivity of hydrocarbons. Phys Chem Chem Phys. 2014;16(24):12194–201. 189. Mi Y, Shen S, Peng X, Bao H, Liu X, Luo J. Selective electroreduction of CO2 to C2 products over Cu3N-derived Cu nanowires. ChemElec- controlling the catalytic selectivity of hydrocarbons. Phys Chem Chem Phys. 2014;16(24):12194–201. 189. Mi Y, Shen S, Peng X, Bao H, Liu X, Luo J. Selective electroreduction of CO2 to C2 products over Cu3N-derived Cu nanowires. ChemElec- troChem. 2019;6(9):2393–7. 89. Mi Y, Shen S, Peng X, Bao H, Liu X, Luo J. Selective electroreduction of CO2 to C2 products over Cu3N-derived troChem. 2019;6(9):2393–7.i ; ( ) 190. Veenstra FLP, Martín AJ, Pérez-Ramírez J. Nitride-derived copper modified with indium as a selective and highly stable catalyst for the electroreduction of carbon dioxide. Chemsuschem. 2019;12(15):3501. 191. Liang ZQ, Zhuang TT, Seifitokaldani A, Li J, Huang CW, Tan CS, Li Y, De Luna P, Dinh CT, Hu Y, et al. Copper-on-nitride enhances the stable electrosynthesis of multi-carbon products from co 2. Nat Commun. 2018;9(1):1–8. 192. Yin Z, Chao Yu, Zhao Z, Guo X, Shen M, Li N, Muzzio M, Junrui Li H, Liu HL, et al. Cu3N nanocubes for selective electrochemical reduction of CO2 to ethylene. Nano Lett. 2019;19(12):8658–63.f 2 y 193. Alfonso DR, Tafen DN, Kauffmann DR. First-principles modeling in heterogeneous electrocatalysis. Cataly afen DN, Kauffmann DR. First-principles modeling in heterogeneous electrocatalysis. Catalysts. 2018;8(10):424. 194. Peterson AA, Abild-Pedersen F, Studt F, Rossmeisl J, Nørskov JK. How copper catalyzes the electroreduction of carbon dioxide into hydrocarbon fuels. Energy Environ Sci. 2010;3(9):1311–5. References Opportunities and challenges in the electrocatalysis of CO2 and CO reduction using bifunctional surfaces: a theoretical and experimental study of Au–Cd alloys. J Catal. 2016;343:215–31. 176. Calle-Vallejo F, Koper MT. Accounting for bifurcating pathways in the screening for CO2 reduction catalysts. ACS Catal. 2017;7(10):7346–51. 176. Calle-Vallejo F, Koper MT. Accounting for bifurcating pathways in the screening for CO2 reduction catalysts. ACS Catal. 2017;7(10):7346–51. 177. Nie X, Luo W, Janik MJ, Asthagiri A. Reaction mechanisms of CO2 electrochemical reduction on Cu(111) determined with density func- tional theory. J Catal. 2014;312:108–22. 77. Nie X, Luo W, Janik MJ, Asthagiri A. Reaction mechanisms of CO2 electrochemical reduction on Cu(111) dete tional theory. J Catal. 2014;312:108–22. Jónsson H. Computational study of electrochemical CO2 reduction at transition metal electrodes. Procedia Comput . 78. Hussain J, Skúlason E, Jónsson H. Computational study of electrochemical CO2 reduction at transition metal el Sci. 2015;51:1865–71. ; 179. Luo J, Fan RQ, Wang T, Gao Y, Liu LZ, Yan SH, Zhang ZH. Evaluation of spent pig litter compost as a peat substitute in soilless growth media. Biol Agric Hortic. 2015;31(4):219–29. g 180. Ledezma-Yanez I, Gallent EP, Koper MT, Calle-Vallejo F. Structure-sensitive electroreduction of acetaldehyde to ethanol on copper and its mechanistic implications for CO and CO2 reduction. Catal Today. 2016;262:90–4. 181. Montoya JH, Peterson AA, Nørskov JK. Insights into cc coupling in CO2 electroreduction on copper electrodes. ChemCatChem. 2013;5(3):737–42. Zanetti A, Broekmann P. Electrochemical reduction of CO2 into multicarbon alcohols on activated Cu mesh catalysts (IL) study. ACS Catal. 2017;7(11):7946–56. 82. Rahaman M, Dutta A, Zanetti A, Broekmann P. Electrochemical reduction of CO2 into multicarbon alcohols on a an identical location (IL) study. ACS Catal. 2017;7(11):7946–56. y 183. Ting LR, García-Muelas R, Martín AJ, Veenstra FL, Chen ST, Peng Y, Per EY, Pablo-García S, López N, Pérez-Ramírez J, Yeo BS. Electrochemi- cal reduction of carbon dioxide to 1-butanol on oxide-derived copper. Angew Chem. 2020;132(47):21258–65. ori Y, Murata A, Takahashi R, Suzuki S. Enhanced formation of ethylene and alcohols at ambient temperature and emical reduction of carbon dioxide at a copper electrode. J Chem Soc Chem Commun. 1988;1:17–9. 184. Hori Y, Murata A, Takahashi R, Suzuki S. Enhanced formation of ethylene and alcohols at ambient temperature and pressure in electro- chemical reduction of carbon dioxide at a copper electrode. J Chem Soc Chem Commun. 1988;1:17–9. rata A, Takahashi R, Suzuki S. References y 2 y ; ( ) 209. Hori Y, Kikuchi K, Suzuki S. Production of co and CH4 in electrochemical reduction of CO2 at metal electrodes in aqueous hydrogencar- bonate solution. Chem Lett. 1985;14(11):1695–8. ; ( ) 210. Kuhl KP, Hatsukade T, Cave ER, Abram DN, Kibsgaard J, Jaramillo TF. Electrocatalytic conversion of carbon dioxide to methane and methanol on transition metal surfaces. J Am Chem Soc. 2014;136(40):14107–13.l 211. Schouten KJ, Gallent EP, Koper MT. The influence of pH on the reduction of CO and CO2 to hydrocarbons on copper electrodes. J Elec- troanal Chem. 2014;716:53–7. Murata A, Hori Y. Product selectivity affected by cationic species in electrochemical reduction of CO2 and CO at a C Soc Jpn. 1991;64(1):123–7. 212. Murata A, Hori Y. Product selectivity affected by cationic species in electrochemical reduction of CO2 and CO at a Cu electrode. Bull Chem Soc Jpn. 1991;64(1):123–7. 213 Singh MR Kwon Y Lum Y Ager JW III Bell AT Hydrolysis of electrolyte cations enhances the electrochemical reduction of CO over Ag Soc Jpn. 1991;64(1):123–7. 213. Singh MR, Kwon Y, Lum Y, Ager JW III, Bell AT. Hydrolysis of electrolyte cations enhances the electrochemical reduction of CO2 over Ag and Cu J Am Chem Soc 2016;138(39):13006 12 p ; ( ) 213. Singh MR, Kwon Y, Lum Y, Ager JW III, Bell AT. Hydrolysis of electrolyte cations enhances the electrochemical reduction of CO2 over Ag and Cu. J Am Chem Soc. 2016;138(39):13006–12.if 214. Chen LD, Urushihara M, Chan K, Nørskov JK. Electric field effects in electrochemical CO2 reduction. ACS Catal. 2016;6(10):7133–9.f . Chen LD, Urushihara M, Chan K, Nørskov JK. Electric fie if 2 215. Resasco J, Chen LD, Clark E, Tsai C, Hahn C, Jaramillo TF, Chan K, Bell AT. Promoter effects of alkali metal c reduction of carbon dioxide. J Am Chem Soc. 2017;139(32):11277–87.i 216. Akhade SA, McCrum IT, Janik MJ. The impact of specifically adsorbed ions on the copper-catalyzed electroreduction of CO2. J Electrochem Soc. 2016;163(6):F477–84. 217. Varela AS, Wen J, Reier T, Strasser P. Tuning the catalytic activity and selectivity of Cu for CO2 electroreduction in the presence of halides. ACS Catal. 2016;6(4):2136–44.fl 218. Ghiringhelli LM, Vybiral J, Levchenko SV, Draxl C, Scheffler M. Big data of materials science: critical role of the descriptor. Phys Rev Lett. 2015;114(10):1–5. 219. Peterson AA, Christensen R, Khorshidi A. Addressing uncertainty in atomistic machine learni 2017;19(18):10978–85. 220. References gy ssell AE. Electrocatalysis: theory and experiment at the interface. Phys Chem Chem Phys. 2008;10(25):3607–8. y y p y y 196. Saravanan K, Basdogan Y, Dean J, Keith JA. Computational investigation of CO2 electroreduction on tin oxide and predictions of Ti, V, Nb and Zr dopants for improved catalysis. J Mater Chem A. 2017;5(23):11756–63. Vol:1 :.(123456789 3 Discover Chemical Engineering \| https://doi.org/10.1007/s43938-022-00009-y (2022) 2:2 Review Review 197. Saravanan K, Gottlieb E, Keith JA. Nitrogen-doped nanocarbon materials under electroreduction operating conditions and implications for electrocatalysis of CO2. Carbon. 2017;111:859–66. y 2 198. Koper MTM. Theory of multiple proton–electron transfer reactions and its implications for electrocatalysis 198. Koper MTM. Theory of multiple proton–electron transfer reactions and its implications for electrocatalysis. Chem Sci. 2013;4(7):2710–23. 199. Koper MTM. Activity volcanoes for the electrocatalysis of homolytic and heterolytic hydrogen evolution. J Solid State Electrochem. 2016;20(4):895–9. 199. Koper MTM. Activity volcanoes for the electrocatalysis of homolytic and heterolytic hydrogen evolution. J Solid State Electrochem. 2016;20(4):895–9. 200. Abild-Pedersen F, Greeley J, Studt F, Rossmeisl J, Munter TR, Moses PG, Skúlason E, Bligaard T, Nørskov JK. Scaling properties of adsorp- tion energies for hydrogen-containing molecules on transition-metal surfaces. Phys Rev Lett. 2007;99(1):016105. 201. Montemore MM, Will Medlin J. Scaling relations between adsorption energies for computational screening and design of catalysts. Catal Sci Technol. 2014;4(11):3748–61. 202. Greeley J. Theoretical heterogeneous catalysis: scaling relationships and computational catalyst design. An 2016;7(1):605–35. tical heterogeneous catalysis: scaling relationships and computational catalyst design. Annu Rev Chem Biomol . 203. Govindarajan N, García-Lastra JM, Meijer EJ, Calle-Vallejo F. Does the breaking of adsorption-energy scaling relations guarantee enhanced electrocatalysis? Curr Opin Electrochem. 2018;8:110–7. y p 204. Calle-Vallejo F, Krabbe A, García-Lastra JM. How covalence breaks adsorption-energy scaling relations and solvation restores them. Chem Sci. 2016;8(1):124–30. 205. Shin H, Ha Y, Kim H. 2D covalent metals: a new materials domain of electrochemical CO2 conversion with broken scaling relationship. J Phys Chem Lett. 2016;7(20):4124–9. y 206. Li Y, Sun Q. Recent advances in breaking scaling relations for effective electrochemical conversion o 2016;6(17):1–19. 207. Khorshidi A, Violet J, Hashemi J, Peterson AA. How strain can break the scaling relations of catalysis. Nat Catal. 2018;1(4):263–8.fi 07. Khorshidi A, Violet J, Hashemi J, Peterson AA. How st g y 208. Cheng MJ, Kwon Y, Head-Gordon M, Bell AT. Tailoring metal-porphyrin-like active sites on graphene to improv tivity of electrochemical CO2 reduction. J Phys Chem C. 2015;119(37):21345–52. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Ulissi ZW, Tang MT, Xiao J, Liu X, Torelli DA, Karamad M, Cummins K, Hahn C, Lewis NS, Jaramillo TF, Chan K, Nørskov JK. Machine- learning methods enable exhaustive searches for active bimetallic facets and reveal active site motifs for CO2 reduction. ACS Catal. 2017;7(10):6600–8. ) ao M, Gao W, Jiang Q. Mechanistic insights into the unique role of copper in CO2 electroreduction reactions. Chem ):387–93. 221. Liu SP, Zhao M, Gao W, Jiang Q. Mechanistic insights into the unique role of copper in CO2 electroreduct 2017;10(2):387–93. 222. Ma X, Li Z, Achenie LE, Xin H. Machine-learning-augmented chemisorption model for CO2 electroreduction catalyst screening. J Phys Chem Lett. 2015;6(18):3528–33. ; ( ) 223. Zhong M, Tran K, Min Y, Wang C, Wang Z, Dinh C-T, De Luna P, Zongqian Yu, Rasouli AS, Brodersen P, Sun S, Voznyy O, Tan C-S, Askerka M, Che F, Liu M, Seifitokaldani A, Pang Y, Lo S-C, Ip A, Ulissi Z, Sargent EH. Accelerated discovery of CO2 electrocatalysts using active machine learning. Nature. 2020;581(7807):178–83. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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https://openalex.org/W3010028600	https://escholarship.org/content/qt7575g9sk/qt7575g9sk.pdf?t=pzvuti	English	null	Bone and mineral disorders in pre-dialysis CKD	International urology and nephrology	2,008	cc-by	9,895	UC Irvine UC Irvine Previously Published Works Title Bone and mineral disorders in pre-dialysis CKD Permalink https://escholarship.org/uc/item/7575g9sk Journal International Urology and Nephrology, 40(2) ISSN 0924-8455 Authors Kovesdy, Csaba P Kalantar-Zadeh, Kamyar Publication Date 2008-06-01 DOI 10.1007/s11255-008-9346-7 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Bone and mineral disorders in pre-dialysis CKD Permalink https://escholarship.org/uc/item/7575g9sk Journal International Urology and Nephrology, 40(2) ISSN 0924-8455 Authors Kovesdy, Csaba P Kalantar-Zadeh, Kamyar Publication Date 2008-06-01 DOI 10.1007/s11255-008-9346-7 Copyright Information This work is made available under the terms of a Creative Comm License, availalbe at https://creativecommons.org/licenses/by/4. Peer reviewed UC Irvine UC Irvine Powered by the California Digital Library University of California eScholarship.org eScholarship.org Int Urol Nephrol (2008) 40:427–440 DOI 10.1007/s11255-008-9346-7 REVIEW Bone and mineral disorders in pre-dialysis CKD Csaba P. Kovesdy Æ Kamyar Kalantar-Zadeh Received: 15 January 2008 / Accepted: 29 January 2008 / Published online: 27 March 2008 Springer Science+Business Media B.V. 2008 Received: 15 January 2008 / Accepted: 29 January 2008 / Published online: 27 March 2008 Springer Science+Business Media B.V. 2008 Received: 15 January 2008 / Accepted: 29 January 2008 / Published online: 27 March 2008 Springer Science+Business Media B.V. 2008 Abstract Disorders in calcium, phosphorus, and parathyroid hormone (PTH) are common in chronic kidney disease (CKD) and may be associated with poor outcomes including a higher rate of CKD progression and increased death risk. Although these abnormalities have been examined extensively in patients with CKD stage 5 who are receiving chronic maintenance dialysis, they have not been studied to the same extent at earlier stages of CKD, in spite of the much larger numbers of patients in the early CKD population. We summarize the available literature on outcomes associated with bone and mineral disorders in patients with CKD not yet receiving maintenance dialysis. We have reviewed novel data linking ﬁbro- blast growth factor 23 (FGF-23) to phosphorus and vitamin D homeostasis. More rapid CKD progression is linked to hyperphosphatemia and its associated hyperparathyroidism and vitamin D deﬁciency. Hence, hyperphosphatemia may play a central role in the diverse disorders characterizing CKD. We provide a brief overview of the available treatment recom- mendations for bone and mineral disorders, with an emphasis on areas needing further research. Keywords Chronic kidney disease Parathyroid hormone Hyperphosphatemia Mortality FGF-23 1,25-Dihydroxy-cholecalciferol Keywords Chronic kidney disease Parathyroid hormone Hyperphosphatemia Mortality FGF-23 1,25-Dihydroxy-cholecalciferol Abbreviations CKD Chronic kidney disease PTH Parathyroid hormone FGF-23 Fibroblast growth factor 23 MHD Maintenance hemodialysis MBD Mineral and bone disorder SHPT Secondary hyperparathyroidism GFR Glomerular ﬁltration rate MDRD Modiﬁcation of diet in renal disease CV Cardiovascular AASK African American study of hypertension and kidney Disease 1,25(OH)2D 1,25-Dihydroxycholecalciferol ESRD End stage renal disease C. P. Kovesdy (&) Division of Nephrology, Salem VA Medical Center, 1970 Roanoke Boulevard, Salem, VA 24153, USA e-mail: csaba.kovesdy@va.gov C. P. Kovesdy University of Virginia, Charlottesville, VA, USA K. Kalantar-Zadeh Harold Simmons Center for Kidney Disease Research and Epidemiology, Torrance, CA 90502, USA K. Kalantar-Zadeh Division of Nephrology and Hypertension, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA 90502, USA K. Bone and mineral disorders in pre-dialysis CKD Kalantar-Zadeh David Geffen School of Medicine at UCLA, Torrance, CA 90502, USA C. P. Kovesdy (&) Division of Nephrology, Salem VA Medical Center, 1970 Roanoke Boulevard, Salem, VA 24153, USA e-mail: csaba.kovesdy@va.gov Abbreviations CKD Chronic kidney disease PTH Parathyroid hormone FGF-23 Fibroblast growth factor 23 MHD Maintenance hemodialysis MBD Mineral and bone disorder SHPT Secondary hyperparathyroidism GFR Glomerular ﬁltration rate MDRD Modiﬁcation of diet in renal disease CV Cardiovascular AASK African American study of hypertension and kidney Disease 1,25(OH)2D 1,25-Dihydroxycholecalciferol ESRD End stage renal disease C. P. Kovesdy University of Virginia, Charlottesville, VA, USA K. Kalantar-Zadeh Harold Simmons Center for Kidney Disease Research and Epidemiology, Torrance, CA 90502, USA K. Kalantar-Zadeh Division of Nephrology and Hypertension, Los Angeles Biomedical Research Institute at Harbor-UCLA Medica Center, Torrance, CA 90502, USA 12 428 Int Urol Nephrol (2008) 40:427–440 The kidneys play a pivotal role in this system as the main organs responsible for phosphorus excretion, and abnormalities affecting phosphorus are one of the centerpieces of CKD-MBD. The kidneys play a pivotal role in this system as the main organs responsible for phosphorus excretion, and abnormalities affecting phosphorus are one of the centerpieces of CKD-MBD. The FGF-23 axis The FGF-23 axis As the glomerular ﬁltration rate (GFR) decreases several changes occur that affect phosphorus balance, the most important ones being a decrease in calcitriol level due to deﬁcient 1a hydroxylation [7] (and with consequently lower intestinal calcium absorption, hypocalcemia, and stimulation of PTH production) and a decrease in the ﬁltered amount of phosphorus (with consequent hyperphosphatemia, hypocalcemia and stimulation of PTH and ﬁbroblast growth factor- 23 [FGF-23] production) [8 9]. The higher PTH levels will enhance urinary clearance of phosphorus by lowering proximal tubular reabsorption, thus ensuring normal plasma levels, albeit at the expense of secondary hyperparathyroidism (the classical trade-off hypothesis [10]), and also a higher FGF- 23 level, which in itself inhibits the 1-a hydroxylation of 25-OH-vitamin D, resulting in further lowering of calcitriol levels and more stimulation of PTH production (Fig. 1) [11, 12]. This regulatory mech- anism is unable to compensate for phosphorus retention once the GFR falls below approximately 40 ml/min; this is when a subtle rise in serum phosphorus occurs, albeit mostly without manifest hyperphosphatemia [5]. Frank hyperphosphatemia becomes common once patients with CKD reach the need for dialysis where the lack of substantial kidney function combined with the inefﬁciency of thrice weekly dialysis treatments in facilitating phos- phorus clearance [13] result in a persistent positive phosphate balance unless the amount of absorbed phosphorus is diminished. This review examines the available literature on the association between abnormalities of CKD-MBD and outcomes in the CKD population. While out- comes associated with abnormalities in phosphorus, calcium, and PTH metabolism, including the novel link via FGF-23 will be discussed separately, they often occur simultaneously, and need to be addressed as such in clinical practice. Treatment recommenda- tions in CKD are limited by the paucity of clinical trials, but suggestions for future research are provided based on potential beneﬁts suggested by observa- tional data. Introduction Disorders of mineral and bone metabolism are common in patients with chronic kidney disease (CKD) [1] and have been implicated as a novel risk factor in the high mortality seen in patients receiving maintenance hemodialysis (MHD) therapy [2, 3]. Several abnormalities are grouped under the currently preferred term of CKD mineral and bone disorder (MBD), [4] including secondary hyperparathyroidism (SHPT), renal osteodystrophy, disorders of vitamin D metabolism, hyperphosphatemia, hypo- and hyper- calcemia, and vascular calciﬁcation. While several of the abnormalities characterizing CKD-MBD develop during earlier stages of CKD, the outcomes associ- ated with these abnormalities, along with the potential beneﬁts of their treatment, are much less thoroughly studied in this patient population. The few studies that examined CKD-MBD at earlier stages of CKD have indicated that the adverse outcomes associated with hyperphosphatemia and SHPT in CKD stage 5 are also present at these earlier stages. Furthermore, in patients with CKD who are not yet on dialysis (hereinafter referred to as CKD) mortality is not the only outcome of interest; in these patients the impact of various risk factors on progression of CKD deserves to be assessed separately. The FGF-23 axis Outcomes associated with serum phosphorus levels in CKD Phosphorus is an essential building block of the human body as a component of the bony skeleton, adenosine triphosphate, nucleic acids, phospholipid membranes and blood and urinary buffers [5]. A complex regula- tory system ensures the maintenance of phos- phorus homeostasis under usual circumstances [6]. Much attention has focused on hyperphosphatemia and its consequences in dialysis patients [3], even though they represent only a minority of all patients with CKD [14]. The lesser attention devoted to this issue in patients with CKD may be explained by the 123 Int Urol Nephrol (2008) 40:427–440 429 Mortality and phosphorus level in CKD Fig. 1 Mechanism of action whereby ﬁbroblast growth factor- 23 and parathyroid hormone participate in the regulation of phosphorus and activated vitamin D metabolism Following several studies showing a signiﬁcant association between hyperphosphatemia and mortal- ity in dialysis patients [2, 3], three studies have examined the same issue in patients with CKD, and one in a non-CKD population (Table 1). Kestenbaum et al. [16] examined 3,490 US veterans with CKD and showed that higher phosphorus was associated with higher mortality. The second study was a secondary analysis from the Modiﬁcation of Diet in Renal Disease (MDRD) study: Menon et al. [17] examined 839 mostly non-diabetic patients and showed an association between higher phosphorus and all-cause and cardiovascular (CV) mortality, but the associations were not statistically signiﬁcant. The third study, by Voormolen et al. [18], examined 448 patients with CKD stage 4–5 and found a hazard ratio for all-cause mortality of 1.62 (95% CI: 1.02–2.59) associated with a 1-mg/dl higher phosphorus level. The fourth study was by Tonelli et al. [19], who examined 4,127 participants with normal kidney function enrolled in the Cholesterol and Recurrent Events study, and showed that higher plasma phos- phorus level was associated with higher all-cause mortality, CV mortality, fatal or non-fatal myocardial infarction, and new onset congestive heart failure. The putative mechanism(s) behind the observed associations could be the calciﬁcation-inducing effects of phosphorus on the vascular bed [15, 20], or the concomitant deleterious effects of other factors linked to hyperphosphatemia, such as secondary hyperparathyroidism [3]. Fig. Outcomes associated with serum phosphorus levels in CKD 1 Mechanism of action whereby ﬁbroblast growth factor- 23 and parathyroid hormone participate in the regulation of phosphorus and activated vitamin D metabolism lack of available data sources, but also by the differences in the bone–mineral milieu between patients with CKD and those on dialysis: as detailed above, serum phosphorus levels in patients with CKD are in general much lower until CKD reaches stage 5. In vitro data suggest that the phenotypic transforma- tion of aortic smooth muscle cells into osteoblast-like cells that is thought to be involved in the soft tissue and cardiovascular calciﬁcation mediated by hyperphos- phatemia occurs at ambient phosphorus concentrations of about 6 mg/dl and above [15]. Such elevated plasma phosphorus levels are unusual in CKD, and it is hence possible that the cardiovascular effects of hyperphos- phatemia are more subtle at earlier stages of CKD, and thus more difﬁcult to detect. A distinct challenge in CKD is the presence of competing end points: while mortality is still of major interest, progression of CKD is also regarded as a separate end point and is in fact the main focus of nephrologists’ clinical practice. Phosphorus and progression of CKD Three observational studies have examined the asso- ciation between higher phosphorus and progression of CKD (Table 1). We examined the association between phosphorus level and the incidence of dialysis or doubling of serum creatinine in 985 male US veterans with CKD and found that higher phosphorus was associated with a higher incidence of the renal end point (Fig. 2); a 1-mg/dl higher phosphorus level was associated with an adjusted hazard ratio of 1.29 (95% CI: 1.12–1.48, P \ 0.001) [21]. A second study by Norris et al. Phosphorus and progression of CKD [22] examined risk factors for progression of CKD in 1,094 black patients enrolled in the African American Study of 12 3 3 430 Int Urol Nephrol (2008) 40:427–440 Table 1 Outcomes associated with plasma phosphorus level in chronic kidney disease End point Study reference Number of patients Kidney function Main results Comments All-cause mortality [16] 3,490 Mean creatinine clearance 47.2 ml/min A 1-mg/dl higher PO4 level associated with a hazard ratio for all-cause mortality of 1.23 (95% CI: 1.12–1.36) Patients were mostly males and mostly Caucasian [17] 839 Mean GFR: 33 ± 12 ml/min/ 1.72 m2 A 1-mg/dl higher PO4 level associated with a hazard ratio for all-cause mortality of 1.10 (95% CI: 0.86–1.40); and for CV mortality of 1.27 (0.94–1.73) Low mortality rate, limited external validity [18] 448 Mean estimated GFR: 13 ± 5.4 ml/min/1.72 m2 A 1-mg/dl higher PO4 level associated with a hazard ratio for all-cause mortality of 1.62 (95% CI: 1.02–2.59) Only 6.7% of patients died [19] 4,127 Normal or nearly normal kidney function (no overall mean value available) A 1-mg/dl higher PO4 level associated with a hazard ratio for all-cause mortality of 1.27 (95% CI: 1.02–1.58) Also showed higher associations for congestive heart failure and cardiovascular events Progression of CKD [21] 985 Mean estimated GFR: 37.0 ± 17.5 ml/min/1.72 m2 A 1-mg/dl higher PO4 level associated with a hazard ratio of renal composite outcome of 1.29 (95% CI: 1.12–1.48) All patients were males [22] 1,094 Mean GFR: 46.4 ± 13.6 ml/min/ 1.72 m2 A 0.3-mg/dl higher PO4 level associated with a hazard ratio of renal composite outcome of 1.07 (95% CI: 1.00–1.15) Association maintained when composite of renal events and death was examined as the end point [18] 448 Mean estimated GFR: 13 ± 5.4 ml/min/1.72 m2 A 1-mg/dl higher PO4 level associated with a 0.178 ml/min/month higher progression (95% CI: 0.082–0.275) Examined ordinary least squares regression slopes of estimated GFR as a marker of progression GFR: glomerular ﬁltration rate 12 Int Urol Nephrol (2008) 40:427–440 431 Fig. Phosphorus and progression of CKD 2 Hazard ratio (95% conﬁdence interval) of the compos- ite outcome of end stage renal disease and doubling of serum creatinine associated with quartiles of serum phosphorus, unadjusted and after adjustment for age, race, systolic and diastolic blood pressure, diabetes, smoking status, estimated glomerular ﬁltration rate, serum albumin, calcium, bicarbonate, blood urea nitrogen, hemoglobin, 24-h urine protein, and use of calcium-containing phosphate binders and angiotensin-con- verting enzyme inhibitors/angiotensin II receptor blockers. The group with serum phosphorus 3.3–3.8 mg/dl served as a reference. Based on data presented in Schwarz et al. [21] lowering these outcomes, but this would have to be proven in clinical trials ﬁrst. Strategies to lower plasma phosphorus in CKD include dietary phosphate restriction and the appli- cation of medications that inhibit the intestinal absorption of phosphorus. Dietary protein restriction (with concomitant restriction of phosphate intake) is already one of the strategies applied to alleviate progression of CKD [30]. Medications that inhibit the absorption of phosphorus include phosphate binders (calcium, magnesium, iron and lanthanum salts, and sevelamer hydrochloride) and inhibitors of intestinal mucosal phosphate transport (nicotinamide [31]) (Table 2). None of these medications has been formally approved for therapy of hyperphosphatemia in CKD; thus, their ‘‘off-label’’ use would be based on data and experience drawn mostly from dialysis patients. It is also worth remembering that the application of either one of the above treatments should be applied with the understanding that there is currently no consensus about what an ideal plasma phosphorus level should be in CKD. The K/DOQI (Kidney Disease Outcomes Quality Initiative) guide- lines on bone and mineral disorders recommend a plasma phosphorus concentration of 2.7–4.5 mg/dl [8], which in fact corresponds to what is currently regarded as the ‘‘normal’’ range of plasma phospho- rus, but it does not address the results of more recent studies that suggest a graded increase in the risk of adverse outcomes associated with higher levels of phosphorus, even within this ‘‘normal’’ range (Table 1). One could also argue that it is not only the plasma phosphorus level that should serve as a therapeutic target in CKD, but also the plasma PTH level and/or the amount of phosphorus excreted in the urine. Further research is warranted to clarify these issues. Another issue worth discussing is the safety of the calcium containing medications in CKD; we will discuss this topic in the following section. Fig. Phosphorus and progression of CKD 2 Hazard ratio (95% conﬁdence interval) of the compos- ite outcome of end stage renal disease and doubling of serum creatinine associated with quartiles of serum phosphorus, unadjusted and after adjustment for age, race, systolic and diastolic blood pressure, diabetes, smoking status, estimated glomerular ﬁltration rate, serum albumin, calcium, bicarbonate, blood urea nitrogen, hemoglobin, 24-h urine protein, and use of calcium-containing phosphate binders and angiotensin-con- verting enzyme inhibitors/angiotensin II receptor blockers. The group with serum phosphorus 3.3–3.8 mg/dl served as a reference. Based on data presented in Schwarz et al. [21] Hypertension and Kidney Disease (AASK) and found phosphorus level to be one of several independent predictors of progressive CKD. The third study by Voormolen et al. [18] described an association between higher serum phosphorus levels and faster decline in renal function (by examining slopes of estimated GFR) in 432 patients with CKD. The plausibility of these ﬁndings is strengthened by the results of studies in patients with CKD [23, 24] and in laboratory animals [25–27], which showed an atten- uation of the progression of CKD after dietary restriction of phosphorus. Several mechanisms could be responsible for the observed associations: renal parenchymal calciﬁcation, a deleterious effect of SHPT, hemodynamic alterations, and derangements in cellular energy metabolism have been suggested as plausible explanations [27–29]. Serum calcium level and calcium intake in CKD The rationale for lowering phosphorus level in CKD is not necessarily to treat frank hyperphosphatemia (given the relative infrequency of it), but rather to treat secondary hyperparathyroidism. Given the asso- ciation of higher phosphorus levels with mortality and progression of CKD, it is also possible that lowering plasma phosphorus may be beneﬁcial in Before the early-to-mid 1980s hyperphosphatemia was managed with aluminum-containing phosphorus bind- ers and dietary phosphorus restriction [32]. Following the emergence of data on the untoward consequences of aluminum-based binders (dementia, refractory anemia, and osteomalacia) [33, 34], calcium-based 12 3 Int Urol Nephrol (2008) 40:427–440 432 Table 2 Phosphorus binders that may be used off-label in a chronic kidney disease population not on dialysis Pros Cons Calcium carbonate (TumsTM) Most inexpensive, antacid properties useful for reﬂux and peptic ulcer disease High calcium load Calcium acetate (PhosLoTM) Relatively inexpensive, less GI calcium absorption compared with Ca carbonate May contribute to calcium load and worsens vascular calciﬁcation Aluminum hydroxide (AmphogelTM) Most effective/potent binder, inexpensive Aluminum toxicity. Should not be used as maintenance treatment Sevelamer-HCl (RenagelTM) Calcium and metal-free binder; may have ancillary beneﬁts beyond phosphorus control, such as lipid lowering, uric acid lowering, anti-inﬂammatory effect Expensive, may worsen metabolic acidosis and hyperchlorhydria, GI symptoms such as diarrhea. High pill burden Sevelamer carbonate (RenVelaTM) Calcium and metal-free binder; may have ancillary beneﬁts beyond phosphorus control (see above), no metabolic acidosis. Improved GI tolerance Probably expensive. Limited/no post- marketing experience Lanthanum carbonate (FosrenolTM) No calcium load. Low pill burden Long-term safety of lanthanum accumulation unknown. Not allowed in liver disease. Chalky taste Magnesium based (MagnebindTM) No calcium load. Anti-constipating Potential for hypermagnesemia, diarrhea Trivalent iron-containing binders No calcium load Limited information available Niceritrol (inhibitors of intestinal phosphate transport) Different mechanism of action, inexpensive Poorly tolerated. Limited data available as a binder Compared with MHD patients, much less is known about the role of calcium intake in CKD. A recent small clinical trial in patients with CKD compared coronary calciﬁcation in untreated hyperphosphatemic patients and in patients treated with calcium carbonate or sevelamer hydrochloride [46]. This study showed less progression of coronary calciﬁcation with calcium carbonate compared with untreated patients, but the progression was lowest in the group treated with sevelamer hydrochloride; this latter group also showed no signiﬁcant changes in blood lipid levels [46]. Secondary hyperparathyroidism in CKD 3); Elevated PTH levels develop early in the course of CKD and progressively rise with advancing stages of this disease [1, 48–53]. SHPT develops as a result of a combination of events: deﬁciency of 1,25-dihy- droxycholecalciferol (1,25(OH)2D) [1, 7], decreased expression of the vitamin D receptor [54] and the calcium-sensing receptor [55], hyperphosphatemia [56], hypocalcemia [57], and PTH resistance [58] are believed to contribute to it. More recently, ﬁbroblast growth factor 23 (FGF-23) has emerged as a new regulator that may also play an important role in PTH regulation (Fig. 1) [11]. In addition to these CKD-speciﬁc factors, PTH levels are also inﬂuenced by various demographic [59, 60], anthro- pometric [61], and co-morbidity characteristics [62]. Secondary hyperparathyroidism is associated with a variety of complications including bone disease [63–66], uremic pruritus [67], refractory anemia [68], and cognitive and sexual dysfunction [69, 70]. In patients receiving MHD, SHPT has been associated with increased cardiovascular morbidity [48, 71–73] and mortality [2, 3]. The association of SHPT with mortality or with progression of kidney disease in CKD has not been well characterized. In a recent study of 515 male patients with CKD stages 3–5 (none receiving MHD), higher PTH level was associated with increased all-cause mortality (Fig. 3); The mechanism behind the higher mortality observed in patients with higher PTH remains speculative. Elevated PTH levels have been shown to cause a wide range of cardiovascular, metabolic, hematologic, and immunologic abnormalities. These include lower cardiac contractility, myocardial cal- cium deposition, hypertrophy and ﬁbrosis and vascular calciﬁcation, [78] mitral annular calciﬁca- tion, [79] impaired insulin sensitivity [80] and glucose intolerance, [81] abnormal lipid metabolism, [82] bone marrow ﬁbrosis [68] with ineffective erythropoiesis [83–85], and abnormal immune func- tion [86]. Animal models showed that PTH increases myocardial calcium content and adversely affects energy utilization in myocardial tissue [87] and that it could play a role in myocardial ﬁbrosis [88], impaired vasodilatation [89], and blood pressure-independent wall thickening of the intramyocardial arterioles [90]. In a population-based cross-sectional study, a higher PTH level was predictive of coronary heart disease [91]. It is important to emphasize that the link between SHPT and mortality in CKD remains associative; a cause–effect relationship can only be proven by clinical trials showing a beneﬁt from lowering PTH levels. Thus far, we are unaware of any plans for such trials. Fig. Serum calcium level and calcium intake in CKD It thus appears from this study that untreated hyperphospha- temia portends a poor prognosis, which can be improved by the administration of a calcium-contain- ing phosphate binder. The ﬁnding of an even better outcome associated with using a non-calcium-con- taining binder may suggest that calcium could have had an additional deleterious effect that diminished its beneﬁt of phosphate lowering. Further studies would be needed to clarify outcomes in the context of using other phosphate binders, including calcium-contain- ing products with a lower absorbable calcium-load, and also different non-calcium-containing binders in order to differentiate between the potential effects of some binders that are unrelated to phosphate lowering [47]. medications became the binders of choice. These agents also offered the added beneﬁt of further suppressing parathyroid hormone production [35]. Subsequently, vascular calciﬁcation was described to be prevalent in MHD, and it was found to be associated with poor survival in these patients [36, 37]. Once vascular calciﬁcation was found to be associated with higher serum calcium level and higher calcium intake [37–39], and once higher serum calcium levels were also associated with higher mortality [2, 3] in CKD patients receiving MHD, the use of calcium-containing binders in this patient population diminished, although their role continues to be a matter of intense debate [40]. Contrary to studies of patients receiving MHD, observational studies in CKD have in general failed to establish an association between higher serum calcium levels and coronary calciﬁcation [41–45]. The reason for the discrepant ﬁndings between studies in CKD and those examining MHD patients are unclear. One explanation could be the better calcium homeostasis provided by residual kidney function and the lack of additional calcium intake from dialysate sources in CKD; hypercalcemia is in fact uncommon, even in advanced stages of CKD [1]. 123 Int Urol Nephrol (2008) 40:427–440 433 Secondary hyperparathyroidism in CKD a serum intact PTH level of [65 pg/ml (compared with B65 pg/ml) was associated with an adjusted hazard ratio for all cause mortality of 1.59 (95% CI: 1.02–2.49) [74]. Interestingly, the higher risk of mortality in this study was already evident at PTH levels above the upper limit of the normal range ([65 pg/ml). Currently, the K-DOQI bone and min- eral metabolism guidelines recommend an intact PTH level of 70–110 pg/ml for patients with CKD stage 4 [8]. This is an opinion-based guideline, and the results of the above study suggest that higher PTH levels, even within this range, could be associated with worse mortality; further research will be needed to clarify what the ideal treatment targets should be for SHPT. The lack of a lower threshold level for mortality risk in the above study is also in contrast to studies in MHD patients, in which the association of PTH level with mortality was U-shaped [3, 75]. A signiﬁcant proportion of the higher mortality seen with lower PTH levels in MHD patients was, however, related to confounding by markers of malnutrition and inﬂammation [3], which themselves have been linked to higher mortality [76, 77]. Elevated PTH levels develop early in the course of CKD and progressively rise with advancing stages of this disease [1, 48–53]. SHPT develops as a result of a combination of events: deﬁciency of 1,25-dihy- droxycholecalciferol (1,25(OH)2D) [1, 7], decreased expression of the vitamin D receptor [54] and the calcium-sensing receptor [55], hyperphosphatemia [56], hypocalcemia [57], and PTH resistance [58] are believed to contribute to it. More recently, ﬁbroblast growth factor 23 (FGF-23) has emerged as a new regulator that may also play an important role in PTH regulation (Fig. 1) [11]. In addition to these CKD-speciﬁc factors, PTH levels are also inﬂuenced by various demographic [59, 60], anthro- pometric [61], and co-morbidity characteristics [62]. Secondary hyperparathyroidism is associated with a variety of complications including bone disease [63–66], uremic pruritus [67], refractory anemia [68], and cognitive and sexual dysfunction [69, 70]. In patients receiving MHD, SHPT has been associated with increased cardiovascular morbidity [48, 71–73] and mortality [2, 3]. The association of SHPT with mortality or with progression of kidney disease in CKD has not been well characterized. In a recent study of 515 male patients with CKD stages 3–5 (none receiving MHD), higher PTH level was associated with increased all-cause mortality (Fig. Treatment of SHPT in CKD omplex pathophysiology of SHPT (vide supra) several options for correcting this abnormality. ave already discussed the potential role of horus lowering in the treatment of SHPT in the n about phosphorus control; this beneﬁt could ectly related to a phosphorus-lowering effect in se of non-calcium-containing medications, with ded calcium-related effect provided by calcium- ning binders. Treatment of SHPT remains, heless, an ‘‘off-label’’ indication for phosphate medications. In common practice secondary parathyroidism in MHD has been managed by ovision of supplemental calcium (in the form of upplementation and through dialysate calcium) In spite of the above plethora of various therapeutic options, presently only activated vitamin D and its analogs are explicitly approved as a treatment for SHPT in CKD. Administration of activated vitamin D and its analogs in MHD patients has been associated with improved survival compared with patients not receiving such treatments [3, 106, 107]. Similar studies in patients with CKD have not been available until recently. In a study of 520 male patients with CKD the administration of 0.25 mcg/day of oral calcitriol was associated with signiﬁcantly better all- cause mortality, even after adjustment for multiple potential confounders [108]. The multivariable adjusted incidence rate ratio of all-cause mortality in 3 Recommended supplementation schedule for 25(OH) n D deﬁciency and insufﬁciency in CKD stages 3 and 4. d from the National Kidney Foundation Clinical Practice Guidelines for Bone Metabolism and Disease in Chronic Kidney Disease [8] 25(OH) level, nmol/l) Deﬁnition Ergocalciferol dose (Vitamin D2) ) Severe vitamin D deﬁciency 50,000 IU/week orally 9 12 weeks; then monthly OR 500,000 IU as single intramuscular dose 12–37) Mild vitamin D deﬁciency 50,000 IU/week 9 4 weeks, then 50,000 IU/month (40–75) Vitamin D insufﬁciency 50,000 IU/month orally In spite of the above plethora of various therapeutic options, presently only activated vitamin D and its analogs are explicitly approved as a treatment for SHPT in CKD. Administration of activated vitamin D and its analogs in MHD patients has been associated with improved survival compared with patients not receiving such treatments [3, 106, 107]. Similar studies in patients with CKD have not been available until recently. In a study of 520 male patients with CKD the administration of 0.25 mcg/day of oral calcitriol was associated with signiﬁcantly better all- cause mortality, even after adjustment for multiple potential confounders [108]. Progression of CKD and SHPT The effect of SHPT on the progression of CKD is unclear. Animal studies have suggested that PTH may have an effect on podocytes [92, 93], which express PTH receptors [94, 95]. In a remnant kidney model PTH accelerated progression of CKD in animals that received a high protein diet, and parathyroidectomy attenuated the increase in serum creatinine [96]. Data in humans on the role of SHPT in the progression of CKD are even sparser. The study by Kovesdy et al. [74] detailed above found a signiﬁcant association with a higher MHD initiation rate only in unadjusted models, but this association became nonsigniﬁcant once adjusting for confound- ing variables. No prospective trials have examined whether lowering PTH levels in CKD can retard the progressive loss of kidney function. Mortality and activated vitamin D in CKD Treatment of SHPT in CKD Secondary hyperparathyroidism in CKD 3 Hazard ratios of all-cause mortality associated with different levels of intact parathyroid hormone level, unadjusted (Model 1), and after adjustment for case mix variables (age, race, body mass index, systolic and diastolic blood pressure, smoking status, comorbidity index, diabetes mellitus and use of activated vitamin D, calcium containing and non-calcium containing phosphate binders; Model 2), and case mix variables plus laboratory parameters (estimated glomerular ﬁltration rate, calcium, phosphorus, albumin, cholesterol, hemoglobin, white blood cell count, percentage of lympho- cytes in white blood cells and 24-h urine protein; Model 3). Based on data presented in Kovesdy et al. [74] Fig. 3 Hazard ratios of all-cause mortality associated with different levels of intact parathyroid hormone level, unadjusted (Model 1), and after adjustment for case mix variables (age, race, body mass index, systolic and diastolic blood pressure, smoking status, comorbidity index, diabetes mellitus and use of activated vitamin D, calcium containing and non-calcium containing phosphate binders; Model 2), and case mix variables plus laboratory parameters (estimated glomerular ﬁltration rate, calcium, phosphorus, albumin, cholesterol, hemoglobin, white blood cell count, percentage of lympho- cytes in white blood cells and 24-h urine protein; Model 3). Based on data presented in Kovesdy et al. [74] 12 3 123 Int Urol Nephrol (2008) 40:427–440 434 and active vitamin D; the latter initially in the form of synthetic calcitriol (in the US) and alfacalcidol (in Europe) [97–99], and more recently in the form of various analogs of activated vitamin D such as paricalcitol, doxercalciferol [100], and maxacalcitol [101]. Our armamentarium was further diversiﬁed with the recent introduction of the ﬁrst calcium- sensing receptor agonist, cinacalcet [102]. Yet another therapeutic option could be the supplemen- tation of 25(OH) vitamin D [103], the levels of which are low in patients with CKD [104], and the administration of which was able to suppress PTH production in vitro by virtue of direct stimulation of the vitamin D receptor and a slow tissue-level 1a hydroxylation [105]. Current guidelines recommend routine assessment of 25(OH) vitamin D and its replacement in the case of deﬁciency (Table 3) [8]. Progression of CKD and SHPT Progression of CKD and SHPT Activated vitamin D therapy and progression of CKD treated versus untreated patients was 0.35 (0.23–0.54, P \ 0.001) and for combined death and dialysis initiation it was 0.46 (0.35–0.61); this study also found a non-signiﬁcant trend between calcitriol treat- ment and a lower incidence of end stage renal disease (ESRD) [108]. The above beneﬁts were present in all the studied subgroups, including patients with lower pre-treatment PTH, and higher calcium and phospho- rus levels. Similar ﬁndings were reported in another study examining the association of calcitriol therapy in mostly male patients with CKD [109]. The application of activated vitamin D in CKD has been subject to concerns over their potential to hasten the decline of kidney function [99], which has been attributed to hypercalciuria and nephrocalcinosis, although hyperphosphatemia could also play a role [21]. Studies employing lower (non-hypercalcemic) doses of calcitriol did not show worsened renal outcomes [121]; in fact, one study indicated a tendency towards slower progression of CKD in a group treated with 0.25 mcg/day of calcitriol com- pared with placebo [122]. A study of 520 male patients with CKD examined the association between calcitriol therapy and the incidence of ESRD, and found a tendency towards a lower incidence of ESRD in the calcitriol-treated group [108], which raises the possibility of a renoprotective effect. Such an effect is indeed conceivable since therapy with paricalcitol, a selective vitamin D analog, has been shown to lower proteinuria [123]. The mechanism of a renoprotective effect from activated vitamin D could be related to their inhibition of cell proliferation [124] and inﬂammation [125, 126], or to suppression of renin production [127]. Prospective studies examining the renoprotective effect of activated vitamin D have not yet been performed in patients with CKD. y p The mechanism of action behind the higher survival associated with activated vitamin D therapy in the aforementioned studies remains unclear. As detailed earlier in this article, SHPT in itself is associated with higher cardiovascular morbidity [48, 71] and mortality [2, 3], which could explain why suppression of PTH concentrations with acti- vated vitamin D would lead to lower mortality. The impact of activated vitamin D treatment may never- theless be much wider ranging. Activated vitamin D therapy and progression of CKD The vitamin D receptor is ubiquitous, and its stimulation with vitamin D analogs has been shown to directly impact on the cardiovascular system by inhibiting the production of proteins implicated in arterial calciﬁ- cation [110–112], by stimulating the production of proteins that are inhibitors of arterial calciﬁcation [110, 113], by inhibiting the production of cytokines that are involved in calciﬁcation and atheroma formation [114, 115] and stimulating the production of cytokines that are inhibiting it [116, 117], and by preventing thrombosis [118]. Furthermore, activated vitamin D deﬁciency was associated with higher all- cause and cardiovascular mortality in a large cohort of hemodialysis patients [119], and lower 1,25(OH)2 vitamin D levels have been associated with worsened coronary calciﬁcation [120], also suggesting a PTH- independent link between vitamin D and survival. Treatment of SHPT in CKD The multivariable adjusted incidence rate ratio of all-cause mortality in The complex pathophysiology of SHPT (vide supra) offers several options for correcting this abnormality. We have already discussed the potential role of phosphorus lowering in the treatment of SHPT in the section about phosphorus control; this beneﬁt could be directly related to a phosphorus-lowering effect in the case of non-calcium-containing medications, with an added calcium-related effect provided by calcium- containing binders. Treatment of SHPT remains, nevertheless, an ‘‘off-label’’ indication for phosphate binder medications. In common practice secondary hyperparathyroidism in MHD has been managed by the provision of supplemental calcium (in the form of oral supplementation and through dialysate calcium) Table 3 Recommended supplementation schedule for 25(OH) vitamin D deﬁciency and insufﬁciency in CKD stages 3 and 4. Adapted from the National Kidney Foundation Clinical Practice Guidelines for Bone Metabolism and Disease in Chronic Kidney Disease [8] Serum 25(OH) level, ng/ml (nmol/l) Deﬁnition Ergocalciferol dose (Vitamin D2) \5 (12) Severe vitamin D deﬁciency 50,000 IU/week orally 9 12 weeks; then monthly OR 500,000 IU as single intramuscular dose 5–15 (12–37) Mild vitamin D deﬁciency 50,000 IU/week 9 4 weeks, then 50,000 IU/month 16–30 (40–75) Vitamin D insufﬁciency 50,000 IU/month orally Practice Guidelines for Bone Metabolism and Disease in Chronic Kidney Disease [8] Int Urol Nephrol (2008) 40:427–440 435 References 18. Voormolen N, Noordzij M, Grootendorst DC et al (2007) High plasma phosphate as a risk factor for decline in renal function and mortality in pre-dialysis patients. Nephrol Dial Transplant 22:2909–2916 1. Levin A, Bakris GL, Molitch M et al (2007) Prevalence of abnormal serum vitamin D, PTH, calcium, and phos- phorus in patients with chronic kidney disease: results of the study to evaluate early kidney disease. Kidney Int 71:31–38 19. Tonelli M, Sacks F, Pfeffer M et al (2005) Relation between serum phosphate level and cardiovascular event rate in people with coronary disease. Circulation 112:2627–2633 2. Block GA, Klassen PS, Lazarus JM et al (2004) Mineral metabolism, mortality, and morbidity in maintenance hemodialysis. J Am Soc Nephrol 15:2208–2218 20. Chen NX, O’Neill KD, Duan D, Moe SM (2002) Phos- phorus and uremic serum up-regulate osteopontin expression in vascular smooth muscle cells. Kidney Int 62:1724–1731 3. Kalantar-Zadeh K, Kuwae N, Regidor DL et al (2006) Survival predictability of time-varying indicators of bone disease in maintenance hemodialysis patients. Kidney Int 70:771–780 21. Schwarz S, Trivedi BK, Kalantar-Zadeh K, Kovesdy CP (2006) Association of disorders in mineral metabolism with progression of chronic kidney disease. Clin J Am Soc Nephrol 1:825–831 4. Moe S, Drueke T, Cunningham J et al (2006) Deﬁnition, evaluation, and classiﬁcation of renal osteodystrophy: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int 69:1945–1953 22. Norris KC, Greene T, Kopple J et al (2006) Baseline predictors of renal disease progression in the African American study of hypertension and kidney disease. J Am Soc Nephrol 17:2928–2936 y 5. Yu AS (2004) Renal transport of calcium, magnesium and phosphate. In: Brenner BM (ed) Brenner & Rector’s the kidney, 7th edn. Saunders, Philadelphia 23. Barsotti G, Morelli E, Giannoni A et al (1983) Restricted phosphorus and nitrogen intake to slow the progression of chronic renal failure: a controlled trial. Kidney Int Suppl 16:S278–S284 6. Yangawa N, Nakhoul F, Kurokawa K, Lee DB (1994) Physiology of phosphorus metabolism. In: Narins RG (ed) Maxwell and Kleeman’s clinical disorders of ﬂuid and electrolyte metabolism, 5th edn. McGraw-Hill, pp 307–371 24. Maschio G, Oldrizzi L, Tessitore N et al (1982) Effects of dietary protein and phosphorus restriction on the pro- gression of early renal failure. Kidney Int 22:371–376 7. Portale AA, Booth BE, Tsai HC, Morris RC Jr (1982) Reduced plasma concentration of 1,25-dihydroxyvitamin D in children with moderate renal insufﬁciency. Conclusions The various disorders that characterize CKD MBD are common in CKD. Recent evidence suggests that both hyperphosphatemia and SHPT are associated with deleterious outcomes in CKD, similar to what has been described in patients receiving MHD. The impact of hypercalcemia and calcium intake on outcomes in CKD is still unclear. Lowering plasma phosphorus levels in CKD can be beneﬁcial in treating SHPT, and could become an additional therapy to lower mortality and to alleviate progres- sive loss of kidney function. Treatment of SHPT with calcitriol is associated with lower all-cause mortality, and may also have a renoprotective effect. In spite of the uniformity of the observational studies in MHD and at the earlier stages of CKD, and in spite of the plausible mechanisms of action detailed above, there have been no attempts to examine the beneﬁt of activated vitamin D in randomized controlled trials. It is also unclear, as detailed above, how much of the beneﬁt observed with activated vitamin D therapy can be ascribed to the lowering of PTH, and thus it is unclear whether similar beneﬁts can be expected from other types of therapy. Several questions remain unanswered. It is unclear what the net beneﬁt is from therapeutic agents that treat one aspect of CKD-MBD while worsening another. Such effects could occur when treating 12 3 3 Int Urol Nephrol (2008) 40:427–440 436 hyperphosphatemia with calcium-containing binders (which could have the undesirable effect of hyper- calcemia), or when treating SHPT with activated vitamin D (undesirable effects: hypercalcemia and hyperphosphatemia) or calcium receptor sensitizing agents (undesirable effect with cinacalcet in CKD: hyperphosphatemia [128]). It is also unclear what outcomes are associated with the use of 25(OH) vitamin D in the treatment of SHPT in CKD. More research is desirable to strengthen the conclusions of observational studies linking CKD-MBD to unfavor- able outcomes in CKD, and randomized controlled trials will be needed before any of the therapies mentioned can be recommended as a means of improving mortality or progression of kidney disease in CKD. 8. Eknoyan G, Levin A, Levin N (2003) Bone metabolism and disease in chronic kidney disease. Am J Kidney Dis 42:1–201 9. Gupta A, Winer K, Econs MJ et al (2004) FGF-23 is elevated by chronic hyperphosphatemia. J Clin Endocri- nol Metab 89:4489–4492 10. Bricker NS (1972) On the pathogenesis of the uremic state. An exposition of the ‘‘trade-off hypothesis’’. N Engl J Med 286:1093–1099 11. Conclusions Fukagawa M, Kazama JJ (2006) FGF23: its role in renal bone disease. Pediatr Nephrol 21:1802–1806 12. Gutierrez O, Isakova T, Rhee E et al (2005) Fibroblast growth factor-23 mitigates hyperphosphatemia but accentuates calcitriol deﬁciency in chronic kidney dis- ease. J Am Soc Nephrol 16:2205–2215 13. Mucsi I, Hercz G (1998) Control of serum phosphate in patients with renal failure—new approaches. Nephrol Dial Transplant 13:2457–2460 14. K/DOQI (2002) Clinical practice guidelines for chronic kidney disease: evaluation, classiﬁcation, and stratiﬁca- tion. Am J Kidney Dis 39:S1–S266 15. Jono S, McKee MD, Murry CE et al (2000) Phosphate regulation of vascular smooth muscle cell calciﬁcation. Circ Res 87:E10–E17 Acknowledgements Conﬂict of interest Csaba P. Kovesdy and Kamyar Kalantar-Zadeh have received grants and/or honoraria from Amgen (manufacturer of SensiparTM), Abbott (manufacturer of CalcijexTM and ZemplarTM), Genzyme (manufacturer of HectorolTM, RenagelTM and RenvelaTM), and/or Shire (manufacturer of FosrenolTM). 16. Kestenbaum B, Sampson JN, Rudser KD et al (2005) Serum phosphate levels and mortality risk among people with chronic kidney disease. J Am Soc Nephrol 16:520– 528 17. Menon V, Greene T, Pereira AA et al (2005) Relationship of phosphorus and calcium—phosphorus product with mortality in CKD. Am J Kidney Dis 46:455–463 References Kidney Int 21:627–632 25. Ibels LS, Alfrey AC, Haut L, Huffer WE (1978) Preser- vation of function in experimental renal disease by 12 3 Int Urol Nephrol (2008) 40:427–440 437 dietary restriction of phosphate. N Engl J Med 298: 122–126 dietary restriction of phosphate. N Engl J Med 298: 122–126 and without chronic kidney disease. Kidney Int 68: 1258–1266 26. Karlinsky ML, Haut L, Buddington B et al (1980) Preser- vation of renal function in experimental glomerulone- phritis. Kidney Int 17:293–302 43. Qunibi WY, Abouzahr F, Mizani MR et al (2005) Car- diovascular calciﬁcation in Hispanic Americans (HA) with chronic kidney disease (CKD) due to type 2 diabe- tes. Kidney Int 68:271–277 27. Lumlertgul D, Burke TJ, Gillum DM et al (1986) Phos- phate depletion arrests progression of chronic renal failure independent of protein intake. Kidney Int 29:658– 666 44. Russo D, Palmiero G, De Blasio AP et al (2004) Coro- nary artery calciﬁcation in patients with CRF not undergoing dialysis. Am J Kidney Dis 44:1024–1030 28. Kraus ES, Cheng L, Sikorski I, Spector DA (1993) Effect of phosphorus restriction on renal response to oral and intravenous protein loads in rats. Am J Physiol 264: F752–F759 45. Tomiyama C, Higa A, Dalboni MA et al (2006) The impact of traditional and non-traditional risk factors on coronary calciﬁcation in pre-dialysis patients. Nephrol Dial Transplant 21:2464–2471 29. Ritz E, Gross ML, Dikow R (2005) Role of calcium– phosphorous disorders in the progression of renal failure. Kidney Int Suppl 99:S66–S70 46. Russo D, Miranda I, Ruocco C et al (2007) The pro- gression of coronary artery calciﬁcation in predialysis patients on calcium carbonate or sevelamer. Kidney Int 72(10):1255–1261 30. Klahr S, Levey AS, Beck GJ et al (1994) The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. Modiﬁcation of diet in renal disease study group. N Engl J Med 330: 877–884 47. Shantouf R, Budoff MJ, Ahmadi N et al (2007) Effects of sevelamer and calcium-based phosphate binders on lipid and inﬂammatory markers in hemodialysis patients. Am J Nephrol 28:275–279 31. Sampathkumar K, Selvam M, Sooraj YS et al (2006) Extended release nicotinic acid—a novel oral agent for phosphate control. Int Urol Nephrol 38:171–174 48. De Boer IH, Gorodetskaya I, Young B et al (2002) The severity of secondary hyperparathyroidism in chronic renal insufﬁciency is GFR-dependent, race-dependent, and associated with cardiovascular disease. References J Am Soc Nephrol 13:2762–2769 32. Llach F, Massry SG(1985) On the mechanism of secondary hyperparathyroidism in moderate renal insufﬁciency. J Clin Endocrinol Metab 61:601–606 49. Fajtova VT, Sayegh MH, Hickey N et al (1995) Intact parathyroid hormone levels in renal insufﬁciency. Calcif Tissue Int 57:329–335 33. Salusky IB, Foley J, Nelson P, Goodman WG (1991) Aluminum accumulation during treatment with aluminum hydroxide and dialysis in children and young adults with chronic renal disease. N Engl J Med 324:527–531 50. Martinez I, Saracho R, Montenegro J, Llach F (1997) The importance of dietary calcium and phosphorous in the secondary hyperparathyroidism of patients with early renal failure. Am J Kidney Dis 29:496–502 34. Thurston H, Swales JD (1971) Aluminium and chronic renal failure. Br Med J 4:490 51. Pitts TO, Piraino BH, Mitro R et al (1988) Hyperpara- thyroidism and 1,25-dihydroxyvitamin D deﬁciency in mild, moderate, and severe renal failure. J Clin Endo- crinol Metab 67:876–881 35. Slatopolsky E, Weerts C, Lopez-Hilker S et al (1986) Calcium carbonate as a phosphate binder in patients with chronic renal failure undergoing dialysis. N Engl J Med 315:157–161 52. Reiss E, Canterbury JM, Kanter A (1969) Circulating parathyroid hormone concentration in chronic renal insufﬁciency. Arch Intern Med 124:417–422 36. Braun J, Oldendorf M, Moshage W et al (1996) Electron beam computed tomography in the evaluation of cardiac calciﬁcation in chronic dialysis patients. Am J Kidney Dis 27:394–401 53. St John A, Thomas MB, Davies CP et al (1992) Deter- minants of intact parathyroid hormone and free 1,25- dihydroxyvitamin D levels in mild and moderate renal failure. Nephron 61:422–427 37. Goodman WG, Goldin J, Kuizon BD et al (2000) Coro- nary-artery calciﬁcation in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 342:1478–1483 54. Korkor AB (1987) Reduced binding of [3H]1,25-di- hydroxyvitamin D3 in the parathyroid glands of patients with renal failure. N Engl J Med 316:1573–1577 38. Guerin AP, London GM, Marchais SJ, Metivier F (2000) Arterial stiffening and vascular calciﬁcations in end-stage renal disease. Nephrol Dial Transplant 15:1014–1021 55. Gogusev J, Duchambon P, Hory B et al (1997) Depressed expression of calcium receptor in parathyroid gland tissue of patients with hyperparathyroidism. Kidney Int 51: 328–336 39. London GM, Guerin AP, Marchais SJ et al (2003) Arte- rial media calciﬁcation in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant 18:1731–1740 56. References Kates DM, Sherrard DJ, Andress DL (1997) Evidence that serum phosphate is independently associated with serum PTH in patients with chronic renal failure. Am J Kidney Dis 30:809–813 40. Kovesdy CP, Mehrotra R, Kalantar-Zadeh K (2008) Battleground: chronic kidney disorders mineral and bone disease—calcium obsession, vitamin D, and binder con- fusion. Clin J Am Soc Nephrol 3:168–173 57. Yamamoto M, Igarashi T, Muramatsu M et al (1989) Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messenger RNA in the rat. J Clin Invest 83:1053–1056 41. Mehrotra R, Budoff M, Christenson P et al (2004) Determinants of coronary artery calciﬁcation in diabetics with and without nephropathy. Kidney Int 66:2022–2031 58. Llach F, Massry SG, Singer FR et al (1975) Skeletal resistance to endogenous parathyroid hormone in patients 42. Mehrotra R, Budoff M, Hokanson JE et al (2005) Pro- gression of coronary artery calciﬁcation in diabetics with 12 3 3 Int Urol Nephrol (2008) 40:427–440 438 with early renal failure. A possible cause for secondary hyperparathyroidism. J Clin Endocrinol Metab 41: 339–345 75. Avram MM, Mittman N, Myint MM, Fein P (2001) Importance of low serum intact parathyroid hormone as a predictor of mortality in hemodialysis and peritoneal dialysis patients: 14 years of prospective observation. Am J Kidney Dis 38:1351–1357 59. Fuleihan GE, Gundberg CM, Gleason R et al (1994) Racial differences in parathyroid hormone dynamics. J Clin Endocrinol Metab 79:1642–1647 76. Fouque D, Guebre-Egziabher F (2007) An update on nutrition in chronic kidney disease. Int Urol Nephrol 39:239–246 60. Gupta A, Kallenbach LR, Zasuwa G, Divine GW (2000) Race is a major determinant of secondary hyperparathy- roidism in uremic patients. J Am Soc Nephrol 11: 330–334 77. Mafra D, Farage NE, Azevedo DL et al (2007) Impact of serum albumin and body-mass index on survival in hemodialysis patients. Int Urol Nephrol 39:619–624 61. Kovesdy CP, Ahmadzadeh S, Anderson JE, Kalantar- Zadeh K (2007) Obesity is associated with secondary hyperparathyroidism in men with moderate and severe chronic kidney disease. Clin J Am Soc Nephrol 2:1024– 1029 78. Massry SG, Smogorzewski M (1994) Mechanisms through which parathyroid hormone mediates its delete- rious effects on organ function in uremia. Semin Nephrol 14:219–231 62. Vincenti F, Arnaud SB, Recker R et al (1984) Parathyroid and bone response of the diabetic patient to uremia. Kidney Int 25:677–682 79. References Mazzaferro S, Coen G, Bandini S et al (1993) Role of ageing, chronic renal failure and dialysis in the calciﬁcation of mitral annulus. Nephrol Dial Transplant 8:335–340 80. Chiu KC, Chuang LM, Lee NP et al (2000) Insulin sen- sitivity is inversely correlated with plasma intact parathyroid hormone level. Metabolism 49:1501–1505 63. Hutchison AJ, Whitehouse RW, Boulton HF et al (1993) Correlation of bone histology with parathyroid hormone, vitamin D3, and radiology in end-stage renal disease. Kidney Int 44:1071–1077 81. Wareham NJ, Byrne CD, Carr C et al (1997) Glucose intolerance is associated with altered calcium homeostasis: a possible link between increased serum calcium concen- tration and cardiovascular disease mortality. Metabolism 46:1171–1177 64. Malluche HH, Ritz E, Lange HP et al (1976) Bone his- tology in incipient and advanced renal failure. Kidney Int 9:355–362 65. Rix M, Andreassen H, Eskildsen P et al (1999) Bone mineral density and biochemical markers of bone turn- over in patients with predialysis chronic renal failure. Kidney Int 56:1084–1093 82. Gadallah MF, el-Shahawy M, Andrews G et al (2001) Factors modulating cytosolic calcium. Role in lipid metabolism and cardiovascular morbidity and mortality in peritoneal dialysis patients. Adv Perit Dial 17:29–36 66. Sherrard DJ, Hercz G, Pei Y et al (1993) The spectrum of bone disease in end-stage renal failure—an evolving disorder. Kidney Int 43:436–442 83. Meytes D, Bogin E, Ma A et al (1981) Effect of para- thyroid hormone on erythropoiesis. J Clin Invest 67:1263–1269 67. Massry SG, Popovtzer MM, Coburn JW et al (1968) Intractable pruritus as a manifestation of secondary hyperparathyroidism in uremia. Disappearance of itching after subtotal parathyroidectomy. N Engl J Med 279: 697–700 84. Neves PL, Trivino J, Casaubon F et al (2002) Elderly patients on chronic hemodialysis: effect of the secondary hyperparathyroidism on the hemoglobin level. Int Urol Nephrol 34:147–149 68. Rao DS, Shih MS, Mohini R (1993) Effect of serum parathyroid hormone and bone marrow ﬁbrosis on the response to erythropoietin in uremia. N Engl J Med 328:171–175 85. Neves PL, Trivino J, Casaubon F et al (2006) Elderly patientsonchronichemodialysiswithhyperparathyroidism: increase of hemoglobin level after intravenous calcitriol. Int Urol Nephrol 38:175–177 86. Massry SG, Alexiewicz JM, Gaciong Z, Klinger M (1991) Secondary hyperparathyroidism and the immune system in chronic renal failure. Semin Nephrol 11: 186–201 69. Bogicevic M, Stefanovic V (1988) Relationship between parathyroid hormone and pituitary-testicular axis in patients on maintenance hemodialysis. Exp Clin Endo- crinol 92:357–362 70. References Am J Physiol 270:F186– F191 111. Drissi H, Pouliot A, Koolloos C et al (2002) 1,25-(OH)2- vitamin D3 suppresses the bone-related Runx2/Cbfa1 gene promoter. Exp Cell Res 274:323–333 112. Virdi AS, Cook LJ, Oreffo RO, Trifﬁtt JT (1998) Mod- ulation of bone morphogenetic protein-2 and bone morphogenetic protein-4 gene expression in osteoblastic cell lines. Cell Mol Biol (Noisy-le-grand) 44:1237–1246 95. Endlich N, Nobiling R, Kriz W, Endlich K (2001) Expression and signaling of parathyroid hormone-related protein in cultured podocytes. Exp Nephrol 9:436–443 113. Fraser JD, Otawara Y, Price PA (1988) 1,25-Di- hydroxyvitamin D3 stimulates the synthesis of matrix gamma-carboxyglutamic acid protein by osteosarcoma cells. Mutually exclusive expression of vitamin K- dependent bone proteins by clonal osteoblastic cell lines. J Biol Chem 263:911–916 96. Shigematsu T, Caverzasio J, BonjourJP (1993) Parathyroid removal prevents the progression of chronic renal failure induced by high protein diet. Kidney Int 44:173–181 97. Baker LR, Abrams L, Roe CJ et al (1989) 1,25(OH)2D3 administration in moderate renal failure: a prospective double-blind trial. Kidney Int 35:661–669 114. Panichi V, De Pietro S, Andreini B et al (1998) Calcitriol modulates in vivo and in vitro cytokine production: a role for intracellular calcium. Kidney Int 54:1463–1469 98. Bianchi ML, Colantonio G, Campanini F et al (1994) Calcitriol and calcium carbonate therapy in early chronic renal failure. Nephrol Dial Transplant 9:1595–1599 99. Goodman WG, Coburn JW (1992) The use of 1,25-di- hydroxyvitamin D3 in early renal failure. Annu Rev Med 43:227–237 115. Staeva-Vieira TP, Freedman LP (2002) 1,25-di- hydroxyvitamin D3 inhibits IFN-gamma and IL-4 levels during in vitro polarization of primary murine CD4 + T cells. J Immunol 168:1181–1189 100. Martin KJ, Gonzalez EA, Gellens M et al (1998) 19-Nor- 1-alpha-25-dihydroxyvitamin D2 (Paricalcitol) safely and effectively reduces the levels of intact parathyroid hor- mone in patients on hemodialysis. J Am Soc Nephrol 9:1427–1432 116. Barrat FJ, Cua DJ, Boonstra A et al (2002) In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines. J Exp Med 195:603–616 101. Kazama JJ, Maruyama H, Narita I, Gejyo F (2005) Maxacalcitol is a possible less phosphatemic vitamin D analog. Ther Apher Dial 9:352–354 117. Boonstra A, Barrat FJ, Crain C et al (2001) 1alpha,25- Dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells. J Immunol 167:4974–4980 102. References Tonner DR, Schlechte JA (1993) Neurologic complica- tions of thyroid and parathyroid disease. Med Clin North Am 77:251–263 87. Baczynski R, Massry SG, Kohan R et al (1985) Effect of parathyroid hormone on myocardial energy metabolism in the rat. Kidney Int 27:718–725 71. Fellner SK, Lang RM, Neumann A et al (1991) Para- thyroid hormone and myocardial performance in dialysis patients. Am J Kidney Dis 18:320–325 88. Amann K, Ritz E (1997) Cardiac disease in chronic uremia: pathophysiology. Adv Ren Replace Ther 4: 212–224 p y 72. London GM (2002) Left ventricular alterations and end- stage renal disease. Nephrol Dial Transplant 1 [17 Sup- pl]:29–36 89. Kosch M, Hausberg M, Vormbrock K et al (2000) Impaired ﬂow-mediated vasodilation of the brachial artery in patients with primary hyperparathyroidism improves after parathyroidectomy. Cardiovasc Res 47:813–818 73. Tseke P, Grapsa E, Stamatelopoulos K et al (2006) Atherosclerotic risk factors and carotid stiffness in elderly asymptomatic HD patients. Int Urol Nephrol 38:801–809 90. Amann K, Tornig J, Flechtenmacher C et al (1995) Blood-pressure-independent wall thickening of intramy- ocardial arterioles in experimental uraemia: evidence for a permissive action of PTH. Nephrol Dial Transplant 10:2043–2048 74. Kovesdy CP, Ahmadzadeh A, Anderson JE, Kalantar- Zadeh K (2008) Outcomes associated with secondary hyperparathyroidism in men with moderate to severe chronic kidney disease. Kidney Int (in press) 12 3 Int Urol Nephrol (2008) 40:427–440 439 91. Kamycheva E, Sundsfjord J, Jorde R (2004) Serum parathyroid hormone levels predict coronary heart dis- ease: the Tromsø study. Eur J Cardiovasc Prev Rehabil 11:69–74 vitamin D and mortality in chronic kidney disease. Arch Intern Med 168:397–403 109. Shoben AB, Rudser KD, de Boer IH et al (2007) Oral cal- citriol use and mortality in non-dialyzed patients with chronickidney disease [abstract]. J Am Soc Nephrol18:98A 92. Ichikawa I, Humes HD, Dousa TP, Brenner BM (1978) Inﬂuence of parathyroid hormone on glomerular ultraﬁl- tration in the rat. Am J Physiol 234:F393–F401 110. Bellows CG, Reimers SM, Heersche JN (1999) Expres- sion of mRNAs for type-I collagen, bone sialoprotein, osteocalcin, and osteopontin at different stages of osteo- blastic differentiation and their regulation by 1,25 dihydroxyvitamin D3. Cell Tissue Res 297:249–259 93. Marchand GR (1985) Effect of parathyroid hormone on the determinants of glomerular ﬁltration in dogs. Am J Physiol 248:F482–F486 94. Lee K, Brown D, Urena P et al (1996) Localization of parathyroid hormone/parathyroid hormone-related pep- tide receptor mRNA in kidney. 127. Li YC, Kong J, Wei M et al (2002) 1,25-Dihydroxyvi- tamin D(3) is a negative endocrine regulator of the renin- angiotensin system. J Clin Invest 110:229–238 128. Charytan C, Coburn JW, Chonchol M et al (2005) Cin- acalcet hydrochloride is an effective treatment for secondary hyperparathyroidism in patients with CKD not receiving dialysis. Am J Kidney Dis 46:58–67 125. Penna G, Adorini L (2000) 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 164:2405–2411 126. Gysemans C, Van EE, Overbergh L et al (2002) Treat- ment of autoimmune diabetes recurrence in non-obese diabetic mice by mouse interferon-beta in combination with an analogue of 1alpha,25-dihydroxyvitamin-D3. Clin Exp Immunol 128:213–220 References Goodman WG, Hladik GA, Turner SA et al (2002) The calcimimetic agent AMG 073 lowers plasma parathyroid hormone levels in hemodialysis patients with secondary hyperparathyroidism. J Am Soc Nephrol 13:1017–1024 118. Aihara K, Azuma H, Akaike M et al (2004) Disruption of nuclear vitamin D receptor gene causes enhanced thrombogenicity in mice. J Biol Chem 279:35798–35802 103. Taskapan H, Wei M, Oreopoulos DG (2006) 25(OH) vitamin D3 in patients with chronic kidney disease and those on dialysis: rediscovering its importance. Int Urol Nephrol 38:323–329 119. Wolf M, Shah A, Gutierrez O et al (2007) Vitamin D levels and early mortality among incident hemodialysis patients. Kidney Int 72:1004–1013 120. Watson KE, Abrolat ML, Malone LL et al (1997) Active serum vitamin D levels are inversely correlated with coronary calciﬁcation. Circulation 96:1755–1760 104. Saab G, Young DO, Gincherman Y et al (2007) Preva- lence of vitamin D deﬁciency and the safety and effectiveness of monthly ergocalciferol in hemodialysis patients. Nephron Clin Pract 105:c132–c138 121. Bertoli M, Luisetto G, Ruffatti A et al (1990) Renal function during calcitriol therapy in chronic renal failure. Clin Nephrol 33:98–102 105. Ritter CS, Armbrecht HJ, Slatopolsky E, Brown AJ (2006) 25-Hydroxyvitamin D(3) suppresses PTH syn- thesis and secretion by bovine parathyroid cells. Kidney Int 70:654–659 p 122. Coen G, Mazzaferro S, Manni M et al (1994) No accel- eration and possibly slower progression of renal failure during calcitriol treatment in predialysis chronic renal failure. Nephrol Dial Transplant 9:1520 106. Teng M, Wolf M, Ofsthun MN et al (2005) Activated injectable vitamin D and hemodialysis survival: a his- torical cohort study. J Am Soc Nephrol 16:1115–1125 123. Agarwal R, Acharya M, Tian J et al (2005) Antiprotei- nuric effect of oral paricalcitol in chronic kidney disease. Kidney Int 68:2823–2828 107. Tentori F, Hunt WC, Stidley CA et al (2006) Mortality risk among hemodialysis patients receiving different vitamin D analogs. Kidney Int 70:1858–1865 124. Nagakura K, Abe E, Suda T et al (1986) Inhibitory effect of 1 alpha,25-dihydroxyvitamin D3 on the growth of the renal carcinoma cell line. Kidney Int 29:834–840 108. Kovesdy CP, Ahmadzadeh S, Anderson JE, Kalantar- Zadeh K (2008) Association of treatment with activated 12 12 3 3 Int Urol Nephrol (2008) 40:427–440 440 125. Penna G, Adorini L (2000) 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 164:2405–2411 128. References Charytan C, Coburn JW, Chonchol M et al (2005) Cin- acalcet hydrochloride is an effective treatment for secondary hyperparathyroidism in patients with CKD not receiving dialysis. Am J Kidney Dis 46:58–67 126. Gysemans C, Van EE, Overbergh L et al (2002) Treat- ment of autoimmune diabetes recurrence in non-obese diabetic mice by mouse interferon-beta in combination with an analogue of 1alpha,25-dihydroxyvitamin-D3. Clin Exp Immunol 128:213–220 123 123
https://openalex.org/W4213293791	https://www.qeios.com/read/AG7EGI/pdf	English	null	Olfactory Receptor 1G1	Definitions	2,020	cc-by	72	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Olfactory Receptor 1G1 National Cancer Institute National Cancer Institute Qeios ID: AG7EGI · https://doi.org/10.32388/AG7EGI Source National Cancer Institute. Olfactory Receptor 1G1. NCI Thesaurus. Code C34010. Olfactory receptor 1G1 (313 aa, ~35 kDa) is encoded by the human OR1G1 gene. This protein is involved in odorant binding and ligand-dependent signal transduction. Qeios ID: AG7EGI · https://doi.org/10.32388/AG7EGI 1/1
https://openalex.org/W3201859555	https://link.springer.com/content/pdf/10.1007/s13143-021-00257-y.pdf	English	null	Atmospheric Correction of True-Color RGB Imagery with Limb Area-Blending Based on 6S and Satellite Image Enhancement Techniques Using Geo-Kompsat-2A Advanced Meteorological Imager Data	Asia-Pacific journal of atmospheric sciences	2,021	cc-by	12,512	Online ISSN 1976-7951 Print ISSN 1976-7633 Online ISSN 1976-7951 Print ISSN 1976-7633 https://doi.org/10.1007/s13143-021-00257-y Asia-Pacific Journal of Atmospheric Sciences (2022) 58:333–352 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract This study aims for producing high-quality true-color red-green-blue (RGB) imagery that is useful for interpreting various environmental phenomena, particularly for GK2A. Here we deal with an issue that general atmospheric correction methods for RGB imagery might be breakdown at high solar/viewing zenith angle of GK2A due to erroneous atmospheric path lengths. Additionally, there is another issue about the green band of GK2A of which centroid wavelength (510 nm) is different from that of natural green band (555 nm), resulting in the unrealistic RGB imagery. To overcome those weakness of the RGB imagery for GK2A, we apply the second simulation of the satellite signal in the solar spectrum radiative transfer model look- up table with improved information considering altitude of the reflective surface to reduce the exaggerated atmospheric cor- rection, and a blending technique that mixed the true-color imagery before and after atmospheric correction which produced a naturally expressed true-color image. Consequently, the root mean square error decreased by 0.1–0.5 in accordance with the solar and view zenith angles. The green band signal was modified by combining it with a veggie band to form hybrid green which adjust centroid wavelength of approximately 550 nm. The original composite of true-color RGB imagery is dark; therefore, to brighten the imagery, histogram equalization is conducted to flatten the color distribution. High-temporal- resolution true-color imagery from the GK2A AMI have significant potential to provide scientists and forecasters as a tools to visualize the changing Earth and also expected to intuitively understand the atmospheric phenomenon to the general public. Keywords Atmospheric correction · Look-up table · True-color imagery · Radiative transfer model · Rayleigh scattering · Solar Zenith angle Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and Satellite Image Enhancement Techniques Using Geo‑Kompsat‑2A Advanced Meteorological Imager Data Minsang Kim1 · Jun‑Hyung Heo2 · Eun‑Ha Sohn2 Received: 27 April 2021 / Revised: 20 August 2021 / Accepted: 31 August 2021 © The Author(s) 2021 / Published online: 1 October 2021 1 Korea Ocean Satellite Center, Korea Institute of Ocean Science and Technology, Busan Metropolitan City 49111, Korea Responsible Editor: Myoung Hwan Ahn. 2 National Meteorological Satellite Center, Korea Meteorological Administration, Jincheon‑gun 27803, Korea * Jun‑Hyung Heo jhheo89@korea.kr Vol.:(0123456789) 1 3 Korean Meteorological Society 1 Introduction Rayleigh scattering effect. The effect of Rayleigh scatter- ing is inversely proportional to wavelength; thus, the blue band—which is the shortest band—is the most affected. As a result, true-color RGB images contain bluish grays (Miller et al. 2016). Atmospheric radiance is interrupted through absorption, scattering, and diffraction along the atmospheric path. Of these processes, scattering has the most dominant influ- ence in the visible bandwidth and is represented by the To improve the quality of a visible band under the influence of Rayleigh scattering, radiative transfer mod- els (RTMs) have been applied in several studies (Gordon 1993; Rahman and Dedieu 1994; Fukushima et al. 1998; Berka et al. 1999; Richter et al. 2006). RTMs can be used to characterize the atmospheric effects of surface radiation signals as measured by satellite sensors. They have sev- eral advantages (He et al. 2019). For instance, they are not limited to a specific region or satellite sensor because they input the atmospheric conditions, geometrical information, and sensory characteristics of areas where the atmospheric Responsible Editor: Myoung Hwan Ahn. Vol.:(0123456789) 1 3 Korean Meteorological Society 334 M. Kim et al. 2006). In particular, for stationary satellites, the full-disk area covered includes regions at a high solar zenith angle (SZA) of over 70°. In terms of data utilization, accurate surface reflection data are required (Ruddick et al. 2014; Lee et al. 2015). Overcorrection problems occur in several RTMs for polar orbits (Wang 2016) and geostationary sat- ellites (Miller et al. 2016), which commonly assume a flat atmosphere (Adler-Golden et al. 1999; Qu et al. 2003; Ver- mote et al. 2006). Recently, RTMs have begun to consider the effects of the Earth’s curvature as a pseudo-spherical approximation (He et al. 2018). Validation has shown that the model results are comparable to benchmarks (He et al. 2018) showing that the effects of Earth’s curvature increase rapidly with SZA, for SZAs of 75°, 80°, and 85°. These results indicate that curvature effects should be considered in high-accuracy atmospheric correction. The Rayleigh scat- tering LUT has also verified that it shows a significant bias at high SZAs (He et al. 2018). correction is to be performed. Moreover, RTM methods are known to be more accurate in simulating atmospheric effects than the empirical line method or improved dark-object sub- traction (Zhou et al. 2011). The second simulation of a satellite signal in the solar spectrum (6S) (Vermote et al. 1 Introduction 2006) and moderate resolu- tion atmospheric transmission (MODTRAN) (Adler-Golden et al. 1999) methods have been frequently used in previous research (Richter 1996; Karpouzli and Malthus 2003; Ghu- lam et al. 2004; Sriwongsitanon et al. 2011; Franch et al. 2013). [emphasize why use 6 S rather than other RTM] However, owing to the complexity of their calculations and consequent large processing times, these RTM methods are inefficient in performing standby calibration over a wide area, such as in the case of satellite imaging. To address this problem, several studies have applied the look-up table (LUT) method; this is an array-based method that replaces runtime calculations with simple array indexing (Liang et al. 2001; Lyapustin et al. 2011; Dorji and Fearns 2018). This study aimed to produce high-quality, true-color RGB images. One important process is that of correcting the sensor-measured radiance of channels affected by Ray- leigh scattering through the atmosphere. Rayleigh scatter- ing is dominant within the visible and near-infrared bands; hence, its effects need to be mitigated. We built an LUT that is computed using a 6 S RTM, to convert the radiance to the atmospherically corrected reflectance. Our LUT applies a minimum curvature surface (MCS) technique to augment the LUT in terms of geometric parameters. Inspection of the resulting LUT shows that the atmospheric correction coefficients dramatically increase over SZAs and view zenith angles (VZAs) of 70°. This caused the reflectance near the limb area exceeds the correction values. To mitigate this reflectance, we applied limb correction according to the SZA and VZA. The GK2A AMI, which has a visual area specifi- cation similar to that of the AHI sensor in Himawari-8, con- tains a green band centered at 510 nm. The band is slightly shifted toward the blue band compared to the 550 nm found in many other sensors (MODIS, Landsat, and VIIRS) (Miller et al. 2016; Broomhall et al. 2019). For this reason, the veg- etation in the true-color RGB images is browner and the bare ground is redder than that of the aforementioned sen- sors. One solution to this problem is hybrid-green adjust- ment, which combines the green visible band with the near- infrared band (870 nm) to mimic green grass vegetation. We applied hybrid green instead of the original green band. True-color RGB imagery incorporating atmospheric cor- rection shows up dark. One remedial method is histogram equalization. 1 3 Korean Meteorological Society 1 Introduction This method brightens the image by expanding the narrow color distribution. True-color RGB imagery is useful in detection and analysis. I hi d ib h h d A number of previous studies have attempted to estimate surface albedo (the atmospheric reflectance for isotropic light) using various satellite sensors, such as the Advanced Very High Resolution Radiometer (Csiszar and Gutman 1999; Strugnell and Lucht 2001), the Moderate Resolution Imaging Spectroradiometer (MODIS) (Schaaf et al. 2002), and the Spinning Enhanced Visible and InfraRed Imager of the Meteosat (Geiger et al. 2008). Compared to the stud- ies of polar satellites, research into geostationary satellite orbits is sparse; however, recently launched geostationary satellites such as the Geo-Kompsat-2 A (GK2A) Advanced Meteorological Imager (AMI) (Kim et al. 2021), Hima- wari-8 Advanced Himawari Imager (AHI) (Bessho et al. 2016), Fengyun-4 Advanced Geosynchronous Radiation Imager (AGRI) (Yang et al. 2017), and GOES-16 Advanced Baseline Imager (ABI) (Schmit et al. 2016) are capable of providing higher temporal, spatial, and spectral resolutions. Depending on their sensor, various methods are applied to produce true-color imagery. In the case of AHI, Rayleigh correction is conducted using RTM modified by the National Aeronautics and Space Administration (NASA) SeaDAS and various image enhancement techniques such as hybrid green, and Simple Hybrid Contrast Stretch (SHCS) were applied (Miller et al. 2016). AGRI also applied image enhancement technique SHCS method (Miller et al. 2016). Since the ABI sensor does not have green band, green-like band is gen- erated by combining red, blue, and vegetation band (Bah et al. 2018). They are expected to be used for estimating reflectance, and an increasing amount of research is being undertaken into them. As described by Vermote et al. (2006), several RTMs mentioned in the previous paragraph are limited because they do not assume a spherical atmosphere; hence, it difficult to apply them to make limb observations (Vermote et al. In this paper, we describe the atmosphere-corrected, true- color RGB imaging procedure from GK2A AMI. Section 2 describes the data used in this study and the methodology for Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… 335 Ocean, and Meteorological Satellite (COMS), performing meteorological and space-weather observation tasks using the AMI. The AMI exhibits a superior observational per- formance to the COMS in terms of spatial, temporal, and spectral resolutions (Kim et al. 2021). obtaining the atmospherically corrected visible bands and image enhancements. 1 Introduction The preliminary results of applying each process—along with their respective final products— are described in Section 3. Section 4 discusses the limita- tions and future works. The paper is concluded in Section 5. In particular, the augmented array of channels—the number of available channels was expanded from five to 16—has led to the improvement of satellite measurement capacities (Table 1). The composite of red, green, and blue visible light channels enable true-color imagery; this represents a milestone improvement over the gray-color imagery previously available. Three water-vapor channels facilitate the detection of water-vapor signals at different heights, and the various infrared channels detect atmos- pheric gases and subtle changes in convective signals. 2.1.1 GK2A AMI GEO-KOMPSAT-2 A is a geostationary meteorologi- cal satellite that was launched on December 5, 2018 and began operation on July 24, 2019. It was designed to take over the meteorological functions of the Communication, Spatial and temporal resolutions were also enhanced. The red channel has a resolution of 0.5 km, the two other visible channels and the 860 nm channel have a resolution of 1 km, and the infrared channels have 2 km spatial reso- lution. The observation cycle of the AMI has a duration of 10 min for the full disk and 2 min for the Korean Peninsula region. Its payload covers a full disk area centered at 0°N, 128.3°E; this includes Asia and Oceania, which contain various land-cover types and diverse climate phenomena. Table 1 GEO-KOMPSAT-2 A channel specifications, center of wave- length, bandwidth, and resolution (Kim et al. 2021) GK2A chan- nel specifica- tion GK2A band # Center of wavelength (μm) Bandwidth (μm) Reso- lution (km) Visible 1 0.47 0.43–0.48 1 2 0.51 0.50–0.52 1 3 0.64 0.63–0.66 0.5 Near Infrared 4 0.86 0.85–0.87 1 5 1.37 1.37–1.38 2 6 1.61 1.60–1.62 2 Water Vapor 7 3.83 3.74–3.96 2 8 6.2 6.06–6.42 2 9 6.9 6.89–7.01 2 Infrared 10 7.3 7.26–7.43 2 11 8.6 8.44–8.76 2 12 9.6 9.54–9.72 2 13 10.4 10.25–10.61 2 14 11.2 11.08–11.32 2 15 12.3 12.15–12.45 2 16 13.3 13.21–13.39 2 Table 1 GEO-KOMPSAT-2 A channel specifications, center of wave- length, bandwidth, and resolution (Kim et al. 2021) Table 2 compares the full width at half-maximum (FWHM) responses and the spatial resolutions of GEO (AMI, AHI, AGRI, and ABI) sensors for specific bands of focus. There is only a subtle difference between AMI and AHI, because they use the same sensor (Bessho et al. 2016). AGRI and ABI has no green band, which means that it must take advantage of near-band values to obtain imaginary green values (Schmit et al. 2016). On the other hand, the AMI and AHI feature a green band; however, this signal is slightly blue-shifted, which results in another imaginary green value, referred to as “hybrid green” (Miller et al. 2016). Outside of the visible range, the AMI features a cirrus band (1.4 μm); however, the cloud parti- cle size band (2.2 μm) is absent. Both are included in the AHI and ABI. baseline imager (ABI) (Schmit et al. 1 3 Korean Meteorological Society 2.2 Methodology The European Centre for Medium-Range Weather Fore- casts (ECMWF) has produced the Copernicus Atmosphere Monitoring Service (CAMS) dataset. CAMS also offers an atmospheric analysis service, which focuses on atmos- pheric composition, including aerosols, chemical species, and greenhouse gases (Massart et al. 2016; Inness et al. 2019). The CAMS analysis is produced using the ECM- WF’s four-dimensional variational (4DVar) system (Enge- len and McNally 2005) within the Integrated Forecasting System (IFS; version CY42r1 for 2016 and CY43r1 for 2017), which is one of the world’s leading operational global weather-prediction systems. The transport of trac- ers such as CO2 is assessed online by the IFS model con- currently with the meteorological forecast. Because the semi-Lagrangian advection scheme in the IFS does not conserve mass, a global mass fixer is applied to restore mass conservation to the global budget (Agust-Panareda et al. 2014). The IFS model used in CAMS has a horizon- tal resolution of approximately 40 km and 137 vertical levels. Further information about the IFS model can be found online (https://www.ecmwf.int/en/forecasts/docum entation-and-support/changes-ecmwf-model/ifs-docum entation). The forecast contains information pertaining to gases in the lowest layer of the atmosphere (troposphere) and the ozone higher up (stratosphere). It also contains data about desert dust, sea salt, organic matter, black car- bon, and sulfate particles (aerosols). The initial conditions of the forecasts (analyses) are obtained by combining a previous forecast with satellite observations of the aero- sol, ozone, carbon monoxide, nitrogen dioxide, and sul- fur dioxide levels, via a process called data assimilation. In this study, we used a three-parameter geostationary environmental monitoring spectrometer, which measures six-hourly instantaneous values of the total column ozone (TCO), total column water vapor (TPW), and total aerosol optical depth (AOD) at 550 nm from the CAMS dataset, interpolating them onto a 0.25° longitude × 0.25° latitude grid (Table 3). This dataset was re-projected to the GK2A full-disk region. Figure 1 illustrates the procedure of atmospherically correct- ing true-color imagery. The sensor-measured reflectance is reduced by atmospheric scattering. The Rayleigh scattering effect is inversely proportional to wavelength, thus visible channels needed to be corrected accordingly. Without reduc- tion, we constructed an LUT by considering geometric and atmospheric conditions, using the 6 S RTM to correct atmos- pheric effects in visible channels. f Inspection of the created LUT showed that the atmos- pheric correction coefficients were dramatically increased over 70° of SZA and VZA (Miller et al. 2016). 2.2 Methodology As a result, the reflectance near the limb area exceeded the correction values. To mitigate reflectance, we applied limb correction according to the SZA and VZA.f The GK-2 A green-channel wavelength (510 nm) differs from the green grass vegetation wavelength (550 nm) taken from the spectral database of NASA’s Advanced Spaceborne Thermal Emission and Reflection Radiometer. One solution is the hybrid-green method, which combines the visible light band with the near-infrared band (870 nm) to mimic green grass vegetation. We applied hybrid green instead of the original green band. True-color RGB imagery incorporating atmospheric cor- rection shows up dark. One remedial method is histogram equalization. This method aims to increase the brightness of the image by expanding the narrow color distribution. True-color RGB imagery is useful for detection and analysis. 2.1.1 GK2A AMI 2016) sensor in full width at half-maximum (FWHM), and spatial resolution at nadir for selected visible and near-infrared bands Table 2 Comparison of the GEO-KOMPSAT-2 A advanced meteoro- logical imager (AMI) (Kim et al. 2021), Himwari-8 advanced Hima- wari imager (AHI) (Bessho et al. 2016), Advanced Geosynchronous Radiation Imager (AGRI) (Yang et al. 2017), and GOES-R advanced baseline imager (ABI) (Schmit et al. 2016) sensor in full width at half-maximum (FWHM), and spatial resolution at nadir for selected visible and near-infrared bands Sensor AMI AHI AGRI ABI No. FWHM(μm) Res. (km) No. FWHM(μm) Res. (km) No. FWHM (μm) Res. (km) No. FWHM (μm) Res. (km) Blue 1 0.43–0.48 1.0 1 0.43–-0.48 1.0 1 0.45–0.49 1.0 1 0.45–-0.49 1.0 Green 2 0.50–0.52 1.0 2 0.50–0.52 1.0 - - - - - - Red 3 0.63–0.66 0.5 3 0.63–0.66 0.5 2 0.55–0.75 0.5 2 0.59–0.69 0.5 Vege. 4 0.85–0.87 1.0 4 0.85–0.87 1.0 3 0.75–0.90 1.0 3 0.85–0.89 1.0 1 3 Korean Meteorological Society 336 M. Kim et al. 2.1.2 ECMWF CAMS Near‑real‑time Data 2.1.2 ECMWF CAMS Near‑real‑time Data 2.1.2 ECMWF CAMS Near‑real‑time Data 2.2 Methodology 1 3 Korean Meteorological Society 2.2.1 6SV2.1 The 6S is a basic RTM; it has been used for the calcula- tion of LUTs in the satellite atmospheric correction algo- rithms developed by Vermote et al. (2006). It is designed to simulate the reflection of solar radiation from the atmos- phere–surface coupling system over a wide range of air, spectrum, and geometric conditions. This enables accu- rate simulation of satellite observations, consideration of the elevated targets, and modeling of the composite atmosphere of realistic molecules and aerosols. 6S is a highly accurate radiative transfer model considering wide range of atmospheric conditions. Compared with Simpli- fied Method for the Atmospheric Correction (SMAC), 6S showed better performance [Proud et al. 2010]. The lat- est updates include a public release of its vector version (6SV), which considers the Stoke’s parameter and polari- zation contribution; this version is based on the successive orders of scattering (SOS) approximations. The accuracy of radiative transfer (RT) calculations can be varied by changing the number of calculation angles and parameters (Vermote et al. 2006). The 6S model atmosphere consists of several layers, and the model solves the RT equations 1 3 Korean Meteorological Society Table 3 European Centre for Medium-Range Weather Forecasts (ECMWF) Copernicus Atmosphere Monitoring Service (CAMS) data characteristics Parameters [unit] Spatial resolution (km) Temporal resolution Total column ozone [ atm −cm] 12.5 Daily Total precipitable water [ gcm−2] 12.5 Daily Aerosol optical depth at 550 nm 12.5 Daily Table 3 European Centre for Medium-Range Weather Forecasts (ECMWF) Copernicus Atmosphere Monitoring Service (CAMS) data characteristics 1 Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… 337 Fig. 1 Flowchart of the atmospheric correction process for true-color imagery Fig. 1 Flowchart of the atmospheric correction process for true-color imagery (1) ρTOA(θs, θv, 휑) = Tg (휃s, 휃v ) × [ 휌R+V + T↓(휃s )T↑(휃v ) 휌s 1 −S휌s ] numerically, layer by layer. The final result is a sum of successive computations, which is performed by integrat- ing over the Fourier series decomposition. Unlike other RT codes, the 6S offers an innovative working system. The 6SV calculates surface reflectivity, not remote sens- ing reflectance—which is total surface incident irradiance (downward irradiance) divided by aquatic luminance. In the calculation, the apparent reflectance value is arbitrarily set (assuming 0, 0.5, 1.0 in the model) and the radiance is inversely calculated. 1 3 Korean Meteorological Society 2.2.1 6SV2.1 (3) with the atmospheric correction coefficients: (3) ρatm = xa × L −xb 1 + xc × (xa × L −xb) (3) where ρatm is the atmospherically corrected reflectance; L is the TOA radiance [ Wm−2휇m−1sr−1 ]; and xa, xb, and xc are atmospheric correction coefficients which represent the inverse of the transmittance, the scattering term of the atmosphere, and the spherical albedo, respectively. where ρatm is the atmospherically corrected reflectance; L is the TOA radiance [ Wm−2휇m−1sr−1 ]; and xa, xb, and xc are atmospheric correction coefficients which represent the inverse of the transmittance, the scattering term of the atmosphere, and the spherical albedo, respectively. 1 3 Korean Meteorological Society 2.2.1 6SV2.1 In this process, the 6S model gener- ates the atmospheric background, returning transmittance, and top of atmosphere (TOA) reflectance. It provides cor- rection coefficients to compute the ground reflectance, given a TOA radiance. It was publicly released in May 2005, and the latest version of the 6SV code (6SV2.1), released in June 2015, is now available for download: http://6s.ltdri.org/pages/downloads.html.l (1) s ] with (2) ρs = ρTOA(θs,θv,휑) Tg(휃s,휃v) −휌R+V T↓(휃s )T↑(휃v ) (2) where ρTOA is the TOA reflectance; θs is the SZA; θv is the VZA; 휑 is the relative azimuth angle (RAA); Tg is the gase- ous transmission of atmospheric gases such as H2O, CO2, and O3; 휌R+V is the total reflectance due to molecular and aerosol scattering; T↓(휃s ) and T↑(휃v )represent the atmos- pheric transmittance from sun to target and target to satellite, respectively; and ρs is the atmospheric reflectance for a Lam- bertian, homogeneous target. The 6SV computes all trans- mittance and atmospheric reflectance using the user-defined parameters and subroutines, which contain vertical profiles of atmospheric temperature, pressure, and absorbing gases where ρTOA is the TOA reflectance; θs is the SZA; θv is the VZA; 휑 is the relative azimuth angle (RAA); Tg is the gase- ous transmission of atmospheric gases such as H2O, CO2, and O3; 휌R+V is the total reflectance due to molecular and aerosol scattering; T↓(휃s ) and T↑(휃v )represent the atmos- pheric transmittance from sun to target and target to satellite, respectively; and ρs is the atmospheric reflectance for a Lam- bertian, homogeneous target. The 6SV computes all trans- mittance and atmospheric reflectance using the user-defined parameters and subroutines, which contain vertical profiles of atmospheric temperature, pressure, and absorbing gases The land surface reflectance in the 6SV atmospheric correction mode is calculated using the following equation: 1 3 Korean Meteorological Society 338 M. Kim et al. ▸ Fig. 2 Range and intervals of atmospheric conditions, selected based on the climatology of the ECMWF CAMS data from 2015 to 2018 ▸ according to altitude. This model provides three atmospheric correction coefficients for removing atmospheric effects. The land surface reflectance can be calculated using Eq. 2.2.3 Image Processing A difference in spatial resolution occurs when multi-band datasets are generated from different bands. The 640 nm band has the highest spatial resolution (about 500 m), other visible channels and vegetation channels have a 1 km spa- tial resolution, and infrared channels have a 2 km spatial resolution. To compose true-color imagery, each band is required to have equal spatial resolution; thus, all bands (except the 640 nm band) were downscaled to a 500 m spa- tial resolution. Spline interpolation—a statistical downscal- ing method—was used to sharpen resolution; it is a form of interpolation in which the interpolant is a special kind of piecewise polynomial, called a spline. The spline interpo- lation is preferred over polynomial interpolations because the interpolation errors can be reduced, even when using low-degree polynomials for the spline. Spline interpolation avoids the problem of Runge’s phenomenon, in which oscil- lations occur between points when interpolating with high- degree polynomials. curvature technique produces a clear, smooth surface (Rabah and Kaloop 2013). In this study, it was assumed that the atmospheric correction coefficients would change smoothly in response to the SZA and VZA when all other conditions are held constant. The SZA and VZA are specified starting from 5° with an interval of 0.5° in the final version of the LUT.l After atmospheric correction of the reflectance, the com- posite true-color imagery featured a particular limb region highlighted in red, whereas the uncorrected true-color imagery did not. The red coloration was a result of the blue and green bands used in the RGB composite being overcor- rected with respect to the red band. The 6S was designed using a parallel-plane atmosphere assumption in the RT model (which reduces the Rayleigh scattering effect), and the impact was exaggerated at high zenith angles, such that the Earth’s limb appeared red. It is possible to mitigate over- correction at the limb by reducing the Rayleigh scattering according to the atmospheric pathlengths and storing the level of reflectance in the LUT. RT codes set reflectance points at ground level. Additional height levels were added into the LUT, and the height indexes were defined as in the Table 5. The uncorrected true-color imagery results in brown vegetation and red bare soil (Fig. 3). The explanation for these unexpected colors is that the signal center of the green Fig. 2.2.2 Building a 6SV2.1 LUT In most practical cases, the minimum 1 3 Korean Meteorological Society Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… 339 Table 4 Configuration of second simulation of a satellite signal in the solar spectrum (6S) interpolated look-up table (LUT) Parameters [unit] Min Max Number of segments (interval) Solar zenith angle [°] 0 70 15 (5) View zenith angle [°] 0 70 15 (5) Relative azimuth angle [°] 0 180 19 (10) Total column ozone [ atm −cm] 0.25 0.40 4 (0.05) Total precipitable water [ gcm−2] 0 5 6 (1) Aerosol optical depth at 550 nm 0.01, 0.05, 0.1, 0.2, 0.3 5 Reflectance height [km] 0 10 6 (2) zenith angle, assuming an altitude of reflective surface. To estimate altitude of reflective surface, the brightness tem- perature value of the infrared channel was used since the altitude at which radiation occurs can be inferred from the temperature of the radiation surface. It is applied all regions regardless of latitudes and atmospheric states. 2.2.2 Building a 6SV2.1 LUT The 6SV was developed specifically to determine atmos- pheric corrections for satellite, it allows users to define a down-looking geometry. Despite the direct atmospheric cor- rections of reflectance available with 6S, most studies have performed atmospheric corrections using LUT approaches. Using a LUT can prevent duplicate calculations for identical conditions in limited computational environments. First, for building our LUT, input parameters were set using the following preconditions described in Table 4. For geometric conditions, each SZA and VZA was set between 0° and 80°, in increments of 5°. The RAA had a range from 0° to 180°, in increments of 10°. The range and intervals of atmospheric conditions were selected based on climatology of the ECMWF CAMS data from 2015 to 2018 (Fig. 2). The TCO had a range of 0.25–0.40 atm-cm and was varied in 0.05 atm-cm increments. TPW had a range of 0–5 gcm−2 , and was varied in 1 gcm−2increments. The AOD had a range of 0.01–0.3, with irregular intervals. The reflectance height was segmented between ground level and a height of 10 km, in 2 km intervals. The spectral conditions were configured using the spectral response function of GK2A.fi The three pre-calculated atmospheric correction coeffi- cients were stored in the LUT. To expand the LUT, an MCS technique was applied. The MCS is a mathematical method for constructing smooth surfaces from irregularly spaced data. The surface of minimum curvature corresponds to the minimum of the Laplacian power or—in the alternative for- mulation—satisfies the bi-harmonic differential equation. Physically, it models the behavior of an elastic plate. In the one-dimensional case, the minimum curvature leads to the natural cubic spline interpolation. In the two-dimensional case, a surface can be interpolated with bi-harmonic splines or gridded with an iterative finite-difference scheme (Smith and Wessel 1990). 1 3 Korean Meteorological Society 3.1 Preprocessing For Atmospheric Correction of TOA Reflectance where Fhg is the hybrid-green factor (which varies from zero to one; in this study, the hybrid-green factor was set to 0.13 empirically), and Rgreen and Rvege are the reflectances of the green and vegetation bands, respectively. The hybrid-green factor adjusts the vegetation signal, highlighting green colors and faded red soil. The use of the vegetation band is not appropriate for the surface of oceans because of the strong absorption by water of light in those wavelengths. However, an ocean is relatively sensitive to the blue band and the prac- tical effect of using the hybrid-green method in true-color imagery is more pronounced. Rayleigh scattering exerts a dominant effect along atmos- pheric pathways and is an important feature of visible chan- nels. It is inversely proportional to the fourth power of the wavelength. The upper images in Fig. 4 are uncorrected, and they all exhibit the Rayleigh effect. The blurred regions, when compared with the lower images in the figure, indi- cate that the surface reflectance suffered Rayleigh scattering before the signal reached the satellite. The edges of Earth (which become more pronounced moving from the red to blue columns) experience a larger scattering effect because the longer the optical pathlength from the target to the sat- ellite along the atmospheric path, the greater the Rayleigh scattering effect. Atmospherically corrected imagery, which adjusts for the Rayleigh scattering effects, can achieves the clearer earth imagery exemplified in the lower images of Fig. 4. g y p The original true-color composite imagery appears faint unless color distributions are enhanced. To compensate for indistinguishable imagery, the visual enhancement technique of histogram equalization was implemented. Histogram equalization is an image-processing technique designed to improve contrasts in images by redistributing the color his- togram. A well-separated image has advantages in terms of visibility and sharpness, particularly over blurred or dark images. Histogram equalization method brightens the image by expanding the narrow color distribution. Through this adjustment, the intensities can be better distributed on the histogram utilizing the full range of intensities evenly. This allows for areas of lower local contrast to gain a higher con- trast. Histogram equalization accomplishes this by effec- tively spreading out the highly populated intensity values which use to degrade image contrast. First, reflectance val- ues (0–1) were rescaled to between zero and 255, because the RGB composite imagery contained pixel values within this range. 2.2.3 Image Processing 3 Step 1: GK-2 A full-disk simple composite of native RGB- band true-color image, taken on 2019/12/01 0300 UTC The purpose of cloud height index is to reduce excessive Rayleigh scattering as the optical distance increases at high Table 5 Reflectance height level and range of brightness temperature corresponding to height index Height index Reflectance height level Range of brightness temperature (BT) 0 Ground level (0 km) BT ≥ 260 K 1 2 km 260 K > BT ≥ 250 K 2 4 km 250 K > BT ≥ 240 K 3 6 km 240 K > BT ≥ 230 K 4 8 km 230 K > BT ≥ 220 K 5 10 km 220 K > BT Table 5 Reflectance height level and range of brightness temperature corresponding to height index Fig. 3 Step 1: GK-2 A full-disk simple composite of native RGB- band true-color image, taken on 2019/12/01 0300 UTC 1 3 Korean Meteorological Society 340 M. Kim et al. band (510 nm) in the AMI sensor is slightly blue-shifted compared with that of other sensors (MODIS, VIIRS, etc.), which are centered at 550 nm for the green band. Subtle differences produce color changes in true-color imagery, as described by Miller et al. (2016). performed data-blending at the extremities of the SZA and VZA, to remove this reddening edge and achieve true-color imagery after the atmospheric correction process. The cor- rected reflectance data were blended gradually according to the zenith angles, becoming less corrected toward the edge of the limb. The blending factor was calculated in terms of the SZA and VZA, it was linearly decreased from 1.0 to 0.0 over zenith angles from 75° to 90° and 65° to 85°, respectively. performed data-blending at the extremities of the SZA and VZA, to remove this reddening edge and achieve true-color imagery after the atmospheric correction process. The cor- rected reflectance data were blended gradually according to the zenith angles, becoming less corrected toward the edge of the limb. The blending factor was calculated in terms of the SZA and VZA, it was linearly decreased from 1.0 to 0.0 over zenith angles from 75° to 90° and 65° to 85°, respectively. To correct this unrealistic green color, Miller et al. 3 Results (4) Hybrid green = Rgreen × (1 −Fhg ) + Rvege × Fhg (4) 2.2.3 Image Processing (2016) suggested a hybrid green, which is a composite of the origi- nal green band and a vegetation band that is sensitive to chlorophyll signals—this is used to monitor vegetation health. The hybrid green is calculated using the following equation: 1 3 Korean Meteorological Society 3.1 Preprocessing For Atmospheric Correction of TOA Reflectance These rescaled reflectance values were used to compute the cumulative density function (CDF). The last process was to convert the reflectance values by multiplying them with respect to the CDF of each intensity. Figure 5 shows an example of atmospherically corrected GK-2 A full-disk true-color RGB imagery; it indicates that the adjustment for Rayleigh scattering removes much of the atmospheric haze, yields sharper contrasts between the cloud and surface features, and enhances the overall surface detail. Despite the atmospheric corrections, red regions remain present where the atmospheric pathlength is long. This is a result of overcorrection; clouds and aerosols at high zenith angles exert an influence on the visible channel, depending on their height and opacity. The Rayleigh correction does not account for these clouds and aerosols; instead, it assumes a 6S RTM. To resolve this problem, Section 3.2 describes how the LUT factors in target elevation when calculating TOA reflectance. l The reddening of Earth’s limb is a result of overcorrec- tion in the green and blue bands. This is consistent with the results of Lee et al. (2015), who found that the atmospheric correction-coefficient values computed in 6S were dramati- cally increased above a 60° zenith angle. This may be due to limitations of the RT code at high angles, and these limi- tations are difficult to quantify without the corresponding The pixel values at high SZA or VZA can become very large and unrealistic, owing to overcorrection by the 6S RTM. Because the correction breaks down nonlinearly for very long atmospheric pathlengths near the Earth’s limb, the corrected imagery features a reddening edge (Miller et al. 2016). Using the uncorrected reflectance datasets, we Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… 341 Fig. 4 Effect of atmospheric correction on the GK-2 A full-disk visible-band imagery. Upper images represent uncorrected band imagery and lower images represent corrected band imagery Fig. 4 Effect of atmospheric correction on the GK-2 A full-disk visible-band imagery. Upper images represent uncorrected band imagery and lower images represent corrected band imagery Fig. 5 Step 2: example of atmospherically corrected GK-2 A full-disk true-color RGB imagery, same date with Fig. 3 separately and using them in atmospheric pathlength correc- tions, the reddening of the Earth’s limb can be reduced. 3.1 Preprocessing For Atmospheric Correction of TOA Reflectance The 10.4-µm BT used to estimate the reflecting surface height is only approximate; however, the results show that atmos- pheric correction is improved at high zenith angles. 1 3 Korean Meteorological Society Fig. 7 Step 4: comparison of GK-2 A using (a) simple com- posite of native RGB and (b) hybrid RGB on Australia, taken on 2019/11/06 0300 UTC 3.2 Image Processing Figure 7 shows the effects of attenuation in green true-color on the Australia region. The native true-color imagery exag- gerates the reddish soil and dark grass; this is because the vegetation has a peak reflectance of around 550 nm due to the presence of chlorophyll, which is why most of the other sensors’ green bands target this peak (Broomhall et al. 2019). However, the true-color imagery supplemented by hybrid- green color showed more realistic vegetation and a brown soil color. The true-color image applied with hybrid green in Fig. 7 is similar to the MODIS Terra Rayleigh-corrected RGB images with its 555 nm green band. Several other works using the Himawari-8 AHI true-color RGB images applied with hybrid green (Miller et al. 2016; Broomhall et al. 2019) are also consistent with the result from GK2A . Figure 7 shows the effects of attenuation in green true-color on the Australia region. The native true-color imagery exag- gerates the reddish soil and dark grass; this is because the vegetation has a peak reflectance of around 550 nm due to the presence of chlorophyll, which is why most of the other sensors’ green bands target this peak (Broomhall et al. 2019). However, the true-color imagery supplemented by hybrid- green color showed more realistic vegetation and a brown soil color. The true-color image applied with hybrid green in Fig. 7 is similar to the MODIS Terra Rayleigh-corrected RGB images with its 555 nm green band. Several other works using the Himawari-8 AHI true-color RGB images applied with hybrid green (Miller et al. 2016; Broomhall et al. 2019) are also consistent with the result from GK2A . Figure 8 shows the result of applying histogram equali- zation to a Rayleigh-corrected, hybrid green-applied true- color RGB image. The original image on the left-hand side is dim and has poor contrast, whereas the right-hand image is brighter and has better contrast (Fig. 8). The color dis- tributions of R, G, and B before histogram equalization Fig. 5 Step 2: example of atmospherically corrected GK-2 A full-disk true-color RGB imagery, same date with Fig. 3 in situ measurements. It may be possible to improve the correction at the limb by reducing the angular increment in the LUT (Broomhall et al. 2019). Figure 6 shows the effects of correcting for atmospheric pathlength by considering target elevation in the LUT. Fig. 6 Step 3: effect of altitude assignment in look-up table (LUT): (a) fixed at ground level, and (b) applied height using IR brightness temperature (BT) of 10.4 μm 1 3 Korean Meteorological Society 3.2 Image Processing By computing target elevation 1 3 Korean Meteorological Society 342 M. Kim et al. Fig. 6 Step 3: effect of altitude assignment in look-up table (LUT): (a) fixed at ground level, and (b) applied height using IR brightness temperature (BT) of 10.4 μm Fig. 7 Step 4: comparison of GK-2 A using (a) simple com- posite of native RGB and (b) hybrid RGB on Australia, taken on 2019/11/06 0300 UTC Fig. 8 Step 5: comparison of GK-2 A using (a) atmos- pherically corrected true-color RGB and (b) atmospherically corrected and enhanced true- color RGB imagery taken on 2020/01/06 0100 UTC Fig. 6 Step 3: effect of altitude assignment in look-up table (LUT): (a) fixed at ground level, and (b) applied height using IR brightness temperature (BT) of 10.4 μm ng a different center (not ation applied, the color is method is effective in f dark images. As a result, tween cloud surfaces and characteristics of various oreover, for clouds (which e detailed representation utility of this imagery for orrected true-color RGB ected imagery near the are more consistent for the Earth’s limb in comparison to the MODIS imagery; however, in the Rayleigh scattering-corrected true-color imagery, errors increase exponentially along long opti- cal trajectories through the atmosphere. In the blending stage of our proposed procedure, the proportion is varied linearly from 100 to 0 % for SZAs between 80° and 90°. Most edge effects that had not been removed by the previ- ous pathlength corrections were reduced. The procedure also resulted in a more realistic transition between Earth and space than the atmospherically corrected true-color RGB imagery. Figure 9 presents the final true-color image, in which all of the aforementioned techniques have been applied. Fig. 8 Step 5: comparison of GK-2 A using (a) atmos- pherically corrected true-color RGB and (b) atmospherically corrected and enhanced true- color RGB imagery taken on 2020/01/06 0100 UTC the Earth’s limb in comparison to the MODIS imagery; however, in the Rayleigh scattering-corrected true-color imagery, errors increase exponentially along long opti- cal trajectories through the atmosphere. In the blending stage of our proposed procedure, the proportion is varied linearly from 100 to 0 % for SZAs between 80° and 90°. Most edge effects that had not been removed by the previ- ous pathlength corrections were reduced. The procedure also resulted in a more realistic transition between Earth and space than the atmospherically corrected true-color RGB imagery. 3.2 Image Processing Figure 9 presents the final true-color image, in which all of the aforementioned techniques have been applied. the Earth’s limb in comparison to the MODIS imagery; however, in the Rayleigh scattering-corrected true-color imagery, errors increase exponentially along long opti- cal trajectories through the atmosphere. In the blending stage of our proposed procedure, the proportion is varied linearly from 100 to 0 % for SZAs between 80° and 90°. Most edge effects that had not been removed by the previ- ous pathlength corrections were reduced. The procedure also resulted in a more realistic transition between Earth and space than the atmospherically corrected true-color RGB imagery. Figure 9 presents the final true-color image, in which all of the aforementioned techniques have been applied. were narrow, with each color having a different center (not shown). With histogram equalization applied, the color distribution becomes uniform, this method is effective in improving the visual appearance of dark images. As a result, the difficulties of distinguishing between cloud surfaces and the ocean are alleviated, and the characteristics of various surface types can be visualized. Moreover, for clouds (which are highly reflective areas), a more detailed representation is possible, thereby improving the utility of this imagery for assessing cloud textures. In Fig. 9, the atmospherically corrected true-color RGB imagery is blended with uncorrected imagery near the limb. The uncorrected AMI data are more consistent for 343 Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… Fig. 9 Final step: final true-color imagery applying all the aforemen- tioned techniques same date with Fig. 3 sharply as it exceeds 70 degrees. This is because the scatter- ing effect of the original band becomes stronger as the SZA increases. The RMSE of the reflectance difference due to atmospheric correction becomes more noticeable with the transition from the red channel to the blue channel. This result is consistent with the stronger scattering effect toward the blue channel. Blending is a method designed to improve images in areas with SZA greater than 70 degrees, which not only mitigates red areas due to atmospheric over correction at high zenith angle, but also helps with natural representation. As a result, Fig. 11 shows the effect of blending process that reduces the rapid increase in RMSE when only atmospheric correction is applied. 3.2 Image Processing Similar to the application of atmospheric correction effect, the variability according to the season is not large, and the scattering effect increases as the channel wavelength decreases, hence RMSE increases.f Figure 12 shows the averaged RMSE of the difference between the original and atmospheric-corrected reflectance (blue) and difference between the original and blended reflectance after atmospheric correction (red) as a func- tion of VZA. The improvement by atmospheric correction becomes more noticeable as VZA increases. The reason is that the longer the optical path is, the more pronounced is the effect caused by the atmosphere. The improvement effect increases with the blue band. In VZA, the effect of the reduc- tion due to blending varies from band to band and season to season, but it reduces RMSE, as a result, helps improve the quality of images. The averaged RMSE (observation-based reflectivity-atmospheric-corrected reflectance, and atmos- pheric-corrected reflectance-blended reflectance) shows the improvement of the true-color RGB imagery. Although the improvement due to atmospheric correction is not applied in all areas and therefore, the blending technique is applied at high angles, the reflectance error due to the Rayleigh scat- tering is improved. As a result, clear results can be obtained in the actual image. Fig. 9 Final step: final true-color imagery applying all the aforemen- tioned techniques same date with Fig. 3 3.3 Robust Result for Atmospheric correction of GK2A visible bands To obtain robust results for atmospheric correction of the GK2A visible band with visual true-color RGB inspection, we quantitatively examined the results of atmospheric cor- rection based on 6S LUT along with various SZA and VZA conditions. Figure 10(a) and (b) show an example image of the spatial distribution of SZA and VZA at 2020.07.07 03 UTC. Because a geostationary satellite is situated in a fixed location, VZA does not change with time. However, in the case of SZA, it changes greatly depending on the sea- son or time; therefore, in this study, we used the reflectance of 03 UTC in the four seasons. We sampled approximately 121 million pixels per image on four channels of GK2A. Figure 10(c) and (d) show the SZA and VZA distributions of the sampled pixels for quantitative evaluation of reflectance. The distributions are non-uniform for both SZA and VZA. The majority of the samples exist around 40 degrees and the number of samples decreases with distance away from the peak degrees. 1 3 Korean Meteorological Society 3.4 Illustrative Examples True-color imagery is designed to represent Earth in a man- ner similar to how normal human color vision represents it. This real-color imagery helps in identifying surface types (e.g., land, ocean, ice, and snow) and atmospheric proper- ties such as cloud, fog, dust, and ash; it produces impor- tant information by condensing three bands into one image, with the advantage of allowing easy communication of this information. Therefore, it is useful in enabling people to intuitively interpret weather phenomena without special training, and it can be very helpful to both forecasters and the general public. Figure 11 shows the averaged root mean square error (RMSE) of the difference between the original and atmos- pheric-corrected reflectance (blue) and difference between the original and blended reflectance after atmospheric cor- rection (red) as a function of SZA for each band per sea- son. Although there are slight differences depending on the season, the reflectance RMSE range for all sampled data is 0–0.5. In general, the RMSE due to atmospheric correction increases with the SZA. In particular, the RMSE increases There are several examples of true-color RGB imagery being employed to consider weather events. In satellite 1 3 Korean Meteorological Society 1 3 Korean Meteorological Society M. Kim et al. 344 Fig. 10 Spatial distribution of (a) SZA and (b) VZA at 2020.07.07 03 UTC. Histogram of (c) SZA and (d) VZA of sample pixels Fig. 10 Spatial distribution of (a) SZA and (b) VZA at 2020.07.07 03 UTC. Histogram of (c) SZA and (d) VZA o observation, the texture of fog is flat and edges of the clouds are sharp. Nevertheless, the cloud environments at low levels cannot be evaluated because the satellite detects energy from reflecting or radiating surfaces. For the same reason, fog is difficult to distinguish from low cloud, because they both generate optically thick and warm cloud textures. GK2A RGB fog imagery is used to detect fog and low-level cloud. This imagery uses different composites of red, green, and blue channels for day and night; however, fog and low cloud are colored as cyan. One method of distinguishing fog from low cloud is that of animating cloud patterns. The move- ment of fog is relatively slow compared with that of low cloud, and the surface texture of fog is flat. Figure 13 shows an example of true-color and RGB fog images taken of the Korean Peninsula. 1 3 Korean Meteorological Society 3.4 Illustrative Examples The fog and lower clouds are located at the West Sea and the Liaodong Peninsula; they are distin- guishable from the high, thick clouds over the Shandong Peninsula (red box). However, it is difficult to discriminate low-level cloud using the GK2A fog imagery, because they are represented by similar colors and shapes. In the true- color RGB imagery, they have different external cloud sur- faces; fog has a smooth surface and is opaque owing to its optical thickness. The animated image facilitates effective classification because the fog is more static than the low- level cloud. Another example is that of sea-ice images (Fig. 14). The GK2A RGB snow and fog image (right) represents snow and sea-ice in red. There is a large quantity of snow over land, and sea-ice is widespread over the Sea of Okhotsk (red area). This is also found in true-color imagery. Sea-ice has 1 3 Korean Meteorological Society Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… 345 Fig. 11 Averaged RMSE of the difference between atmospheric corrected and blended reflectance on the original reflectance as a function of SZA for each band of (a) spring, (b) summer, (c) fall, and (d) winter season Fig. 11 Averaged RMSE of the difference between atmospheric corrected and blended reflectance on the original reflectance as a function of SZA for each band of (a) spring, (b) summer, (c) fall, and (d) winter season the same color as cloud; however, it differs in terms of its shape and movements, sea-ice does not undergo changes of area coverage during the daytime. (red box) which is the most dangerous and destructive fea- ture of typhoon and clear shape of convective structure. Right part of Fig. 16 also shows typhoon MAYSAK in enhanced color IR imagery. It highlights convective struc- ture by repeating color and gray scale compared with tradi- tional IR color scheme so in this figure, the structure of the typhoon and eyewall is more clearly separated. Yellow dust is a weather phenomenon that occurs in desert areas in China and Mongolia, and in the middle Yel- low River, where it is generated by strong winds or terrain and falls to the surface during long-distance transportation. The Fig. 15 shows the example of yellow dust in true-color imagery. The red box indicates yellow dust which represents as yellow and brown color and it travels with clouds. 3.4 Illustrative Examples The Fig. 15 on the right is dust RGB from GK-2 A which shows yellow dust as red and hot pink color at the same time with true-color RGB imagery. 1 3 Korean Meteorological Society 4.1 6S Parameterization for Building an LUT The 6S uses sensitive RT code to predict a satellite signal at the satellite level for a Lambertian surface. Several param- eters are required for it to make good estimations. However, Figure 16 shows the typhoon MAYSAK (2020) in true- color imagery and enhanced color IR imagery from GK2A. Example of true-color imagery shows well-defined eyewall 1 3 Korean Meteorological Society M. Kim et al. 346 Fig. 12 Averaged RMSE of the difference between atmospheric corrected blended reflectance on the original reflectance as a function of VZA for each band of (a) spring, (b) summer, (c) fall, and (d) winter season Fig. 12 Averaged RMSE of the difference between atmospheric corrected blended reflectance on the original reflectance as a function of VZA for each band of (a) spring, (b) summer, (c) fall, and (d) winter season maritime, urban, and desert). By implementing surface-type information, reflectance can be calculated using the coef- ficients derived from the LUT. Models differ through using different processes and atmospheric profiles. 6S calculates reflectance in terms of a bidirectional reflectance distribution function (BRDF) model. Numerous BRDF subroutines in 6S require a parameter for computing reflectance. Utilization of the 6SV2.1’s detailed, in-built atmosphere models (which can more effectively address latitudinal and seasonal differ- ences in the global atmosphere) can increase accuracy and may be implemented using the same LUT structure. accurate model construction takes a lot of time; thus, a bal- ance between simplicity and elaboration is required when setting the model configuration. Fortunately, the 6S model contains pre-prepared atmospheric conditions, which enable the specification of seasons (summer and winter), latitudi- nal zone (tropical, middle-latitude, and subarctic), the US standard atmosphere model (US62), and user-defined condi- tions; thus, it is possible to improve the model’s ability with- out requiring the in-depth consideration of seasonality, the selection of a suitable molecular atmosphere model, or the frequent provision of input atmosphere data. During the pro- cess of atmospheric correction, an LUT that contains season- ally and latitudinally separated coefficients may give more accurate solutions. Furthermore, the conditions are sensitive to the aerosol model used; 6S sets “no aerosol” for simplic- ity and offers a number of land-type options (continental, The computational costs involved in constructing an LUT introduces limitations to the design parameter con- ditions. 1 3 Korean Meteorological Society 1 3 Korean Meteorological Society 4.1 6S Parameterization for Building an LUT 16 Example of typhoon MAYSAK (2020) in East China Sea for true-color (left) and enhanced IR imagery (right), taken on 2020/09/01 0200 UTC 6S transmittance from that of the MODTRAN—is small, at around 3.5 %. This is a result of the different assumptions used in calculating the light scattering process (Lee et al. 2020). desirable whenever comparisons are to be made with data acquired under different atmospheric or geometric con- ditions. The absolute atmospheric correction of optical, remotely sensed data relies on RT codes. Several RT codes are available with different features; however, the most popular RT codes are 6S and MODTRAN. Callieco and Dell’Acqua (2011), compared two RT codes under identi- cal geometric and atmospheric profile conditions. Their results showed that the mean relative difference between the simulated transmittances—obtained by subtracting the 4.1 6S Parameterization for Building an LUT One solution to this problem is to interpolate the LUT using an appropriate method, the MCS technique is one such validated method; it improves the quality of surface 1 3 Korean Meteorological Society 347 Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… Fig. 13 Example of fog in the Korean Peninsula for true-color (left) and fog (right) RGB imagery, taken on 2020/02/11 0300 UTC Fig. 13 Example of fog in the Korean Peninsula for true-color (left) and fog (right) RGB imagery, taken on 2020/02/11 0300 UTC Fig. 14 Example of sea-ice in the East Asia for true-color (left) and day-snow-fog (right) RGB imagery, taken on 2020/02/20 0300 UTC Fig. 14 Example of sea-ice in the East Asia for true-color (left) and day-snow-fog (right) RGB imagery, taken on 2020/02/20 0300 UTC Fig. 14 Example of sea-ice in the East Asia for true-color (left) and day-snow-fog (right) RGB imagery, ta reflectance data, by interpolating the 6S LUT for applica- tion to the Himawari-8 AHI. Specifically, at high SZAs (greater than 75° SZA), improvements of 45.1 %, 39.6 %, 19.8 %, and 12.57 % in the relative root mean square errors were observed for Channels 1–4, respectively (Li et al. 2019). This method was here proposed for application to the GK2A AMI, and the results obtained were—when the atmospheric coefficients increased exponentially with the SZA and VZA—also consistent with those of the linearly interpolated method. The trends of the atmospheric coef- ficients also confirm that, for GK2A, xa is dependent only on the SZA, and xb is dependent on both the SZA and VZA (not shown); this corresponds with the results of Li et al. (2019) for Himawari-8 AHI.f Correction of the atmospheric distortion effects caused by molecular and particulate scattering and absorption is 1 3 Korean Meteorological Society M. Kim et al. 348 Fig. 15 Example of dust in the East Asia for true-color (left) and dust (right) RGB imagery, taken on 2021/03/15 0200 UTC Example of dust in the East Asia for true-color (left) and dust (right) RGB imagery, taken on 2021/03/15 0200 UTC Fig. 16 Example of typhoon MAYSAK (2020) in East China Sea for true-color (left) and enhanced IR imagery (right), taken on 2020/09/01 0200 UTC n MAYSAK (2020) in East China Sea for true-color (left) and enhanced IR imagery (right), taken on 2020/09/01 Fig. 1 3 Korean Meteorological Society 4.3 Image Enhancement At the extremities of SZA and VZA, data are blended for aesthetic reasons (Miller et al. 2016; Broomhall et al. 2019) applied this method—although in a slightly edited form—by moving the input-uncorrected RGB composite of the visible band into the infrared band. Overcorrected reflectance—which produces a red edge—can be blended with BT at the solar transition region. As exemplified by Broomhall et al. (2019), the blended imagery at high SZAs effectively removed the red edge along the Earth’s limb. Figure 17 displays the result of blending using a BT of 10.4 μm for GK2A. The visible imagery is blended with black at the limb, with the propor- tion varying linearly from 100 to 0 % for VZAs between 75° and 90°. Some discussions remain around Rayleigh scattering at high zenith angles. This region contains very long optical paths, and high cloud can lead to further erroneous correc- tion. The blending of Earth’s limb regions entails the dif- ficult task of discerning which region suffers the largest Rayleigh scattering effect. Broomhall et al. (2019) used a BT of 10.4 μm and alternatively applied another blending, instead of an uncorrected visible composite; this removed red edges and improved continuous nighttime scenes. Fig- ure 17 shows an example of application of this method to GK2A. Although there is already a natural darkening around Earth’s edge, brownish shadows may still appear in some regions where the SZA is large. This imagery technique has an additional advantage in that it provides visually continuous information during night- time. The histogram equalization enhances color contrasts by redistributing the color histogram. The effect of histogram equalization is a distinctive brightening of the true-color images. However, the color distribution may be narrowed by the amount of light in the sample, such as during sunset or sunrise. Figure 18 shows a true-color scene at various times. The solar noon case shows Earth with a refined color distribu- tion. As expected, it depicts ocean, land, cloud, and vegetation well. Images taken at other times—such as during sunrise and sunset—are low in brightness; in these, almost all features except clouds are shaded. The lower panels show the histo- grams of normalized reflectance—for pixel values between 0 and 255—before (dotted) and after (line) applying histogram equalization. The shape of the histogram before equalization in the solar noon case differs from the others, which exhibit wider distributions. 4.2 Limitation of 6S RTM Earth has a spherical-shell atmosphere (SSA); however, almost all RT codes—including those of 6SV—are based on a parallel-plane atmosphere model, which assumes that 1 3 Korean Meteorological Society 349 Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… the Earth’s surface and atmosphere are flat. In this case, RTMs tend to overcorrect the TOA reflectance above SZAs and VZAs of 70°; this issue is exacerbated when the two zenith angles approach one another. To resolve this problem, the Monte Carlo solver is an ideal approach, outperforming the SOS approximation (used in 6SV) or discrete ordinate (DISORT) code solver (used in MODTRAN, PSTAR, etc.); however, this method is not suitable for constructing an LUT because the computational cost is excessively high. Recently, an RT solver previously used in SOS and DISORT has been developed for improving the SSA model through pseudo- correction (Callieco and Dell’Acqua 2011; He et al. 2018). The atmospheric correction could succeed for high zenith angles, where the RT code alters the SSA structure model. the Earth’s surface and atmosphere are flat. In this case, RTMs tend to overcorrect the TOA reflectance above SZAs and VZAs of 70°; this issue is exacerbated when the two zenith angles approach one another. To resolve this problem, the Monte Carlo solver is an ideal approach, outperforming the SOS approximation (used in 6SV) or discrete ordinate (DISORT) code solver (used in MODTRAN, PSTAR, etc.); however, this method is not suitable for constructing an LUT because the computational cost is excessively high. Recently, an RT solver previously used in SOS and DISORT has been developed for improving the SSA model through pseudo- correction (Callieco and Dell’Acqua 2011; He et al. 2018). The atmospheric correction could succeed for high zenith angles, where the RT code alters the SSA structure model. The solar radiation directly reflected by water surfaces is computed exactly using the Snell–Fresnel laws in the 6S; how- ever, the sun-glint problems remain unresolved. System vicari- ous calibration (SVC) enables relative radiometric calibration to be achieved for satellite ocean color sensors. This could minimize uncertainty in the water-leaving radiance measured from TOA radiance and resolve the problems cause by sun glint (Zibordi et al. 2015). 4.3 Image Enhancement After histogram equalization was applied, their shape became very similar. This suggests that corrective color redistribution may be possible; however, the input dataset has a low performance in discriminating subtle differences in the reflecting surface type. Fig. 17 Result of blending using a brightness temperature (BT) of 10.4 μm for GK2A. The visible imagery is blended with black at the limb, with the proportion varying linearly from 100–0 % for VZAs between 75° and 90° same date with Fig. 8 1 3 Korean Meteorological Society 5 Conclusions Fig. 17 Result of blending using a brightness temperature (BT) of 10.4 μm for GK2A. The visible imagery is blended with black at the limb, with the proportion varying linearly from 100–0 % for VZAs between 75° and 90° same date with Fig. 8 This paper described the production of atmospherically cor- rected, true-color GK2A RGB imagery. The main process is separated into absolute atmospheric correction and relative 1 3 Korean Meteorological Society 1 3 Korean Meteorological Society 350 M. Kim et al. Fig. 18 Example of true-color imagery and frequency plot of reflectance before (dotted) and after (line) histogram equalization Fig. 18 Example of true-color imagery and frequency plot of reflectance before (dotted) and after (line) histogram equ Fig. 18 Example of true-color imagery and frequency plot of reflectance before (dotted) and after (line) histogram equalization atmospheric correction, based on image-processing tech- niques. LUTs represent a practical approach to performing atmospheric corrections that require the processing of large quantities of data (GK2A produces 22,000 × 22,000 and 11,000 × 11,000 pixel scenes of 0.5 and 1 km resolutions every 10 min, respectively). an appropriate elevation model and cloud-height product; thus, many more RT computations would need to be per- formed (a unique set for each selected pathlength), and an extra dimension would be required in the LUT structure. It is unlikely that the visible appearance of the true-color imagery would be significantly improved by including path- length as an extra variable in the atmospheric correction process, except perhaps for the highest VZAs and SZAs. To quantitatively confirm the effect of atmospheric correction, RMSE was analyzed in accordance with SZA and VZA and found to be in the range of 0–0.5. As SZA increases, the effect of atmospheric scattering is enhanced and the RMSE due to atmospheric correction increases. In particular, RMSE increases significantly as it exceeds 70 degrees. As the scattering is stronger as it goes to the shorter band, the improvement of the reflectivity is noticeable. To generate true-color imagery, all datasets must have an identical spatial resolution; thus, the visible (excluding the 640 nm band), vegetation, and infrared channels were downscaled to 500 m (the spatial resolution of the 640 nm band—the sharpest channel). During statistical downscal- ing, spline interpolation was used to sharpen resolution. 1 3 Korean Meteorological Society g g p doi.org/10.1117/12.366315 Even after atmospheric correction, true-color imagery appeared dark and lacked contrast. Histogram equalization, which adjusts the brightness and contrast of images by redis- tributing the histogram of the RGB composite, was used to produce vivid true-color images. True-color information provides a practical method for interpreting a wide variety of environmental phenomena. High-temporal-resolution true-color imagery from the GK2A AMI provides a tool for scientists and forecasters to visualize the changing Earth and has considerable potential to engage the general public in an intuitive manner. We anticipate that our procedure, with its successful integration of a number of sub-processes pertaining to the GK2A AMI, will constitute a significant step in this direction. Agust-Panareda, A, Massart, S, Chevallier, F, Boussetta, S, Balsamo, G, Beljaars, A: Forecasting global atmospheric CO 2. Atmos. Chem. Phys. 14(21), 11959–11983 (2014). https://doi.org/10. 5194/acp-14-11959-2014 Bah, M.K., Gunshor, M.M., Schmit, T.J.: Generation of GOES-16 true color imagery without a green band. Earth Space Sci. 5(9), 549–558 (2018). https://doi.org/10.1029/2018EA000379 Berka, A., Anderson, G.P., Bernstein, L.S., Acharya, P.K., Dothe, H., Matthew, M.W., Adler-Golden, S.M., et al.: MODTRAN4 radia- tive transfer modeling for atmospheric correction. Optical spectro- scopic techniques and instrumentation for atmospheric and space research III. International Society for Optics and Photonics, 3756, pp. 348–353 (1999) Bessho, K., et al.: An introduction to Himawari-8/9—Japan’s new-gen- eration geostationary meteorological satellites. J. Meteor. Soc Jpn. 94(2), 151–183 (2016). https://doi.org/10.2151/jmsj.2016-009 Broomhall, M.A., Majewski, L.J., Villani, V.O., Grant, I.F., Miller, S.D.: Correcting Himawari-8 advanced himawari imager data for the production of vivid true-color imagery. J. Atmos. Ocean. Technol. 36(3), 427–442 (2019) Authors’ Contributions Minsang Kim conceived and designed the experiments; Minsang Kim performed the experiments; Minsang Kim, Jun-Hyung Heo, and Eun-Ha Sohn analyzed the data; Minsang Kim, Jun-Hyung Heo, and Eun-Ha Sohn contributed materials and analysis tools; Minsang Kim and Jun-Hyung Heo wrote the paper; Eun-Ha Sohn managed the whole process of the research. Callieco, F., Dell’Acqua, F.: A comparison between two radiative trans- fer models for atmospheric correction over a wide range of wave- lengths. Int. J. Remote Sens. 32(5), 1357–1370 (2011) Csiszar, I., Gutman, G.: Mapping global land surface albedo from NOAA AHVRR. J. Geophys. Res-Atmos. 104(D6), 6215–6228 (1999). https://doi.org/10.1029/1998JD200090 Dorji, P., Fearns, P.: Atmospheric correction of geostationary Hima- wari-8 satellite data for total suspended sediment mapping: a case study in the coastal waters of Western Australia. ISPRS J. Pho- togramm. Remote Sens. 144, 81–93 (2018). 5 Conclusions An LUT was built considering the geometric parameters (SZA, VZA, and RAA), atmospheric parameters (TCO, TPW, and AOD), and reflectance height from the ground (up to 10 km). The three pre-calculated atmospheric correction coefficients were stored in the LUT. To augment the LUT, an MCS tech- nique was applied. l Each original band that composes a true-color image has a different spatial resolution. To match the resolutions, spline interpolation was used. Downscaled imagery is expected to express coastlines and cloud surfaces in detail. Utilizing the methods of Miller et al. (2016), it is possible to produce a hybrid green band that better matches the peak reflectance of chlorophyll (~ 555 nm). The initial atmospherically cor- rected true-color RGB images contained large, unrealistic values for long atmospheric pathlengths near Earth’s limb, owing to overcorrection by the 6S RTM (Miller et al. 2016). The RTM can be extended to distinguish cloud-top heights. In this case, the pathlength is described for each pixel, which avoids the requirement for pathlength cor- rection during the atmospheric correction process. These enhancements have the potential to improve the accuracy of the Rayleigh-corrected reflectance, particularly at higher SZAs and VZAs. Adding the RTM’s capacity to perform Rayleigh corrections with defined pathlengths would require 1 3 Korean Meteorological Society 351 Atmospheric Correction of True‑Color RGB Imagery with Limb Area‑Blending Based on 6S and… One method of overcoming this difficulty is to blend the atmospherically corrected true-color RGB imagery with uncorrected imagery near the limb. It was found that the quantitative improvement of the reflectivity according to the blending technique increased the correction effect according to the cloud height as the VZA increased. g g p doi.org/10.1117/12.366315 https://doi.org/10. 1016/j.isprsjprs.2018.06.019 Funding This work was funded by the Korea Meteorological Adminis- tration’s Research and Development Program “Technical Development on Weather Forecast Support and Convergence Service using Meteoro- logical Satellites” under Grant (KMA2020-00120). Engelen, R.J., McNally, A.P.: Estimating atmospheric CO 2 from advanced infrared satellite radiances within an operational four- dimensional variational (4d-Var) data assimilation system: results and validation. J. Geophys. Res. 110, 18305 (2005). https://doi. org/10.1029/2005JD005982 Data Availability Not applicable. Code Availability Not applicable. Franch, B., Vermote, E.F., Sobrino, J.A., Fédèle, E.: Analysis of direc- tional effects on atmospheric correction. Remote Sens. Environ. 128, 276–288 (2013) References Adler-Golden, S.M., Matthew, M.W., Bernstein, L.S., Levine, R.Y., Berk, A., Richtsmeier, S.C., Acharya, P.K., et al.: Atmospheric correction for shortwave spectral imagery based on Modtran4. Proc. SPIE 3753 Imaging Spectrometry V: 61–69 (1999). https:// doi org/10 1117/12 366315 1 3 Korean Meteorological Society Declarations Fukushima, H., Higurashi, A., Mitomi, Y., Nakajima, T., Noguchi, T., Tanaka, T., Toratani, M.: Correction of atmospheric effect on ADEOS/OCTS ocean color data: algorithm description and evaluation of its performance. J. Oceanogr. 54(5), 417–430 (1998) Conflicts of Interest/Competing Interests The authors declare no con- flict of interest. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, 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/. Geiger, B., Carrer, D., Franchisteguy, L., Roujean, J.-L., Meurey, C.: Land surface albedo derived on a daily basis from Meteosat sec- ond generation observations. IEEE Trans. Geosci. Remote Sens. 46(11), 3841–3856 (2008). https://doi.org/10.1109/TGRS.2008. 2001798 Ghulam, A., Qin, Q., Zhu, L.: 6S model-based atmospheric correction of visible and near-infrared data and sensitivity analysis. Acta Sci. Nat. Univ. Pekin. 40, 611–618 (2004) Gordon, H.R.: Radiative transfer in the atmosphere for correction of ocean color remote sensors. In Ocean Colour: Theory and appli- cations in a decade of CZCS experience, edited by Barale V. and Schlittenhardt P.M. Eurocourses: Remote Sens. 3, 33–77 (1993) He, T., Zhang, Y., Liang, S., Yu, Y., Wang, D.: Developing land sur- face directional reflectance and albedo products from geostation- ary GOES-R and Himawari data: theoretical basis, operational 1 3 Korean Meteorological Society 352 M. Kim et al. implementation, and validation introducing the next generation advanced baseline imager on GOES-R. Remote Sens. 11(22), 2655 (2019)f Rahman, H., Dedieu, G.: SMAC: a simplified method for the atmos- pheric correction of satellite measurements in the solar spectrum. Remote Sens. 15(1), 123–143 (1994) Richter, R.: Atmospheric correction of DAIS hyperspectral image data. Comput. Geosci. 22(7), 785–793 (1996) He, X., Stamnes, K., Bai, Y., Li, W., Wang, D.: Effects of earth cur- vature on atmospheric correction for ocean color remote sensing. Remote Sens. Environ. AMS-D-15-00230.1 Lee, K.S., Lee, C.S., Seo, M., Choi, S., Seong, N.H., Jin, D., Yeom, J.M., Han, K.S.: Improvements of 6S look-up-table based sur- face reflectance employing minimum curvature surface method. Asia-Pac. J. Atmos. Sci. 56, 1–14 (2020). https://doi.org/10.1007/ s13143-019-00164-3 Smith, W.H.F., Wessel, P.: Gridding with continuous curvature splines in tension. Geophys 55, 293–305 (1990)l Sriwongsitanon, N., Surakit, K., Thianpopirug, S.: Influence of atmos- pheric correction and number of sampling points on the accuracy of water clarity assessment using remote sensing application. J. Hydrol. 401(3–4), 203–220 (2011) Li, H., He, X., Shanmugam, P., Bai, Y., Wang, D., Huang, H.: Radio- metric sensitivity and signal detectability of ocean color satel- lite sensor under high solar zenith angles. IEEE Trans. Geosci. Remote Sens. 57(11), 8492–8505 (2019) Strugnell, N.C., Lucht, W.: An algorithm to infer continental-scale albedo from AVHRR data, land cover class, and field observa- tions of typical BRDFs. J. Clim. 14(7), 1360–1376 (2001). https:// doi.org/10.1175/1520-0442(2001)014 Liang, S., Fang, H., Chen, M.: Atmospheric correction of landsat ETM + land surface imagery. I. methods. IEEE Trans. Geosci. Remote Sens. 39, 2490–2498 (2001). https://doi.org/10.1109/36.964986 Vermote, E., Tanre, D., Deuze, J.L., Herman, M., Morcette, J.-J.: Sec- ond simulation of the satellite signal in the Solar Spectrum-Vector (6SV): an overview. IEEE Transact. Geosci. Remote Sens. 35, 675–686 (2006) Lyapustin, A., Martonchik, J., Wang, Y., Laszlo, I., Korkin, S.: Multi- angle implementation of atmospheric correction (MAIAC): 1. Radiative transfer basis and look-up tables. J. Geophys. Res. Atmos. 116, 3 (2011). https://doi.org/10.1029/2010JD014986 Wang, M.: Rayleigh radiance computations for satellite remote sens- ing: accounting for the effect of sensor spectral response function. Opt. Express 24, 12414–12429 (2016). https://doi.org/10.1364/ OE.24.012414 Massart, S., Agustí-Panareda, A., Heymann, J., Buchwitz, M., Cheval- lier, F., Reuter, M.: Ability of the 4-D-var analysis of the GOSAT BESD XCO 2 retrievals to characterize atmospheric CO 2 at large and synoptic scales. Atmos. Chem. Phys. 16(3), 1653–1671 (2016). https://doi.org/10.5194/acp-16-1653-2016 Yang, J, Zhang, Z, Wei, C, Lu, F, Guo, Q: Introducing the new genera- tion of Chinese geostationary weather satellites, Fengyun-4. Bull. Am. Meteor. Soc. 98(8), 1637–1658 (2017). https://doi.org/10. 1175/BAMS-D-16-0065.1 Miller, S.D., Schmit, T.L., Seaman, C.J., Lindsey, D.T., Gunshor, M.M., Kohrs, R.A., Sumida, Y., Hillger, D.: A sight for sore eyes: the return of true color to geostationary satellites. Bull. Am. Mete- orol. Soc. 97(10), 1803–1816 (2016) Zhou, J., Wang, J., Li, J., Hu, D.: Atmospheric correction of PROBA/ CHRIS data in an urban environment. Int. J. Remote Sens. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Declarations 209, 118–133 (2018) Richter, R., Schlapfer, D., Muller, A.: An automatic atmospheric cor- rection algorithm for visible/NIR imagery. Int. J. Remote Sens. 27(10), 2077–2085 (2006) Inness, A., Ades, M., Agust-Panareda, A., Barré, J., Benedictow, A., Blechschmidt, A.-M.: The CAMS re-analysis of atmospheric com- position. Atmos. Chem. Phys. 19(6), 3515–3556 (2019). https:// doi.org/10.5194/acp-19-3515-2019 Ruddick, K., Neukermans, G., Vanhellemont, Q., Jolivet, D.: Chal- lenges and opportunities for geostationary ocean colour remote sensing of regional seas: a review of recent results. Remote Sens. Environ. 146, 63–76 (2014). https://doi.org/10.1016/j.rse.2013. 07.039 Karpouzli, E., Malthus, T.: The empirical line method for the atmos- pheric correction of IKONOS imagery. Int. J. Remote Sens. 24(5), 1143–1150 (2003) Kim, D., Gu, M., Oh, T.H., Kim, E.K., Yang, H.J.: Introduction of the advanced meteorological imager of Geo-Kompsat-2a: In- orbit tests and performance validation. Remote Sens. 13(7), 1303 (2021). https://doi.org/10.3390/rs13071303 Schaaf, C.B., Gao, F., Strahler, A.H., Lucht, W., Li, X., Tsang, T., Strugnell, N.C.: First operational BRDF, albedo and nadir reflec- tance products from MODIS. Remote Sens. Environ. 83(1–2), 135–148 (2002). https://doi.org/10.1016/S0034-4257(02)00091-3fi Lee, C.S., Yeom, J.M., Lee, H.L., Kim, J.J., Han, K.S.: Sensitivity analysis of 6S-based look-up table for surface reflectance retrieval. Asia-Pac. J. Atmos. Sci. 51, 91–101 (2015). https://doi.org/10. 1007/s13143-015-0062-9 Schmit, T., Griffith, P., Gunshor, M., Daniels, J., Goodman, S., Lebair, W.: A closer look at the ABI on the GOES-R series. Bull. Amer. Meteor. Soc. 98(4), 681–698 (2016). https://doi.org/10.1175/ BAMS-D-15-00230.1 AMS-D-15-00230.1 32(9), 2591–2604 (2011). https://doi.org/10.1080/01431161003698443 Proud, S.R., Fensholt, R., Rasmussen, M.O., Sandholt, I.: (2010). A comparison of the effectiveness of 6S and SMAC in correcting for atmospheric interference of Meteosat Second Generation images. J. Geophys. Res. Atmos. 115(D17) Zibordi, G, Melin, F, Voss, KJ, Johnson, BC, Franz, BA, Kwiatkowska, E: System vicarious calibration for ocean color climate change applications: Requirements for in situ data. Remote Sens. Environ. 159, 361–369 (2015) Qu, Z., Kindel, B.C., Goetz, A.F.: The high accuracy atmospheric correction for hyperspectral data (HATCH) model. IEEE Trans. Geosci. Remote Sens. 41(6), 1223–1231 (2003) 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. Rabah, M, Kaloop, M: The use of minimum curvature surface tech- nique in geoid computation processing of Egypt. Arab. J. Geosci. 6(4), 1263–1272 (2013) 1 3 Korean Meteorological Society
https://openalex.org/W2802628893	https://sat.bntu.by/jour/article/download/1484/1388	Russian	null	Problems of Bridge Construction Longevity and Methods of Its Increas	DOAJ (DOAJ: Directory of Open Access Journals)	2,004	cc-by	2,278	Проф. ЛЯХЕВИЧ Г. Д , аспиранты МАКСИМЕНКО А. А., ПАСТУШКОВ В. Г., ГРЕЧУХИН В. А. Белорусский национальный технический университет тельствует об актуальности проблемы защи ты мостовых конструкций, работающих в ус ловиях агрессивного действия окружающей среды. Результаты обследования мостов Республи ки Беларусь показали, что значительная их часть находится в плохом или неудовлетвори тельном состоянии. Основные проблемы низ кой долговечности мостовых конструкций свя заны с неблагоприятными экологическими ус ловиями, а именно: повышенным содержанием С02, S02, хлоридов, превышающих допусти мые нормы непосредственно на мосту; повы шенной вибрационной нагрузкой в результате увеличения интенсивности движения. Опыт эксплуатации мостов и тоннелей (особенно расположенных в городской черте) показал, что наибольшее коррозионное воздействие на них оказывают хлориды металлов, которые входят в состав реагентов, используемых для обработ ки проезжей части улиц, дорог. Ежегодно в нашей стране для этой цели применяют более 750 тыс. т солевых композиций [1], главными составляющими которых являются: техниче ская соль и сильвинит (побочный продукт про изводства калийных удобрений ПО «Беларусь- калий»). Основной компонент указанных мате риалов - хлористый натрий. Водные растворы этих материалов, а также разбавленные серни стая кислота и углекислота проникают к мосто вым конструкциям через покрытие проезжей части и подвергают их коррозионному разру шению. Это приводит к существенному сокра щению сроков эксплуатации мостов. Ущерб от такого вида коррозии мостовых конструкций в Беларуси не подсчитывался, хотя он весьма существен не только для нашей республики, но и других стран. Так, в США ежегодные потери от коррозии бетонных мостовых конструкций составляют более 150 млн дол. [1]. Это свиде Другой не менее значимой проблемой дол говечности мостовых и тоннельных конструк ций является то, что одновременно с негатив ным влиянием указанных выше веществ на конструкции действует вибрация от проез жающего автомобильного транспорта и пеше ходов. То есть в течение суток и всего периода эксплуатации мостов и тоннелей соли и кисло ты в условиях вибрационного воздействия ин тенсивно корродируют металл и бетон. Про блемой низкой долговечности мостов является и тот факт, что коррозия материалов усилива ется с изменением температуры за счет при родных факторов и в результате выброса отра ботавших газов. Серьезную проблему для дол говечности представляет синергизм действия приведенных выше агрессивных веществ в твердой, жидкой и газовых фазах, положитель ных и отрицательных температур, повышенной влажности, осадков в виде дождя и снега, а также других факторов. Не менее важной про блемой существенного снижения долговечно сти бетонных и железобетонных конструкций представляет их неэффективная гидроизоляция, которая подвергается быстрому старению и разрушению. АРХИТЕКТУРА И СТРОИТЕЛЬСТВО АРХИТЕКТУРА И СТРОИТЕЛЬСТВО Проф. ЛЯХЕВИЧ Г. Д , аспиранты МАКСИМЕНКО А. А., ПАСТУШКОВ В. Г., ГРЕЧУХИН В. А. Белорусский национальный технический университет Проф. ЛЯХЕВИЧ Г. Д , аспиранты МАКСИМЕНКО А. А., ПАСТУШКОВ В. Г., ГРЕЧУХИН В. А. Проф. ЛЯХЕВИЧ Г. Д , аспиранты МАКСИМЕНКО А. А., ПАСТУШКОВ В. Г., ГРЕЧУХИН В. А. В этой связи необходимо рассмотреть неко торые пути повышения долговечности мостов, работающих в неблагоприятных экологических условиях: • повышение коррозионной стойкости ка менных материалов и арматуры, используемых для изготовления мостовых конструкций; • повышение коррозионной стойкости ка менных материалов и арматуры, используемых для изготовления мостовых конструкций; Вестник БИТУ, № 4, 2004 5 Архитектура и строительство • использование оптимального минералоги ческого и полидисперсного состава цементного клинкера; веществ; ПГФ - парогазовая фаза; ТП - твердая поверхность (гидроизоляция); а ^ Ф, о®вхв > ПГФ а ВРХв ~ поверхностные натяжения на границе раздела соответственно: парогазовая фаза - твердая поверхность; водные растворы химиче ских веществ - твердая поверхность; парогазо вая фаза - водные растворы химических ве ществ. • использование оптимального минералоги ческого и полидисперсного состава цементного клинкера; • понижение водоцементного отношения при условии использования эффективных пла стифицирующих добавок; • создание новых технологий приготовления бетонных смесей и созревания цементного камня с применением гидрофобизующих, коль- матирующих и антистарительных веществ; В зависимости от природы твердой поверх ности, концентрации водных растворов хими ческих веществ, а также химического соста ва парогазовой фазы возможны следующие виды смачиваемости твердой поверхности: а) не смачиваемая, краевой угол фв£хв > 90°, ВРХВ a cos(pTn имеет отрицательное значение; б) смачиваемая, краевой угол ср?™ < 90°, а coscpyj-j ^ 0. • создание бетонов сверхплотной микро- и макроструктуры с минимальным количеством капилляров, пустот и других дефектов; при этом Дб > ііц.кам и і?б стремится к і?зап (здесь Дб, Яц.кам> Л3ап - предел прочности на сжатие соот ветственно бетона, цементного камня, заполни теля). Необходимо обратить особое внимание на создание прочной структуры на границе це ментный камень - заполнитель. Здесь хранится ключ к созданию сверхпрочного, и самое глав ное, долговечного бетона; Поверхностные натяжения а ^ ф и ПГФ а ВРХВ значительно меньше, чем поверхностное натя жение а в£хв • В неблагоприятных экологиче- ПГФ ских условиях поверхностные натяжения ПГФ и а врхв стремятся к минимальным значениям. Поверхностное натяжение на границе раздела водные растворы химических веществ - твер дая поверхность в неблагоприятных экологиче- ВРХВ ских условиях о н тп существенно снижается и с н.тп <<; °тп • Условием надежной и долго вечной гидроизоляции является тот факт, что а ? Г должно иметь максимальное значение, а °н.тг? - приближаться-к (Здесь о®™ - поверхностное натяжение в неблагоприятных экологических условиях на границе раздела водные растворы химических веществ - твер- дая поверхность.) Так как с тп имеет наи большее значение для слабополярных твердых поверхностей, решение проблемы надежной и долговечной гидроизоляции сводится к выбору гидроизоляционного материала с наименьшей полярностью. Проф. ЛЯХЕВИЧ Г. Д , аспиранты МАКСИМЕНКО А. А., ПАСТУШКОВ В. Г., ГРЕЧУХИН В. А. • создание новых конструкций из бетона и железобетона, исключающих попадание агрес сивных средств на их поверхность; • обработка поверхности конструкций раз личными антикоррозионными составами; • вторичная защита мостовых конструкций новыми гидроизоляционными материалами (ГИМ) второго и третьего поколений. Учитывая ранее выполненные исследования [2...6], мы остановили свой выбор на наиболее универсальном пути повышения долговечности мостов, а именно вторичной защите их битум но-полимерными материалами с учетом небла гоприятных экологических условий. Так как в радикально изменившихся экологических усло виях на долговечность гидроизоляции и мосто вых конструкций влияют растворы углекисло ты, сернистой кислоты, хлоридов калия, каль ция и натрия, мы рассмотрели смачиваемость поверхности гидроизоляционного материала растворами малой концентрации этих веществ, которая определялась из выражения ПГФ ВРХВ гп*тврхв - атп COSCPrn “ ПГФ ’ а ВРХВ Для исследований нами получено битумно полимерное вяжущее, которое может быть ис пользовано для получения рулонных гидроизо ляционных материалов (оклеенная гидроизоля ция), а также для получения мастик (обмазоч где ср?пХВ “ краевой угол смачивания твердой поверхности водными растворами химических веществ, ВРХВ - водные растворы химических Вестник БИТУ, № 4, 2004 Вестник БИТУ, № 4, 2004 6 Архитектура и строительство которые помещали в водные растворы 0,5 % NaCl; 0,15 % Н2С 03; 0,5 % H2S 0 3 и выдержива ли при 20 °С в течение фиксированного време ни: 1, 2, 3, 4, 5, 6 месяцев, а затем определяли диэлектрическую проницаемость Еш . Анализ данных (рис. 1, 2) показал, что с увеличением продолжительности контактирования водных растворов химических веществ с твердой по верхностью гидроизоляционного материала диэлектрическая проницаемость последнего повышается, а это способствует снижению гид рофобное™ поверхности гидроизоляции, т. е. повышению ее смачиваемости. Как и сле довало ожидать, наименьшее изменение ди электрической проницаемости отмечено при контактировании образцов ГИМ с дистиллиро ванной водой, а наибольшее - с 0,5%-м раство ром NaCl. Например, для ГИМ, контактирую которые помещали в водные растворы 0,5 % NaCl; 0,15 % Н2С 03; 0,5 % H2S 0 3 и выдержива ли при 20 °С в течение фиксированного време ни: 1, 2, 3, 4, 5, 6 месяцев, а затем определяли диэлектрическую проницаемость Еш . Анализ данных (рис. 1, 2) показал, что с увеличением продолжительности контактирования водных растворов химических веществ с твердой по верхностью гидроизоляционного материала диэлектрическая проницаемость последнего повышается, а это способствует снижению гид рофобное™ поверхности гидроизоляции, т. е. повышению ее смачиваемости. Как и сле довало ожидать, наименьшее изменение ди электрической проницаемости отмечено при контактировании образцов ГИМ с дистиллиро ванной водой, а наибольшее - с 0,5%-м раство ром NaCl. Проф. ЛЯХЕВИЧ Г. Д , аспиранты МАКСИМЕНКО А. А., ПАСТУШКОВ В. Г., ГРЕЧУХИН В. А. Таким образом, создание битумно-олигомерного вя жущего более чем в 2 раза улучшило гидроизо лирующие свойства гидроизоляции, а это по зволит существенно повысить долговечность ее и мостовой конструкции. щего в течение пяти месяцев с дистиллирован ной водой, диэлектрическая проницаемость Ет составила 2,7. При контактировании этого же материала с 0,5%-м раствором NaCl диэлек трическая проницаемость £ Б0° составила 7,2, т. е. в течение одного и того же времени ди электрическая проницаемость Е%°0 увеличилась в 2,7 раза. Следовательно, изменение экологии на мостах ведет к существенному увеличению смачиваемости поверхности гидроизоляции и, как результат, к уменьшению ее надежности и долговечности, даже если она выполнена из самых современных гидроизоляционных мате риалов. При контактировании битумных вяжу щих в различных средах в течение пяти меся цев установлено (рис. 2), что диэлектрическая проницаемость битума £ Б00 в 2,8...4,2 раза больше, чем диэлектрическая проницаемость битумно-олигомерного вяжущего £ Б0° . Таким образом, создание битумно-олигомерного вя жущего более чем в 2 раза улучшило гидроизо лирующие свойства гидроизоляции, а это по зволит существенно повысить долговечность ее и мостовой конструкции. битумно-полимерными материалами. Разработ ка современных гидроизоляционных материа лов в значительной степени решает проблему долговечной и надежной защиты мостовых конструкций. Необходимо также существенно доработать конструкцию гидроизоляции, тех нологию профилактики и условия ее эксплуа тации. битумно-полимерными материалами. Разработ ка современных гидроизоляционных материа лов в значительной степени решает проблему долговечной и надежной защиты мостовых конструкций. Необходимо также существенно доработать конструкцию гидроизоляции, тех нологию профилактики и условия ее эксплуа тации. Л И Т Е Р А Т У Р А 1. Минин А. В. Защита мостовых конструкций, рабо тающих в условиях агрессивного действия хлоридов // Материалы 54-й науч.-техн. конф. БГПА. - Мн., 2000. - Ч. 8 .-С . 98. 2. Ляхевич Г. Д. Теоретический анализ структуры и надежности битумно-полимерных материалов, применяе мых для гидроизоляции мостовых и тоннельных конст рукций // Диагностика эксплуатационного состояния автомобильных дорог, новые технологии их ремонта и содержания: Докл. междунар. науч.-техн. конф. - Мн., 1998.-С . 73-78. 2. Ляхевич Г. Д. Теоретический анализ структуры и надежности битумно-полимерных материалов, применяе мых для гидроизоляции мостовых и тоннельных конст рукций // Диагностика эксплуатационного состояния автомобильных дорог, новые технологии их ремонта и содержания: Докл. междунар. науч.-техн. конф. - Мн., 1998.-С . 73-78. 3. Проблемы эксплуатации мостовых конструкций в неблагоприятных экологических условиях / Г. Д. Ляхевич, А. Л. Максименко, В. Г. Пастушков, В. А. Гречухин // Наука - образованию, производству, экономике: Реф. докл. междунар. науч.-техн. конф. - Мн., 2003. - С. 19. 4. Ляхевич Г. Д. Рентгеноскопическое, электронно скопическое и дериватографическое исследование твер дой фазы вяжущих на основе полимерных материалов // Проблемы развития сети и улучшения эксплуатационных качеств автомобильных дорог местного назначения и внутрихозяйственных дорог колхозов и совхозов: Мате риалы респ. науч.-техн. конф. - Мн., 1984. - С. 141-145. Проф. ЛЯХЕВИЧ Г. Д , аспиранты МАКСИМЕНКО А. А., ПАСТУШКОВ В. Г., ГРЕЧУХИН В. А. Например, для ГИМ, контактирую ная гидроизоляция). Оно содержит 85 % нефтя ного битума и 15 % олигомера натурального каучука. Характеристика его следующая: тем пература размягчения по КиШ составляла 92 °С; глубина проникания иглы (0,1 мм при 25 °С) - 37; температура вспышки - плюс 226 °С; температура хрупкости по Фраасу - минус 29 °С; интервал пластичности - 121 °С; адгезия к бетону - 1,24 МПа. В опытах исполь зовался также нефтяной высокоплавкий битум с параметрами: температура размягчения по КиШ составляла 94 °С; глубина проникания иглы (0,1 мм при 25 °С) - 28; температура вспышки - плюс 224 °С; температура хрупко сти по Фраасу - минус 3 °С; интервал пластич ности - 97 °С; адгезия к бетону - 0,18 МПа. Из битумно-олигомерного материала и неф тяного битума готовили образцы 100x100x5 мм, щие. 1. Изменение диэлектрической проницаемости в зависимости от контакта гидроизоляционного материала с вод ными растворами химических веществ: 1 - 0,5%-м раствором NaCl; 2 - 0,5%-м раствором H2S03; 3 - 0,15%-м раст вором Н2С03; 4 - дистиллированной водой щие. 1. Изменение диэлектрической проницаемости в зависимости от контакта гидроизоляционного материала с вод ными растворами химических веществ: 1 - 0,5%-м раствором NaCl; 2 - 0,5%-м раствором H2S03; 3 - 0,15%-м раст вором Н2С03; 4 - дистиллированной водой Рис. 2. Изменение диэлектрической проницаемости высокоплавкого битума в зависимости от продолжительности его контакта с водными растворами химических веществ: 1 - 0,5%-м раствором NaCl; 2 - 0,5%-м раствором H2S03; 3 - 0,15%-м раствором Н2С03; 4 - дистиллированной водой Рис. 2. Изменение диэлектрической проницаемости высокоплавкого битума в зависимости от продолжительности его контакта с водными растворами химических веществ: 1 - 0,5%-м раствором NaCl; 2 - 0,5%-м раствором H2S03; 3 - 0,15%-м раствором Н2С03; 4 - дистиллированной водой Вестник БИТУ, № 4, 2004 7 Архитектура и строительство щего в течение пяти месяцев с дистиллирован ной водой, диэлектрическая проницаемость Ет составила 2,7. При контактировании этого же материала с 0,5%-м раствором NaCl диэлек трическая проницаемость £ Б0° составила 7,2, т. е. в течение одного и того же времени ди электрическая проницаемость Е%°0 увеличилась в 2,7 раза. Следовательно, изменение экологии на мостах ведет к существенному увеличению смачиваемости поверхности гидроизоляции и, как результат, к уменьшению ее надежности и долговечности, даже если она выполнена из самых современных гидроизоляционных мате риалов. При контактировании битумных вяжу щих в различных средах в течение пяти меся цев установлено (рис. 2), что диэлектрическая проницаемость битума £ Б00 в 2,8...4,2 раза больше, чем диэлектрическая проницаемость битумно-олигомерного вяжущего £ Б0° . ВЫВОДЫ 1. Низкая долговечность мостовых конст рукций связана с неблагоприятными экологи ческими условиями и, прежде всего, с агрес сивным воздействием при постоянной вибра ции водных растворов хлорида натрия, двуокисей серы и углерода, с синергизмом дей ствия этих растворов, а также положительными и отрицательными температурами, повышен ной влажностью и другими параметрами. 5. Ляхевич Г. Д., Максименко А. Л., Гречу хин В. А. Теоретическое обоснование выбора гидроизо ляции для мостов, подверженных вибрационному воздей ствию // Наука - образованию, производству, экономике: Реф. докл. междунар. науч.-техн. конф. - Мн., 2003. - С. 19. 6. Ляхевич Г. Д., Максименко А. Л., Пастуш ков В. Г. Теоретические аспекты и экспериментальные исследования адгезионного взаимодействия полидисперс- ной системы «битумно-полимерное вяжущее - цементобе тонная поверхность» // Вестник БИТУ. - 2003. - № 2. - С. 13-17. 2. Наиболее универсальный путь повыше ния долговечности мостов - вторичная защита Вестник БИТУ, № 4, 2004 Вестник БИТУ, № 4, 2004 Вестник БИТУ, № 4, 2004 8
https://openalex.org/W2047135239	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0083917&type=printable	English	null	Molecular Profiling of Single Sca-1+/CD34+,− Cells—The Putative Murine Lung Stem Cells	PloS one	2,013	cc-by	9,194	Abstract Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker- positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD342 and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD342 cells (p = 0.03), whereas mRNA of the mesenchymal marker Pdgfra (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a Pdgfra positive subpopulation within Epcam+/Sca-1+/CD342 epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD342 cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,2 cells in the biology of the murine lung. Citation: Hittinger M, Czyz ZT, Huesemann Y, Maneck M, Botteron C, et al. (2013) Molecular Profiling of Single Sca-1+/CD34+,2 Cells—The Putative Murine Lung Stem Cells. PLoS ONE 8(12): e83917. doi:10.1371/journal.pone.0083917 Editor: Dinender K. Singla, University of Central Florida, United States of America Editor: Dinender K. Singla, University of Central Florida, United States of America Received April 12, 2013; Accepted November 4, 2013; Published December 31, 2013 Received April 12, 2013; Accepted November 4, 2013; Published December 31, 2013 Copyright: 2013 Hittinger et al. Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. nding: The work was supported by German Research Foundation (DFG) grant HI 1101/1 (to M.H.) and the Bavarian Genome Resear A.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Bernhard.polzer@klinik.uni-regensburg.de ¤a Current address: Institute of Neuroradiology, University of Luebeck, Luebeck, Germany ¤b Current address: Institute for Translational Oncology, University of Mainz, Mainz, Germany ¤a Current address: Institute of Neuroradiology, University of Luebeck, Luebeck, Germany ¤b Current address: Institute for Translational Oncology, University of Mainz, Mainz, Germany ¤a Current address: Institute of Neuroradiology, University of Luebeck, Luebeck, Germany ¤b Current address: Institute for Translational Oncology, University of Mainz, Mainz, German . These authors contributed equally to this work. Molecular Profiling of Single Sca-1+/CD34+,2 Cells—The Putative Murine Lung Stem Cells Markus Hittinger1.¤a, Zbigniew T. Czyz1., Yves Huesemann1, Matthias Maneck1,2, Catherine Botteron3, Stephanie Kaeufl1, Christoph A. Klein1,3, Bernhard Polzer1,3* 1 Chair for Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany, 2 Institute of Functional Genomics, University of Regensburg, Regensburg, Germany, 3 Project Group ‘‘Personalized Tumor Therapy’’, Fraunhofer Institute for Experimental Medicine and Toxicology, Regensburg, Germany Introduction However, a more detailed characterization of this subpopula- tion in terms of its protein expression remained challenging. Kim and colleagues [4] identified BASCs as positive for stem cell antigen 1 (Sca-1), surfactant protein C (Sftpc), clara cell secretory protein (Ccsp), and cluster of differentiation 34 (CD34) as well as negative for the platelet endothelial cell adhesion molecule (CD31, a marker of endothelial cells), and protein tyrosine phosphatase, receptor type C (CD45, a marker of hematopoietic cells). More recent studies, however, confirmed Sca-1 and Sftpc but not CD34 as potential marker proteins of BASCs [8–10]. In contrast to the original publication [4], Sca-1+/CD34+/CD452/CD312 cells showed no enhanced proliferation rate after lung injury as it could be demonstrated with Sca-1+/CD342/CD452/CD312 cells [10]. Furthermore, Sca-1+/CD34+/CD452/CD312 cells co- expressed mesenchymal markers thymus cell antigen 1 (CD90) and platelet derived growth factor receptor a (Pdgfra, CD140a) questioning their epithelial origin [8]. The epithelial cell adhesion molecule (Epcam, CD326), a6-Integrin (Itga), ß4-Integrin and The murine lung contains at least 40 morphologically distinct cell types of mesodermal or endodermal origin [1]. It can be subdivided anatomically in three different regions: large airways, bronchioles, and alveoli. Each region is composed of different cell types whose homeostasis is guaranteed by regionally specific progenitor cells [2,3]. Importantly, Sca-1+ cells have been ascribed decisive functions regarding the maintenance and repair of the airways [2]. In injury models, Sca-1+ cells demonstrated resistance to damage and clonal expansion leading to a restoration of previously experimentally depleted epithelial structures [2]. During the last years, considerable efforts have been undertaken to further characterize a specific subgroup of Sca-1+ cells. These cells were named bronchioal- veolar stem cells (BASCs) based on their location at the bronchioalveolar duct junction [4] where they proved to play a key role in distal airway repair [4–7]. PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83917 1 Single Cell Profiling of Lung Stem Cells cluster of differentiation 24 (CD24) were newly introduced as BASC-specific markers [2,9,10]. 15 min at room temperature. Thereafter, cells were washed with PBS, centrifuged und resuspended in PBS to be counted in a Neubauer chamber. Considering the heterogeneity of Sca-1+ murine lung cells, as it could be demonstrated for the group of BASCs, the isolation and subsequent gene expression analysis of single cells may provide an interesting tool for the identification of novel molecular markers in poorly described rare cells [11,12]. Immunofluorescence Staining and Single Cell Isolation Immunofluorescence was performed on one million cells of each lung preparation with antibody concentrations of 10 mg/ml for all stainings. Cells were stained subsequently with either antibodies specific for CD31 (Biozol, clone 390) and Sca-1 (FITC-conjugated, Cedarlane, clone CT-6A/6E) or for CD34 (BD Pharmingen, clone RAM34) and CD45 (FITC-conjugated, Becton Dickinson GmbH, clone 30-F11). Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, code number: 115-166-071) were used to visualize CD31 or CD34 and Alexa 488-conjugated secondary antibodies (Molecular Probes, cat # A-11096) to visualize Sca-1 or CD45, respectively. Isotype controls were applied for all staining procedures as negative controls. Depending on the staining, Propidium iodide (PI, for Sca-1/CD31 staining) or GFP-Annexine (GFP-A, for CD34/CD45 staining) served as markers for apoptotic cells. Lung cells were screened on an 8-field glass slide for Sca-1+ and CD34+ cells (0.256106 cells/field) using an inverted fluorescence microscope (Zeiss, Germany) equipped with a micromanipulator (Eppendorf, Germany). Sca-1+/ CD312/PI2 and CD34+/CD452/GFP-A2 cells were isolated and each single cell was separately transferred into a 0.2 ml reaction tube (1 cell/tube) containing 4 ml of lysis buffer and 0.4 ml of tRNA. Introduction Eventually, this approach could allow better distinction between the different subtypes of Sca-1+ cells and lead to the discovery of novel molecular markers facilitating a better detection and functional evaluation. In this study, we isolated viable Sca-1+ and CD34+ cells on the basis of their protein expression. Following global amplification of single cell mRNA, gene expression profiling was performed to analyze the cell populations at the single cell level. The analysis included markers that have previously been ascribed to BASCs and intended to identify markers that thus far have not been described in Sca-1+ murine lung cells. Whole Transcriptome Amplification (WTA) of Single Cells Whole Transcriptome Amplification (WTA) of Single Cells The transcriptome of each obtained cell was extracted and amplified according to an established protocol [12] with modifi- cations [11]: One microlitre protease/lysis buffer mix (1:20- dilution; Active Motif, Rixensart, Belgium) and 1 ml biotinylated peptide nucleic acids (PNAs; Midi-Kit, Active Motif, dissolved in 400 ml water) were added to each tube for proteolytic digestion. Digestion was performed at 45uC for 10 min, followed by inactivation for 1 min at 70uC and 15 min at 22uC for PNA annealing to the mRNA. mRNA was captured by streptavidine- coated magnetic particles (Active Motif) during 45 min rotation at room temperature. After addition of 10 ml wash buffer 1 (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT and 0.25% Igepal), tubes were placed in a magnetic rack. The supernatants containing the genomic DNA were removed and the beads washed with 20 ml wash buffer 2 (50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT and 0.5% Tween). The supernatants were removed and the beads washed once again with 20 ml wash buffer 1. The mRNA was reverse transcribed using a mix comprising 0.5 mM dNTPs, 30 mM CFL15CN8 primer ([C]n = 15GTCTAGA[Nn = 8]), 15 mM CFL15CT24 primer ([C]n = 15GTCTAGA[T]n = 24), 0.25% Igepal, 10 mM DTT (Invitrogen), the reaction buffer supplied by the manufacturer and 200 U of Superscript II reverse transcriptase (Invitrogen, Karlsruhe) in a final volume of 20 ml. Primers were Animals and Tissue Preparation Animals and Tissue Preparation No animal underwent animal experimentation as defined by the German law (1 7 TierschG). All animals were euthanized and killed before organs were taken. According to German law (1 6 Abs 1 TierschG) there is no requirement for an Ethics vote nor a notification of the local Government for dissection and organ use after the death of an animal. Mice were kept according to the guidelines of the Felasa (Federation for Laboratory Animal Science Associations) and the guideline 2010/63/EU and the ETS123 (APPENDIX A to the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes). Since quantitative gene expression analysis of the isolated Sca- 1+ and CD34+ cells was an important goal of our study, we had to acquire appropriate reference cells for comparison, i.e. pulmonary cells lacking Sca-1 and CD34 expression and not originating from hematopoietic or endothelial cell lines. Therefore, cells of 6 enzymatically digested lungs were double-stained with CD31 and CD45 antibodies in order to collect single cell samples. The same isolation procedure as described above was applied for CD312/CD452 reference cells. To exclude cells expressing Sca- 1 and CD34, specific PCRs on the corresponding amplified transcripts of those genes were performed. Experiments were conducted on 26 Balb/c mice (10 female, 16 male) between 8 and 12 weeks of age. Mice were euthanized with 100% carbon dioxide, and subsequently the thoracic cavity was opened and the great venous vessels were clamped. The right ventricle was incised and a knop canula introduced to perfuse the lungs with 20–30 ml of body-warm saline. After exsanguination, lungs were dissected and collected in Hanks Balanced Salt Solution (HBSS, pH 7.4, Sigma Aldrich). Analytical PCR on Specific Transcripts For quality control of whole transcriptome amplification products, we tested for amplicons of ubiquitously expressed genes Actb (ß-actin) and Gapdh (Glyceraldehyde 3-phosphate dehydroge- nase) by PCR. Only cells with at least one positive result were considered for further analysis. For initial molecular characteriza- tion of isolated cells, PCR on transcripts of Sca-1, CD34, CD45 and CD31 were performed. In order to differentiate between a more epithelial or mesenchymal phenotype of isolated cells, we conducted further PCRs specific for epithelial markers Epcam (Epithelial cell adhesion molecule), Itga (Integrin alpha-6) and Sftpc (Surfactant protein C) and mesenchymal markers CD90 (Thy-1) and Pdgfra (platelet derived growth factor receptor alpha, CD140a), as suggested by McQualter et al. [9]. Specificity of all primers was confirmed by restriction digestion, sequences are depicted in Table S1. Single Cell Profiling of Lung Stem Cells Therefore, 35 ml of PCR mix 1 containing 4 ml buffer 1 (Expand long template, Roche) and 3% deionized formamide was added to each sample., Next, the samples were heat up to 78uC followed by addition of 5.5 ml of the PCR mix 2 (350 mM dNTPs, 1.2 mM CP2 primer (TCAGAATTCATG[C]n = 15) and 5 U Pol Mix (Expand long template)). Forty cycles were run in a MJ research PCR machine: 20 cycles of 15 sec at 94uC, 30 sec at 65uC, 2 min at 68uC and 20 cycles with an elongation of the extension time of 10 sec and a final elongation step of 7 min at 68uC. blocking reagent for nucleic acid hybridization (Roche). Slides were then stained with 16 mg/ml anti-Dig-Cy5 (Jackson Labora- tories) and 18 mg/ml Streptavidin-Cy3 (Jackson Laboratories). In order to remove excess antibody/streptavidin, slides were washed with 46 SSC +0.2% Tween-20. The microarray slides were scanned using the GenePix 4000A Arrayscanner (Molecular Device). The downstream data prepro- cessing and analysis was done in R using the limma package [14]. Raw probe intensities were background corrected by applying the ‘normexpr’ method. Analysis was restricted to Cy5 intensities. Loess normalization was used in M versus A plots of individual Cy5 intensities of a given transcript in a given array and the median Cy5 intensity across all arrays of the same transcript. Log2 ratios were calculated from the normalized intensities and quantile-normalization was subsequently applied across all arrays. All further analysis was based on normalized log ratios. Based on the hybridization date the data set is separated into two main batches of microarrays (early 2007/2008 and late 2009), which degrade further into small sub-batches. We used the ComBat algorithm [15] to adjust for the main batches. Transcripts differentially expressed between the groups were identified using regularized linear model as implemented in the limma package [16]. Transcripts were considered as significantly differentially expressed when their corresponding adjusted P-value was #0.05. Adjustment of P-values as correction for multiple testing was done as proposed by Benjamini et al. [17]. The differential gene expression analysis was used to generate candidates of regulated genes. The data have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and as- signed series accession number GSE52215. Quantitative PCR The differentially expressed genes for Decorin (Dcn), Gelsolin (Gsn) and Esterase D/formylglutathion hydrolase (Esd) were used to validate microarray results in an additional series of specific analytical and quantitative PCRs (qPCR). qPCR was performed using the LightCyclerH 480 instrument (Roche, Mannheim, Germany). The real-time quantification was conducted using LightCyclerH 480 SYBR Green I Master reagent (Roche, Mannheim, Germany). The cycling procedure consisted of an initial denaturation step (5 min at 95uC) followed by 38 cycles of 20 s at 95uC, 15 s at 58uC and 15 s at 72uC. All reactions were run in three replicas in a final volume of 19 ml containing 5 ml of cDNA template and 7 pmol of each forward and reverse primer. Positive and negative controls were included in all qPCR runs. Additionally, to confirm the specificity of reaction, all experiments included melt curve analysis. For each cell group three represen- tative samples were generated by pooling equal amounts of single- cell cDNA samples assigned to a given group (10, 7 or 12 samples for Sca-1+/CD34+, Sca-1+/CD342 and reference cells, respectively). This was done to decrease the measurement noise originating from cell-to-cell variability of gene expression levels. 1006 dilutions of the original cDNA sample representations were used as template for the qPCR. The relative gene expression levels were calculated using the efficiency corrected mathematical model described elsewhere [18]. Quantification was performed in parallel using three different reference genes Actb, Gapdh and Hprt1 (Hypoxan- thine phosphoribosyl transferase 1), in each case giving highly comparable results. Group-wise comparison of relative gene expression levels was performed using 2-tailed Student’s t-test. A value of p,0.05 was considered to indicate a statistically significant difference. Single Cell Profiling of Lung Stem Cells Single Cell Profiling of Lung Stem Cells allowed to anneal at room temperature for 10–15 min before the enzyme was added. Reverse transcription was conducted for 45 min at 44uC in an oven with constant steering. Following reverse transcription, the magnetic cDNA-mRNA-hybrids were washed in 20 ml tailing wash buffer (50 mM KH2PO4 (pH 7), 1 mM DTT, 0.25% Igepal), resuspended in 10 ml tailing buffer (10 mM KH2PO4 (pH 7), 4 mM MgCl2, 0.1 mM DTT, 200 mM dGTP) and then coated with 40 ml PCR oil. Hybrids underwent denaturation at 94uC for 4 min. The tailing reaction was performed at 37uC for 60 min after addition of 10 U terminal deoxynucleotide transferase (TdT; Amersham, Freiburg). TdT was inactivated at 70uC for 5 min. Next, a hot-start PCR was performed. Therefore, 35 ml of PCR mix 1 containing 4 ml buffer 1 (Expand long template, Roche) and 3% deionized formamide was added to each sample., Next, the samples were heat up to 78uC followed by addition of 5.5 ml of the PCR mix 2 (350 mM dNTPs, 1.2 mM CP2 primer (TCAGAATTCATG[C]n = 15) and 5 U Pol Mix (Expand long template)). Forty cycles were run in a MJ research PCR machine: 20 cycles of 15 sec at 94uC, 30 sec at 65uC, 2 min at 68uC and 20 cycles with an elongation of the extension time of 10 sec and a final elongation step of 7 min at 68uC. allowed to anneal at room temperature for 10–15 min before the enzyme was added. Reverse transcription was conducted for 45 min at 44uC in an oven with constant steering. Following reverse transcription, the magnetic cDNA-mRNA-hybrids were washed in 20 ml tailing wash buffer (50 mM KH2PO4 (pH 7), 1 mM DTT, 0.25% Igepal), resuspended in 10 ml tailing buffer (10 mM KH2PO4 (pH 7), 4 mM MgCl2, 0.1 mM DTT, 200 mM dGTP) and then coated with 40 ml PCR oil. Hybrids underwent denaturation at 94uC for 4 min. The tailing reaction was performed at 37uC for 60 min after addition of 10 U terminal deoxynucleotide transferase (TdT; Amersham, Freiburg). TdT was inactivated at 70uC for 5 min. Next, a hot-start PCR was performed. Tissue Digestion and Cell Separation The lungs were finely minced and the tissue was enzymatically digested according to an established protocol [13], with minor modifications. Briefly, each finely minced lung preparation was incubated with 5.4 U/ml collagenase (Roche), 0.03 U/ml dispase (GE Healthcare-Invitrogen) and 2.5 mM CaCl2 for 45 minutes at 37uC. The suspension was then filtered through nylon membranes (BD; 100 mm, 40 mm). Enzymes were inactivated in HBSS/0.2 M EDTA (pH 7.4), cells were centrifuged (1500 rpm, 4uC, 5 min) and resuspended in PBS (pH 7.4). A Percoll gradient (70%) density centrifugation was performed (2050 rpm, 4uC, 20 min) to separate the mononuclear cell fraction. The cell interphase was recovered and washed in PBS. Following a centrifugation step (1500 rpm, 4uC, 10 min) cells were resuspended in PBS (pH 7.4). Finally, viable cells were identified by Trypan blue assay and counted in a Neubauer chamber. For FACS analysis, the lung preparations of 5 mice were finely minced. The tissue was enzymatically digested with collagenase (Sigma; 1.5 mg/ml) for 30 minutes at 37uC. The enzymes were inactivated with PBS/BSA (Sigma; 2.5 g/100 ml). The suspension was filtered through a cell strainer (Greiner Bio-One; 40 mm), centrifuged (300 g, 10 min, 4uC) and resuspended in 5 ml red blood cell lysis solution (16) (Miltenyi; order no. 130-094-183) for December 2013 \| Volume 8 \| Issue 12 \| e83917 PLOS ONE \| www.plosone.org 2 FACS Staining For FACS analysis a FACSCanto II (BD Biosciences) was used. Five million cells per animal were blocked with 1 mg Fc-block/ tube and stained with anti-mouse CD31 antibody (PE-conjugated, clone 390; Biolegend, 1 mg/ml), anti-mouse CD34 antibody (FITC-conjugated, clone RAM34; eBioscience, 10 mg/ml), anti- mouse CD45 antibody (PerCP/Cy5.5-conjugated, clone 30-F11; Biolegend, 2.5 mg/ml), anti-mouse Sca-1 antibody (APC-Cy7- conjugated, clone D7; Biolegend, 2.5 mg/ml), anti-mouse EpCAM antibody (eF450-conjugated, clone G8.8; eBioscience, 10 mg/ml) and anti-mouse PDGFRa antibody (APC-conjugated, clone APA5; Biolegend, 10 mg/ml) or corresponding isotype antibodies for 20 min at 4uC. Results were visualized by the FlowJo package (Tree Star Inc., Ashland, OR, USA). Up to one million cells per animal were screened, equally divided for Sca-1/CD31 and CD34/CD45 immunofluorescence (i.e. up to 0.506106 cells for each staining and animal). We identified and isolated 68 Sca-1+/CD312/PI2 cells, the number of obtained cells per mouse ranging from 0 to 10 (mean 4.5, SEM 3.0, Table 1 and Figure S1). Additionally, we detected 58 CD34+/ CD452/GFP-A2 cells, ranging from 0 to 13 in individual mice (mean 3.9; SEM 4.6, Table 1 and Figure S1). All detected cells were isolated and subjected to single cell gene expression analysis following whole transcriptome amplification (WTA). The enzy- matically digested lungs of 6 additional mice yielding up to 1.76106 non-erythrocytic cells (mean 1.36106 cells; SEM 0.326106 cells) were stained with antibodies against CD31 and CD45 resulting in the isolation of 35 control cells (CD312/CD452). All isolated cells were subjected to whole transcriptome amplification (WTA) and WTA quality controlled by expression of Actb and Gapdh. In total, 115/161 (71.4%) samples passed our quality assay and were subjected to further molecular analysis (no difference between cell types, data not shown). Identification of Sca-1+ and CD34+ Cells and Selection for Molecular Analysis In order to isolate Sca-1+ and CD34+ cells 15 Balb/c mice between 8 and 12 weeks of age were sacrificed. Tissue digestion and cell separation started immediately after lung explantation. The number of obtained non-erythrocytic, non-apoptotic cells per experiment ranged from 0.406106 to 5.06106 (mean 1.66106; standard error of the mean (SEM) 1.166106). The number of erythrocytes ranged from 0.206106 to 2.66106 (mean 0.876106; SEM 0.736106), the number of apoptotic cells (i.e. PI+ or GFP-A+ We continued by testing for the presence of transcripts of the proteins targeted by antibodies in immunofluorescence: Sca-1, CD34, CD45 and CD31. Within the two populations of putative BASC, 35 cells completely lacked transcript expression of Sca-1/ CD34, while another 10 cells co-expressed CD31 and/or CD45 (Table 2). We decided to exclude those cells from further analyses which resulted in a cohort of 46 single putative BASCs remaining Figure 1. Flowchart of the experimental setup. Simplified schematic overview on the workflow of single cell isolation and immunofluorescent staining strategy: Cells are divided into two groups after separation and stained with antibodies directed against CD31 and Sca-1 or CD34 and CD45, respectively. In order to gain appropriate reference cells for comparative gene expression analysis, additional lung cells are stained with antibodies directed against CD31 and CD45, and only CD312/CD452 cells are isolated. Propidium iodide (PI) and GFP-Annexine (GFP-A) are applied to exclude apoptotic cells. Genes recently introduced in literature in order to differentiate between epithelial and mesenchymal cells are tested by specific PCR. PCR results enable further subdivision of analyzed cells (Sca-1+/CD34+ cells, Sca-1+/CD342 cells, Sca-12/CD34+ cells). In a final step, selected Sca-1+/ CD34+ cells, Sca-1+/CD342 cells and Sca-12/CD342 reference cells are subjected to comparative gene expression analysis. Results are validated by qPCR of pooled samples. doi:10.1371/journal.pone.0083917.g001 Figure 1. Flowchart of the experimental setup. Simplified schematic overview on the workflow of single cell isolation and immunofluorescent staining strategy: Cells are divided into two groups after separation and stained with antibodies directed against CD31 and Sca-1 or CD34 and CD45, respectively. In order to gain appropriate reference cells for comparative gene expression analysis, additional lung cells are stained with antibodies directed against CD31 and CD45, and only CD312/CD452 cells are isolated. Propidium iodide (PI) and GFP-Annexine (GFP-A) are applied to exclude apoptotic cells. Genes recently introduced in literature in order to differentiate between epithelial and mesenchymal cells are tested by specific PCR. Single Cell Profiling of Lung Stem Cells Single Cell Profiling of Lung Stem Cells cells) ranged from 0.926106 to 4.06106 (mean 1.96106; SEM 1.36106). The experimental setup is outlined in Figure 1. Array Hybridization and Data Analysis y y y Probes of the 29 selected cells were hybridized on Mouse Genome OpArrays (Eurofins MWG Operon; cat # OPMMV4- 05). The arrays contain probes for 16,928 genes and have previously been used for hybridization of single cell WTA products [11]. The amplified single cell cDNA was labeled with 0.05 mM digoxygenin-dUTP (Roche) and 0.05 mM aminodigoxygenin- dCTP (PerkinElmer, Rodgau-Ju¨gesheim) in the presence of 3% formamide, 2.4 mM CP2-BGL primer (TCAGAATT- CATGCCGCCCCCCCGGCCC) and dNTPs (0.35 mM dATP and dGTP, 0.3 mM dTTP and dCTP). Reference cDNA was labeled with biotin-dUTP (Roche) and biotin-dCTP (Invitrogen). Primer sequences were then separated from the cDNA sequences in a subsequent digestion step with 30 U of BglI (Fermentas, St. Leon-Rot), and then purified (QIAquick PCR Purification Kit, QIAGEN, Hilden). Test and reference cDNA were co-precipitat- ed with 0.8 ml polyacrylamide carrier, 0.163 M sodium acetate and 2.56 ethanol (100%). Arrays were pre-hybridized with 56 SSC +0.1% SDS +0.1% BSA at 42uC and hybridized in an Arraybooster hybstation (Implen, Munich) at 42uC overnight. The slides were washed twice at 42uC in 26SSC+0.1% SDS for 5 min, twice at room temperature in 0.16 SSC +0.1% SDS for 10 min, and finally twice at room temperature in 0.16SSC for 2 min 30 s. Unspecific binding of labeled proteins was blocked with 1% December 2013 \| Volume 8 \| Issue 12 \| e83917 3 PLOS ONE \| www.plosone.org Identification of Sca-1+ and CD34+ Cells and Selection for Molecular Analysis PCR results enable further subdivision of analyzed cells (Sca-1+/CD34+ cells, Sca-1+/CD342 cells, Sca-12/CD34+ cells). In a final step, selected Sca-1+/ CD34+ cells, Sca-1+/CD342 cells and Sca-12/CD342 reference cells are subjected to comparative gene expression analysis. Results are validated by qPCR of pooled samples. doi:10 1371/journal pone 0083917 g001 December 2013 \| Volume 8 \| Issue 12 \| e83917 PLOS ONE \| www.plosone.org 4 Single Cell Profiling of Lung Stem Cells Table 1. Non-erythrocytic cells, Sca-1+ and CD34+ cells per lung preparation. Mice non-erythrocytic cells Sca-1+/CD312 cells CD34+/CD452 cells 1 680000 3 0 2 1.526106 4 3 3 920000 4 2 4 1.26106 4 5 5 1.26106 0 0 6 2.46106 5 6 7 2.06106 5 11 8 1.26106 6 12 9 2.46106 3 3 10 400000 3 1 11 520000 3 0 12 1.26106 8 2 13 1.06106 0 13 14 5.06106 10 0 15 2.56106 10 0 doi:10.1371/journal.pone.0083917.t001 Table 1. Non-erythrocytic cells, Sca-1+ and CD34+ cells per lung preparation. Mice non-erythrocytic cells Sca-1+/CD312 cells CD34+/CD452 cells 1 680000 3 0 2 1.526106 4 3 3 920000 4 2 4 1.26106 4 5 5 1.26106 0 0 6 2.46106 5 6 7 2.06106 5 11 8 1.26106 6 12 9 2.46106 3 3 10 400000 3 1 11 520000 3 0 12 1.26106 8 2 13 1.06106 0 13 14 5.06106 10 0 15 2.56106 10 0 doi:10.1371/journal.pone.0083917.t001 both protein and mRNA level in 19 of 24 Sca-1+/CD312/PI2 cells (79.2%) and CD34 expression could be detected on protein and mRNA level in 15 of 22 CD34+/CD452/GFP-A2 cells (68.2%), thus showing a positive correlation between protein and transcript level in the majority of putative BASCs. According to the detected mRNA transcripts after single cell WTA, cells could be grouped either as Sca-1+/CD34+ (n = 17), Sca-1+/CD342 (n = 22) or Sca-12/CD34+ (n = 7). Interestingly, simultaneous expression of Sca-1 and CD34 could be detected in 13/22 single cells isolated after CD34-staining (59.1%) and only in 4/24 (16.7%) single cells isolated after Sca-1- staining, resulting in a significantly higher prevalence of cells showing mRNA transcripts of both markers Sca-1 and CD34 in the group of CD34+/CD452/GFP-A2 cells (Fisher’s exact test, p = 0.005, Table 3). Identification of Novel Molecular Markers in Putative BASCs To further analyze the isolated cells, we selected 17 putative BASCs (10 Sca-1+/CD34+ cells and 7 Sca-1+/CD342 cells) and 12 reference cells (Sca-12/CD342/CD312/CD452) for hybridization on Mouse Genome OpArrays (Eurofins MWG Operon). Here, we decided to compare microarray data of two of the three cell groups independently with each other. doi:10.1371/journal.pone.0083917.t001 for downstream analyses. Likewise, among the tested pulmonary reference cells we excluded one sample expressing Sca-1 and two samples positively tested for the presence of CD45 transcripts resulting in a cohort of 21 cDNA libraries of Sca-12/CD342 cells. First, we analysed data of Sca-1+/CD34+ cells and the selected pulmonary reference cells only, which resulted in detection of significant changes in expression levels of 107 genes (adjusted p- value ,0.05 each, Figure 2A, Table S2). Interestingly, we could not find any differentially expressed genes between the groups of Sca-1+/CD342 cells and the reference cells (Sca-12/CD342). In turn, comparative gene expression analysis of Sca-1+/CD34+ and Sca-1+/CD342 cells identified 8 differentially expressed genes (adjusted p-value ,0.05 each, Table S3). Identification of Sca-1+ and CD34+ Cells and Selection for Molecular Analysis On the other hand, the group of Sca-1+/ CD312/PI2 cells showed a higher prevalence for cells positive for Sca-1 transcripts only, an expression pattern that matched 15/ 24 Sca-1+/CD312/PI2 cells and 7/22 and CD34+/CD452/GFP- A2 cells, respectively (Chi Square test, p = 0.04, Table 3). These results indicate the existence of different subpopulations within the isolated fractions of cells. Correlation between Protein and mRNA Expression in Single Cells Next, we assessed the correlation between protein staining and PCR results for Sca-1 and CD34 in the group of putative BASCs (Table 3). In total, 24/46 cells were isolated as Sca-1+/ CD312/PI2 and 22/46 cells as CD34+/CD452/GFP-A2 using immunofluorescent staining (Figure 1). Direct comparison re- vealed that Sca-1 expression could be detected simultaneously at In general, gene expression in single cells is subjected to stochastic fluctuations [19]. While in high-dimensional data (as microarrays) normalization generally uses many if not all probes, relative quantification in quantitative PCR (qPCR) experiments relies on stable expression levels of individual genes. This especially holds true for some house-keeping genes, as e.g. Actb or Gapdh, which renders reliable relative quantification using such an approach at the single cell level questionable [20,21]. Although specific genes can be expressed at a stable level between individual single cells of a certain cell type, this approach is not feasible for stochastic fluctuations [19]. While in high-dimensional data (as microarrays) normalization generally uses many if not all probes, relative quantification in quantitative PCR (qPCR) experiments relies on stable expression levels of individual genes. This especially holds true for some house-keeping genes, as e.g. Actb or Gapdh, which renders reliable relative quantification using such an approach at the single cell level questionable [20,21]. Although specific genes can be expressed at a stable level between individual single cells of a certain cell type, this approach is not feasible for Table 2. PCR results of corresponding transcripts in Sca-1+/ CD31- and cells CD34+/CD45- cells. PCR result Immunofluorescence Sca-1 CD31 CD34 CD45 Sca-1+/CD312/PI2 CD34+/CD452/GFP-A2 N N + – – – 15 7 + - + – 4 13 - - + – 5 2 + - + + 2 1 + – – + 3 0 – – + + 2 1 – – – + 12 10 – + – – 1 0 – – – – 7 3 + + – + 0 1 – + – + 0 2 doi:10.1371/journal.pone.0083917.t002 Table 3. Distribution of PCR-based Sca-1/CD34 expression in isolated putative BASCs. Immunofluorescence Sca-1+ CD34+ mRNA transcripts Sca-1+/CD34+ 4 13 17 Sca-1+/CD342 15 7 22 Sca-12/CD34+ 5 2 7 24 22 46 doi:10.1371/journal.pone.0083917.t003 PLOS ONE \| www.plosone.org 5 December 2013 \| Volume 8 \| Issue 12 \| e83917 Table 2. PCR results of corresponding transcripts in Sca-1+/ CD31- and cells CD34+/CD45- cells. Correlation between Protein and mRNA Expression in Single Cells PCR result Immunofluorescence Sca-1 CD31 CD34 CD45 Sca-1+/CD312/PI2 CD34+/CD452/GFP-A2 N N + – – – 15 7 + - + – 4 13 - - + – 5 2 + - + + 2 1 + – – + 3 0 – – + + 2 1 – – – + 12 10 – + – – 1 0 – – – – 7 3 + + – + 0 1 – + – + 0 2 doi:10.1371/journal.pone.0083917.t002 Table 3. Distribution of PCR-based Sca-1/CD34 expression in isolated putative BASCs. Table 3. Distribution of PCR-based Sca-1/CD34 expression in isolated putative BASCs. Table 3. Distribution of PCR-based Sca-1/CD34 expression in isolated putative BASCs. Immunofluorescence Sca-1+ CD34+ mRNA transcripts Sca-1+/CD34+ 4 13 17 Sca-1+/CD342 15 7 22 Sca-12/CD34+ 5 2 7 24 22 46 doi:10.1371/journal.pone.0083917.t003 December 2013 \| Volume 8 \| Issue 12 \| e83917 doi:10.1371/journal.pone.0083917.t002 December 2013 \| Volume 8 \| Issue 12 \| e83917 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 5 Single Cell Profiling of Lung Stem Cells Figure 2. Gene expression microarray analysis of isolated single cells. Panel A: The comparison CD34+ cells and pulmonary reference (Sca-12/CD342/CD312/CD452) cells showed 107 differentially expre from Sca-1+/CD34+ subpopulation (red), Sca-1+/CD342 subpopulation (blue) and pulmonary reference cell against differentially expressed genes Dcn, Esd and Gsn. The selected genes not only show significant di also represent different subpopulations of proteins. Error bars indicate standard deviation of the mean ca values are calculated by relative quantification against housekeeping gene Actb and illustrated in (expression value = 1.0) on a logarithmic scale. All comparisons between different groups, as determine different expression levels (student’s t-test, * indicating p,0.05). doi:10.1371/journal.pone.0083917.g002 Figure 2. Gene expression microarray analysis of isolated single cells. Panel A: The comparison of microarray expression profiles of Sca-1+/ CD34+ cells and pulmonary reference (Sca-12/CD342/CD312/CD452) cells showed 107 differentially expressed genes. Panel B: Pools of analyzed cells from Sca-1+/CD34+ subpopulation (red), Sca-1+/CD342 subpopulation (blue) and pulmonary reference cells (green) were analyzed by quantitative PCR against differentially expressed genes Dcn, Esd and Gsn. The selected genes not only show significant differences regarding to their expression, but also represent different subpopulations of proteins. Error bars indicate standard deviation of the mean calculated for analyzed triplicates. Expression values are calculated by relative quantification against housekeeping gene Actb and illustrated in comparison to pulmonary reference cells (expression value = 1.0) on a logarithmic scale. Single Cell Analysis of Epithelial and Mesenchymal Transcripts Allows Further Delineation of Sca-1+/CD34+,2 Subpopulations To further clarify the epithelial or mesenchymal commitment of isolated cells, we checked expression of mesenchymal (CD90, Pdgfra) and epithelial transcripts (Epcam, Itga and Sftpc) as recently described [8]. For this purpose, we analyzed all 46 putative BASCs and 21 pulmonary reference cells by analytical PCR (Table S4). Combined protein and cDNA analysis of Sca-1 and CD34 in our study showed a highly positive correlation between staining and transcript detection (79.2% for Sca-1 and 68.2% for CD34, respectively). In addition, our approach enabled further classifica- tion in different subpopulations characterized by Sca-1 and CD34 transcript expression. Analyzed cells consisted of two larger groups of Sca-1+/CD342 (n = 22) and Sca-1+/CD34+ (n = 17) cells and a smaller group of Sca-12/CD34+ (n = 7) cells. While statistical analysis revealed no differences in the expression frequency of mesenchymal markers in the group of putative BASCs compared to reference cells, significant differences were detected between Sca-1+/CD342 cells (n = 22) and Sca-1+/ CD34+ cells (n = 17, Figure 3A). Here, we noted that the epithelial transcript Epcam was exclusively expressed in some Sca-1+/CD342 cells (6/22 cells; p = 0.03, Fisher’s exact test). In comparison, the mesenchymal transcript Pdgfra was more frequently expressed by Sca-1+/CD34+ cells (9/17 cells), although 4/22 Sca-1+/CD342 cells also expressed transcripts of the gene (p = 0.04, Fisher’s exact test). Interestingly, while the putative epithelial marker Sftpc was frequently expressed in both major cell types, it could be detected more frequently in Sca-1+/CD34+ than Sca-1+/CD342 cells (11/ 17 vs. 7/22 single cells; p = 0.04, Chi-square test). Itga and CD90 were rarely expressed in single cells of either cell type. Within the small group of Sca-12/CD34+ cells, 2/7 cells co-expressed epithelial and mesenchymal markers, whereas another 2/7 cells expressed mesenchymal markers only (Table S4). The remaining three cells were negative for both mesenchymal and epithelial markers. To further investigate the expression profiles of the different cell types, we performed microarray-based gene expression analysis of single cell WTA products, including 10 Sca-1+/CD34+ cells, 7 Sca- 1+/CD342 cells, and 12 Sca-12/CD342 pulmonary reference cells. Comparisons between the different subgroups revealed 107 genes differentially expressed in Sca-1+/CD34+ cells when compared to the reference cells, whereas no differentially expressed genes could be detected by comparing Sca-1+/CD342 cells with reference cells. The comparison between the two Sca-1+ cell subgroups (Sca-1+/ CD34+ vs. Sca-1+/CD342) yielded 8 differentially expressed genes. Discussion In this study we investigated the gene expression profile of single Sca-1+/CD312/PI2 and CD34+/CD452/GFP-A2 cells that were detected by immunofluorescence and subsequently sub-divided into groups based on the protein expression of Sca-1 and CD34, as well as mRNA expression of subgroup-specific markers. For validation, we chose transcripts for Decorin (Dcn), and Gelsolin (Gsn), which were differentially expressed between Sca-1+/ CD34+ and reference cells, as well as Esterase D/formylglutathion hydrolase (Esd), which was in addition differentially expressed between Sca-1+/CD34+ and Sca-1+/CD342 cells. Besides the differential expression, the rationale for the selection of the three chosen transcripts was that they represent different functional groups of genes. As expected, cells from the Sca-1+/CD34+ subpopulation expressed very high levels of all three transcripts in comparison to cells of the other two groups (student’s t-test, p,0.05). If comparing Sca-1+/CD342 cells with pulmonary reference samples, Dcn and Gsn were expressed in significantly higher levels in Sca-1+/CD342 cells than Sca-12/CD342 cells, while Esd was expressed at significantly lower level in Sca-1+/ CD342 cells than in pulmonary reference cells as determined by quantitative PCR (student’s t-test, p,0.05, Figure 2B). Sca-1 and CD34 have both been ascribed to bronchioalveolar stem cells (BASCs), a rare population of long-living, regional fixed, robust cells residing at the bronchioalveolar duct junction that have been sparsely investigated so far. Only few markers were established in previous studies mainly applying FACS analysis, which enables a rapid screening of very large cell numbers and subsequent comparison between cell populations as defined by applied protein markers. This approach shows high sensitivity and at the same time an elevated risk to isolate false-positive cells, which may impair the detection of extremely rare cells [22]. In this study, we intentionally utilized a microscope-based approach to isolate single cells to analyze cell-to-cell heterogeneity in poorly characterized cells. Considering the low incidence of putative BASCs in lung tissue, we believe that this approach is the method of choice for the molecular analysis of such extremely rare and poorly characterized cells. Beyond that, our workflow additionally allows direct assessment of morphology and viability of the target cells, thereby reducing the risk of contamination with false-positive cells or cell debris to an absolute minimum. Single Cell Profiling of Lung Stem Cells poorly described cell populations. Therefore, we decided to pool the single cell samples of a pre-defined subgroup for qPCR analyses to validate the results of the microarray analyses. Correlation between Protein and mRNA Expression in Single Cells All comparisons between different groups, as determined by quantitative PCR, showed significantly different expression levels (student’s t-test, * indicating p,0.05). doi:10.1371/journal.pone.0083917.g002 Figure 2. Gene expression microarray analysis of isolated single cells. Panel A: The comparison of microarray expression profiles of Sca-1+/ CD34+ cells and pulmonary reference (Sca-12/CD342/CD312/CD452) cells showed 107 differentially expressed genes. Panel B: Pools of analyzed cells from Sca-1+/CD34+ subpopulation (red), Sca-1+/CD342 subpopulation (blue) and pulmonary reference cells (green) were analyzed by quantitative PCR against differentially expressed genes Dcn, Esd and Gsn. The selected genes not only show significant differences regarding to their expression, but also represent different subpopulations of proteins. Error bars indicate standard deviation of the mean calculated for analyzed triplicates. Expression values are calculated by relative quantification against housekeeping gene Actb and illustrated in comparison to pulmonary reference cells (expression value = 1.0) on a logarithmic scale. All comparisons between different groups, as determined by quantitative PCR, showed significantly different expression levels (student’s t-test, * indicating p,0.05). doi:10.1371/journal.pone.0083917.g002 December 2013 \| Volume 8 \| Issue 12 \| e83917 PLOS ONE \| www.plosone.org 6 December 2013 \| Volume 8 \| Issue 12 \| e83917 Single Cell Analysis of Epithelial and Mesenchymal Transcripts Allows Further Delineation of Sca-1+/CD34+,2 Subpopulations Validation by quantitative PCRs for the genes Dcn, Gsn and Esd confirmed the microarray results. During the validation of the microarray results we intentionally abstained from performing qPCR analysis directly on single cell WTA products due to stochastic variation in expression level, a phenomenon that has been repeatedly reported before [19,23–25]. Cell-to-cell heterogeneity in gene expression, which is also detectable in our data sets, may represent stochastic transcriptional bursts that are generated by intrinsic on-off transitions of the corresponding genes occurring at irregular intervals [23,25]. Fluctuations in levels of individual transcripts also affect the housekeeping genes, whose expression serves as reference in qPCR analyses [20,21]. Stable reference is indispensable to reliably quantify relative expression levels of genes of interest. Higher stability in gene expression levels facilitating more reliable downstream analysis can be achieved only by pooling single cell samples representing the same cell population, thereby averaging the stochastic variability of transcript levels in individual cells especially for reference genes. In order to confirm the PCR results we performed FACS analysis of 5 additional digested lung explants using antibodies directed against Sca-1, CD34, CD45, CD31, Epcam and Pdgfra. Within the population of CD452/CD312 cells, we could detect 3.61% of Sca-1+/CD342 cells and 14.3% of Sca-1+/CD34+ cells in mean (Figure 3B). We proceeded to look at Epcam and Pdgfra expression in these two subpopulations of putative BASCs. As previously described, the majority of Sca-1+/CD342 cells expressed Epcam, while Pdgfra was expressed in the Sca-1+/ CD34+ subpopulation. However, in good correlation to our PCR results, we found a significant subpopulation of Epcam+/Pdgfra+ cells within the subpopulation of Sca-1+/CD342 cells (Figure 3B and C). December 2013 \| Volume 8 \| Issue 12 \| e83917 PLOS ONE \| www.plosone.org 7 Figure 3. Expression of epithelial and mesenchymal markers in Sca-1+/CD34+,2 cells. Panel A: The expression of epithe mesenchymal transcripts was tested by analytical PCR and is illustrated in a hierarchical cluster heatmap. The analysis shows that the majorit 1+/CD34+ cells (light grey) show similar marker expression as Sca-12/CD34+ cells (white), while all Sca-1+/CD342 cells (dark grey) are locate second branch. Red squares indicate specific bands in analytical PCR, black squares indicate negative PCR results. Panel B: FACS analysi EpCAM+/Pdgfra+ subpopulation within Sca-1+/CD342/CD312/CD452 cells. For each of 5 mice, 56106 murine lung cells were isolated fr explants and stained with antibodies directed against Sca-1, CD34, CD31, CD45, Pdgfra and Epcam. References 9. McQualter JL, Yuen K, Williams B, Bertoncello I (2010) Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung. Proc Natl Acad Sci U S A 107: 1414–1419. 1. Fine A (2009) Breathing life into the lung stem cell field. Cell Stem Cell 4: 468– 469. 2. Chen H, Matsumoto K, Brockway BL, Rackley CR, Liang J, et al. (2012) Airway epithelial progenitors are region specific and show differential responses to bleomycin-induced lung injury. Stem Cells 30: 1948–1960. 10. Teisanu RM, Lagasse E, Whitesides JF, Stripp BR (2009) Prospective isolation of bronchiolar stem cells based upon immunophenotypic and autofluorescence characteristics. Stem Cells 27: 612–622. 3. Rawlins EL, Okubo T, Xue Y, Brass DM, Auten RL, et al. (2009) The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell 4: 525–534. 11. Hartmann CH, Klein CA (2006) Gene expression profiling of single cells on large-scale oligonucleotide arrays. Nucleic Acids Res 34: e143. 11. Hartmann CH, Klein CA (2006) Gene expression profiling 4. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, et al. (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121: 823–835. 12. Klein CA, Seidl S, Petat-Dutter K, Offner S, Geigl JB, et al. (2002) Combined transcriptome and genome analysis of single micrometastatic cells. Nat Biotechnol 20: 387–392. 13. Summer R, Kotton DN, Sun X, Ma B, Fitzsimmons K, et al. (2003) Side population cells and Bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol 285: L97–104. 5. Nolen-Walston RD, Kim CF, Mazan MR, Ingenito EP, Gruntman AM, et al. (2008) Cellular kinetics and modeling of bronchioalveolar stem cell response during lung regeneration. Am J Physiol Lung Cell Mol Physiol 294: L1158– 1165. 14. Smyth GK, Speed T (2003) Normalization of cDNA microarray data. Methods 31: 265–273. 6. Qian S, Ding JY, Xie R, An JH, Ao XJ, et al. (2008) MicroRNA expression profile of bronchioalveolar stem cells from mouse lung. Biochem Biophys Res Commun 377: 668–673. 15. Johnson WE, Li C, Rabinovic A (2007) Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 8: 118–127. 7. Tiozzo C, De Langhe S, Yu M, Londhe VA, Carraro G, et al. (2009) Deletion of Pten expands lung epithelial progenitor pools and confers resistance to airway injury. Am J Respir Crit Care Med 180: 701–712. Acknowledgments We thank Isabell Blochberger and Siegfried Rein for excellent technical assistance Table S4 Detected mRNA transcripts in isolated single cells. Our results support an epithelial commitment of Sca-1+/CD342 cells – in contrast to the Sca-1+/CD34+ cells that displayed an increased incidence of mesenchymal marker expression. However, taking into account the novel subpopulation of Sca-1+/ CD342/EpCAM+/Pdgfra+ cells we postulate that single cell isolation and transcription profiling on rare cells represents a sensitive approach to obtain molecular data of single cells of interest. In this study, we showed on single murine lung cells that this approach has the power to deliver novel molecular markers that could contribute to a better understanding of the cellular Single Cell Analysis of Epithelial and Mesenchymal Transcripts Allows Further Delineation of Sca-1+/CD34+,2 Subpopulations On the other hand, an exclusive expression of the novel markers in mesenchymal cells has not been proven so far. Esd has been ascribed an important role in intracellular detoxification [32] making tumor cells more resistant against harmful substances [33]. We selected three genes from the list of differentially expressed genes for qPCR validation coding for proteins of different functional groups (Dcn, Gsn, and Esd), representing extracellular matrix components, intracellular/membrane-bound proteins and cytoplasmic metabolic proteins, respectively. Antiproliferative or tissue stabilizing effects as well as the promotion of cellular motility and invasion have been demonstrated for Gelsolin and Decorin [26–31]. Both markers have been previously assigned to pulmo- nary fibroblasts. Consequently, their enhanced expression in Sca- 1+/CD34+ cells would be in line with mesenchymal commitment. On the other hand, an exclusive expression of the novel markers in mesenchymal cells has not been proven so far. Esd has been ascribed an important role in intracellular detoxification [32] making tumor cells more resistant against harmful substances [33]. We then searched for transcripts of recently described epithelial (Epcam, Itga, Sftpc) and mesenchymal (CD90, Pdgfra) markers [8] by analytical PCRs and found a frequent expression of Pdgfra in Sca- 1+/CD34+ single cells. Interestingly, almost 80% of these cells additionally showed expression of Sftpc, described as an epithelial marker, while only 14% of Sftpc-expressing Sca-1+/CD342 cells showed coexpression of Pdgfra. Moreover, expression of the epithelial transcript Epcam was restricted to the group of Sca-1+/ CD342 cells (and one Sca-12/CD34+ cells), while expression of the mesenchymal marker Pdgfra could be detected in only four of the analyzed Sca-1+/CD342 cells. In FACS analysis, we could confirm a significant subpopulation of Epcam+/Pdgfra+ cells within the Sca-1+/CD342/CD452/CD31- cells (Figure 3). Author Contributions Conceived and designed the experiments: MH CAK BP. Performed the experiments: MH ZTC YH CB SK. Analyzed the data: MM CAK BP. Wrote the paper: MH BP. Conceived and designed the experiments: MH CAK BP. Performed the experiments: MH ZTC YH CB SK. Analyzed the data: MM CAK BP. Wrote the paper: MH BP. Table S1 Primer sequences. (DOC) Table S1 Primer sequences. (DOC) g g [ ] We then searched for transcripts of recently described epithelial (Epcam, Itga, Sftpc) and mesenchymal (CD90, Pdgfra) markers [8] by analytical PCRs and found a frequent expression of Pdgfra in Sca- 1+/CD34+ single cells. Interestingly, almost 80% of these cells additionally showed expression of Sftpc, described as an epithelial marker, while only 14% of Sftpc-expressing Sca-1+/CD342 cells showed coexpression of Pdgfra. Moreover, expression of the epithelial transcript Epcam was restricted to the group of Sca-1+/ CD342 cells (and one Sca-12/CD34+ cells), while expression of the mesenchymal marker Pdgfra could be detected in only four of the analyzed Sca-1+/CD342 cells. In FACS analysis, we could confirm a significant subpopulation of Epcam+/Pdgfra+ cells within the Sca-1+/CD342/CD452/CD31- cells (Figure 3). Table S2 Differentially expressed genes (Sca1+/CD34+ vs Reference cells). (DOC) Single Cell Analysis of Epithelial and Mesenchymal Transcripts Allows Further Delineation of Sca-1+/CD34+,2 Subpopulations While Sca-1+/CD342 cells consistently Single Cell Profiling of Lung St PLOS ONE \| l 8 D b 2013 \| V l 8 \| I 12 \| Single Cell Profiling of Lung Stem Cells Figure 3. Expression of epithelial and mesenchymal markers in Sca-1+/CD34+,2 cells. Panel A: The expression of epithelial and mesenchymal transcripts was tested by analytical PCR and is illustrated in a hierarchical cluster heatmap. The analysis shows that the majority of Sca- 1+/CD34+ cells (light grey) show similar marker expression as Sca-12/CD34+ cells (white), while all Sca-1+/CD342 cells (dark grey) are located in the second branch. Red squares indicate specific bands in analytical PCR, black squares indicate negative PCR results. Panel B: FACS analysis reveals EpCAM+/Pdgfra+ subpopulation within Sca-1+/CD342/CD312/CD452 cells. For each of 5 mice, 56106 murine lung cells were isolated from lung explants and stained with antibodies directed against Sca-1, CD34, CD31, CD45, Pdgfra and Epcam. While Sca-1+/CD342 cells consistently showed December 2013 \| Volume 8 \| Issue 12 \| e83917 PLOS ONE \| www.plosone.org 8 Single Cell Profiling of Lung Stem Cells Epcam expression, Pdgfra expression was predominantly found in Sca-1+/CD34+ cells. However, Sca-1+/CD342/Epcam+ cells could be divided in two major subpopulations defined by Pdgfra expression. Relative quantification is given for corresponding selected subpopulation as indicated by arrows. Panel C: Scatter plots of the detected cell populations for mouse 5, only. doi:10.1371/journal.pone.0083917.g003 Epcam expression, Pdgfra expression was predominantly found in Sca-1+/CD34+ cells. However, Sca-1+/CD342/Epcam+ cells could be divided in two major subpopulations defined by Pdgfra expression. Relative quantification is given for corresponding selected subpopulation as indicated by arrows. Panel C: Scatter plots of the detected cell populations for mouse 5, only. doi:10.1371/journal.pone.0083917.g003 heterogeneity and hierarchy in the murine lung. The presented approach may, therefore, help to analyze extremely rare and yet poorly characterized cells (e.g. stem cells) in other fields of biology as well. We selected three genes from the list of differentially expressed genes for qPCR validation coding for proteins of different functional groups (Dcn, Gsn, and Esd), representing extracellular matrix components, intracellular/membrane-bound proteins and cytoplasmic metabolic proteins, respectively. Antiproliferative or tissue stabilizing effects as well as the promotion of cellular motility and invasion have been demonstrated for Gelsolin and Decorin [26–31]. Both markers have been previously assigned to pulmo- nary fibroblasts. Consequently, their enhanced expression in Sca- 1+/CD34+ cells would be in line with mesenchymal commitment. Table S2 Differentially expressed genes (Sca1+/CD34+ vs Reference cells). (DOC) Table S3 Differentially expressed genes (Sca1+/CD34+ vs Sca1+/CD34–). (DOC) 7. Tiozzo C, De Langhe S, Yu M, Londhe VA, Carraro G, et al. (2009) Deletion of Pten expands lung epithelial progenitor pools and confers resistance to airway injury. Am J Respir Crit Care Med 180: 701–712. Supporting Information Figure S1 Immunofluorescence stained single cell sus- pensions of explanted lungs. Panels A–D: The arrow points to a single CD34+/CD452/GFP-Annexin2 cell (Cy3-signal) surrounded by several CD34-negative cells showing fluorescence in FITC channel. Panels E–F: Single Sca-1+/CD312/PI2 cell. (TIF) Table S1 Primer sequences. (DOC) Single Cell Profiling of Lung Stem Cells 19. Raj A, van Oudenaarden A (2008) Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135: 216–226. 28. Shao F, Zhang R, Don L, Ying K (2011) Overexpression of gelsolin-like actin- capping protein is associated with progression of lung adenocarcinoma. Tohoku J Exp Med 225: 95–101. 20. Bengtsson M, Hemberg M, Rorsman P, Stahlberg A (2008) Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. BMC Mol Biol 9: 63. 29. Spence HJ, Johnston I, Ewart K, Buchanan SJ, Fitzgerald U, et al. (2000) Krp1, a novel kelch related protein that is involved in pseudopod elongation in transformed cells. Oncogene 19: 1266–1276. 21. Reiter M, Kirchner B, Muller H, Holzhauer C, Mann W, et al. (2011) Quantification noise in single cell experiments. Nucleic Acids Res 39: e124. 30. Tufvesson E, Westergren-Thorsson G (2003) Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration. J Cell Sci 116: 4857–4864. 22. Alexander CM, Puchalski J, Klos KS, Badders N, Ailles L, et al. (2009) Separating stem cells by flow cytometry: reducing variability for solid tissues. Cell Stem Cell 5: 579–583. 23. Chubb JR, Trcek T, Shenoy SM, Singer RH (2006) Transcriptional pulsing of a developmental gene. Curr Biol 16: 1018–1025. 31. Wiedl T, Arni S, Roschitzki B, Grossmann J, Collaud S, et al. (2011) Activity- based proteomics: identification of ABHD11 and ESD activities as potential biomarkers for human lung adenocarcinoma. J Proteomics 74: 1884–1894. 24. Huh D, Paulsson J (2011) Non-genetic heterogeneity from stochastic partitioning at cell division. Nat Genet 43: 95–100. 32. Lee WH, Wheatley W, Benedict WF, Huang CM, Lee EY (1986) Purification, biochemical characterization, and biological function of human esterase D. Proc Natl Acad Sci U S A 83: 6790–6794. 25. Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4: e309. 26. Bearer EL (1991) Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ. J Cell Biol 115: 1629–1638. 33. Recktenwald CV, Kellner R, Lichtenfels R, Seliger B (2008) Altered detoxification status and increased resistance to oxidative stress by K-ras transformation. Cancer Res 68: 10086–10093. binding by gCap39 and CapZ. J Cell Biol 115: 1629–1638. 27. References 16. Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3. 8. McQualter JL, Brouard N, Williams B, Baird BN, Sims-Lucas S, et al. (2009) Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction. Stem Cells 27: 623–633. 17. Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I (2001) Controlling the false discovery rate in behavior genetics research. Behav Brain Res 125: 279–284. 18. Pfaffl MW (2001) A new mathematical model for relative quantification in real- time RT-PCR. Nucleic Acids Res 29: e45. PLOS ONE \| www.plosone.org 9 December 2013 \| Volume 8 \| Issue 12 \| e83917 December 2013 \| Volume 8 \| Issue 12 \| e83917 33. Recktenwald CV, Kellner R, Lichtenfels R, Seliger B (2008) Altered detoxification status and increased resistance to oxidative stress by K-ras transformation. Cancer Res 68: 10086–10093. 32. Lee WH, Wheatley W, Benedict WF, Huang CM, Lee EY (1986) Purification, biochemical characterization, and biological function of human esterase D. Proc Natl Acad Sci U S A 83: 6790–6794. 28. Shao F, Zhang R, Don L, Ying K (2011) Overexpression of gelsolin-like actin- capping protein is associated with progression of lung adenocarcinoma. Tohoku J Exp Med 225: 95–101. Single Cell Profiling of Lung Stem Cells Romaris M, Heredia A, Molist A, Bassols A (1991) Differential effect of transforming growth factor beta on proteoglycan synthesis in human embryonic lung fibroblasts. Biochim Biophys Acta 1093: 229–233. PLOS ONE \| www.plosone.org December 2013 \| Volume 8 \| Issue 12 \| e83917 PLOS ONE \| www.plosone.org 10
https://openalex.org/W4383498808	https://www.annualreviews.org/doi/pdf/10.1146/annurev-devpsych-120621-043144	English	null	The Functioning of Offspring of Depressed Parents: Current Status, Unresolved Issues, and Future Directions	Annual review of developmental psychology	2,023	cc-by	15,065	Keywords First published as a Review in Advance on July 7, 2023 depressed parents, offspring, psychobiology, brain structure, COVID-19 depressed parents, offspring, psychobiology, brain structure, COVID-19 Ian H. Gotlib,1 Jessica L. Buthmann,1 and Jonas G. Miller2 Annu. Rev. Dev. Psychol. 2023. 5:375–97 Annual Review of Developmental Psychology The Functioning of Offspring of Depressed Parents: Current Status, Unresolved Issues, and Future Directions Annual Review of Developmental Psychology The Functioning of Offspring of Depressed Parents: Current Status, Unresolved Issues, and Future Directions g. Guest (guest) IP: 195.242.1.103 On: Thu, 24 Oct 2024 04:44:48 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 376 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Contents DEPRESSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 MALADAPTIVE PSYCHOBIOLOGICAL CHARACTERISTICS OF OFFSPRING OF DEPRESSED PARENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Psychiatric Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Cognitive Functioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Social Relationships. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Biological Characteristics of Offspring of Depressed Parents . . . . . . . . . . . . . . . . . . . . . . 379 Mediators, Moderators, and Interacting Variables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382 Non-White Participants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384 COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 CONCLUSIONS AND DIRECTIONS FOR FUTURE RESEARCH . . . . . . . . . . . . . 385 Altered Characteristics and Clinical Outcomes in Offspring of Depressed Parents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 Issues of Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 SUMMARY . . . . . . . . . . . . . . . . . . 376 Gotlib • Buthmann • Miller Abstract The Annual Review of Developmental Psychology is online at devpsych.annualreviews.org Although the intergenerational transmission of risk for depression is well documented, the mechanisms and moderators involved in this transmission of risk from depressed parents to their offspring are not clear. In this review, we discuss the progress that has been made over the past two decades in studying offspring of depressed parents and describe the maladaptive char- acteristics of these offspring in a diverse range of domains, including clinical, cognitive, and biological functioning. Despite recent advances in this area, there are unresolved questions that warrant further investigation involving the nature of risk transmission from parent to offspring, the specificity of findings to depression, and the role of factors that often accompany depres- sion. We discuss these issues and offer directions for future research that we believe will move the field forward in gaining a better understanding of the relation between parental depression and altered psychobiological functioning in their offspring. https://doi.org/10.1146/annurev-devpsych-120621- 043144 Copyright © 2023 by the author(s). This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See credit lines of images or other third-party material in this article for license information. 375 Contents DEPRESSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 MALADAPTIVE PSYCHOBIOLOGICAL CHARACTERISTICS OF OFFSPRING OF DEPRESSED PARENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Psychiatric Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Cognitive Functioning . . . . . . . . . . . . Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 Social Relationships. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Biological Characteristics of Offspring of Depressed Parents . . . . . . . . . . . . . . . . . . . . . . 379 Mediators, Moderators, and Interacting Variables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382 Non-White Participants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384 COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 CONCLUSIONS AND DIRECTIONS FOR FUTURE RESEARCH . . . . . . . . . . . . . 385 Altered Characteristics and Clinical Outcomes in Offspring of Depressed Parents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386 Issues of Specificity . DEPRESSION Major depressive disorder (MDD) is among the most prevalent and recurrent of all psychiatric disorders (Am. Psychiatr. Assoc. 2013); almost 20% of the US population, or more than 30 million adults, will experience a clinically significant episode of depression, with most having multi- ple episodes of MDD over their lifetime (Kessler & Wang 2008). Further, rates of MDD and depressive symptoms have been increasing in both children and adults, particularly during the COVID-19 pandemic (Gotlib et al. 2021, Racine et al. 2021) and more strikingly in females than in males (Cent. Dis. Control 2023). MDD has enormous personal, familial, and societal costs, including adverse effects on physical health, interpersonal relationships, educational attainment, and financial security (Kessler & Wang 2008, Luppino et al. 2010). It is not surprising, therefore, that the World Health Organization (2017) has projected that depression will be the single most burdensome and costly disease in the world in terms of disability-adjusted years in the twenty-first century. Given the increasing prevalence and rising costs of depression, the consistency with which researchers have documented adverse effects of parental depression on the functioning of off- spring is especially alarming; in particular, a large literature has confirmed the high rates of depression in offspring with a depressed parent. Specifically, children and adolescents of parents with MDD are two to four times more likely to develop depression than are their peers with no family history of the disorder (Apter-Levy et al. 2013, Goodman & Garber 2017). Further, first episodes of depression appear to be occurring at increasingly younger ages (Lebrun-Harris et al. 2022); indeed, early-onset depression has been found to be associated with pervasive dysfunc- tion over the life course (Hill et al. 2004). Given that over 10% of children in the United States are now exposed to parental depression each year (Ertel et al. 2011), it is clear that these high rates of depression in offspring with a family history of the disorder are a significant public health concern. Although studies documenting high levels of depressive symptoms and an elevated prevalence of diagnosed depression in offspring of depressed parents are important in highlighting a specific adverse consequence of parental MDD, over the past two decades researchers have been building 6 Gotlib • Buthmann • Miller 376 on scientific advances to identify other types of difficulties and forms of altered or maladaptive functioning that characterize offspring of depressed parents. DEPRESSION Thus, in addition to high rates of depression, there is now evidence that offspring of depressed parents are characterized by negative biases in their processing of information, impaired cognitive development, and problem- atic interpersonal relationships and exhibit altered neural function, structure, and connectivity, dysregulated hypothalamic-pituitary-adrenal (HPA) axis functioning, high levels of inflammation, and accelerated biological aging. Several review articles have now been published describing the effects of parental depression on offspring. Perhaps most notably, Goodman & Gotlib (1999) formulated a comprehensive, integrative model of intergenerational transmission of risk for MDD focused on genetic, contex- tual, regulatory, and social-cognitive-demographic factors as mechanisms and moderators of risk and resilience for depression as outcomes in offspring of depressed mothers.1 Goodman & Gotlib drew several conclusions and caveats from their review, noting the lack of research in particular areas and the tenuous links between psychobiological difficulties in offspring of depressed moth- ers and the subsequent development of depression. Although their review was comprehensive, Goodman & Gotlib focused primarily on clinical symptoms and diagnoses as the outcomes of having a depressed mother; they conceptualized other maladaptive characteristics as mediators of the association between maternal depression and offspring depression. More recently, Gotlib et al. (2020) also focused on clinical symptoms and diagnoses in offspring of depressed parents and identified mechanisms that might underlie the intergenerational transmission of risk for depression, underscoring findings that maternal depression is associated with a range of other risk factors such as poverty, marital conflict, and other environmental stressors. To date, however, there has not been a detailed consideration of the range of functioning that appears to be affected in offspring with a family history of depression and how this altered functioning may lead to the development of depression and/or other forms of psychopathology in offspring. The purpose of this article is to synthesize the current literature examining the intergenera- tional effects of parental depression, elucidating the diverse domains and ways in which offspring of depressed parents exhibit maladaptive functioning. We begin by presenting recent data con- cerning symptoms and psychiatric diagnoses in offspring of depressed parents. Next, we describe cognitive and social difficulties in offspring of depressed parents and discuss alterations in their biological functioning. We discuss the likelihood that difficulties or alterations in specific domains do not occur in isolation but instead almost certainly influence each other and interact, leading to higher rates of disorder. 1Given that, as we note above, the prevalence of depression is twice as high in women as it is in men, combined with the fact that most studies of offspring of depressed parents have focused on depressed mothers, it is un- derstandable that Goodman & Gotlib (1999) discussed clinical outcomes in offspring of depressed mothers. Klein et al. (2005), Lewinsohn et al. (2005), and Gutierrez-Galve et al. (2019) examined the effects of maternal versus paternal depression on offspring’s functioning and found that both were associated with adverse ef- fects in offspring and that there were inconsistent differences between the effects of maternal versus paternal depression. In this review we broadly discuss the effects of parental depression on offspring and try to note differences, when available, between maternal and paternal depression. DEPRESSION In this context, therefore, we describe findings involving mediation and/or moderation of the association of parental depression with disorder in offspring and note examples of interactions between different domains of functioning. We also discuss the impor- tance of conducting research with non-White participants and describe findings concerning the effects of the COVID-19 pandemic on pregnancy and the relation between parental depression and offspring functioning. Finally, we raise issues that warrant further investigation and offer di- rections for future research that we think will help make significant progress in understanding 1Given that, as we note above, the prevalence of depression is twice as high in women as it is in men, combined with the fact that most studies of offspring of depressed parents have focused on depressed mothers, it is un- derstandable that Goodman & Gotlib (1999) discussed clinical outcomes in offspring of depressed mothers. Klein et al. (2005), Lewinsohn et al. (2005), and Gutierrez-Galve et al. (2019) examined the effects of maternal versus paternal depression on offspring’s functioning and found that both were associated with adverse ef- fects in offspring and that there were inconsistent differences between the effects of maternal versus paternal depression. In this review we broadly discuss the effects of parental depression on offspring and try to note differences, when available, between maternal and paternal depression. www.annualreviews.org • Functioning of Offspring of Depressed Parents 377 the nature of the relation between parental depression and altered psychobiological functioning in offspring. MALADAPTIVE PSYCHOBIOLOGICAL CHARACTERISTICS OF OFFSPRING OF DEPRESSED PARENTS One of the most consistent findings in clinical psychology and psychiatry is that offspring of depressed parents are at increased risk for developing depression (Hammen 2018). The link between parental depression and increased rates of psychopathology has been reported in children as young as 6 years of age, with over 60% meeting the full criteria for a psychiatric disorder, particularly an affective disorder (Priel et al. 2019). Given the consistency of this finding and our space constraints, we do not go into details of specific studies here but will note that in an exceptional longitudinal study, Weissman and colleagues (1982) significantly advanced our knowledge of the extent of the impact of parental depression on offspring by following a sample of depressed parents, their offspring, and now the offspring’s children for almost 40 years. In 1982, Weissman and her colleagues recruited 91 families with and without a depressed parent, assessed psychopathology in the 276 offspring of these parents, and documented elevated rates of depres- sion in these offspring. In 2005, Weissman et al. assessed the grandchildren of the initial probands and found that, even at a mean age of only 12 years, almost 60% of the grandchildren in families in which two generations had experienced depression already had a psychiatric disorder (Weissman et al. 2005). And in 2021, Weissman et al. reported a 38-year follow-up of these families and found that offspring of depressed parents were at increased risk not only for developing MDD and other psychiatric disorders but also for death by suicide or overdose (Weissman et al. 2021). Similar findings were reported by van Dijk et al. (2021) in the Adolescent Brain Cognitive Development sample and by Josefsson et al. (2019) in a Swedish cohort study. Finally, in the largest study in this area, Gronemann et al. (2023) followed a sample of almost three million people (all Danish citizens born between 1960 and 2003 with known parental identity) from their fifteenth birthday until they were diagnosed with depression, censored, or reached December 31, 2018. They found that for both men and women, exposure to maternal or paternal depression was associated with a two-times higher risk for developing MDD. Further, onset of maternal MDD before age 70 (for female offspring) and before age 30 (for male offspring) was associated with higher risk for MDD. 378 Gotlib • Buthmann • Miller Social Relationships Depression often involves dysfunction in social relationships, difficulties that have also been ob- served in offspring of depressed parents. For example, in a meta-analysis, Barnes & Theule (2019) found that rates of insecure infant attachment were higher in offspring of depressed mothers than in controls. Further, prenatal maternal depressive symptoms have been associated with more peer relationship problems and less prosocial behavior in 4-year-old children (Koutra et al. 2017). Henry et al. (2020) found that symptoms of anxiety and depression were highest in adolescents who experienced more peer relationship stress and whose mothers had more symptoms of depres- sion. Kujawa et al. (2020) found that the quality of the mother–child relationship at age 3 mediated the association of maternal depression with both child peer stress and neural response to social rejection at age 12. Finally, experiences of peer victimization have been found to mediate the as- sociation between maternal depressive symptoms and suicidal ideation in adolescent girls (Tsypes & Gibb 2015). Thus, like depressed adults, offspring of depressed parents appear to consistently experience difficulties in their interpersonal relationships. Cognitive Functioning Depressed persons have consistently been found to have poorer and/or less adaptive cognitive functioning than do neurotypically functioning people. Importantly, similar differences have also frequently been observed in their offspring. For example, in 3- to 5-year-old children, maternal depressive symptoms were associated with a bias toward interpreting puppets as being sad (Martin et al. 2015). Compared with their peers without a family history of disorder, late childhood– to early adolescent–aged children of depressed parents have been found to have a more negative in- terpretation bias (Dearing & Gotlib 2009, Sfärlea et al. 2019), to have more difficulty identifying emotional facial expressions ( Joormann et al. 2010, Székely et al. 2014), and to attend selectively to negative facial expressions ( Joormann et al. 2007, Montagner et al. 2016). Interestingly, bet- ter cognitive functioning has been found to be protective of depressive symptoms in high-risk children. Davidovich et al. (2016) found that 9- to 17-year-old children of depressed parents with better inhibitory control and cognitive flexibility had fewer symptoms of depression than did chil- dren with poorer cognitive performance. Thus, cognitive functioning may offer an opportunity for targeted interventions for children at familial risk for depression. Gotlib • Buthmann • Miller 378 Biological Characteristics of Offspring of Depressed Parents Offspring of depressed parents have consistently been found to exhibit alterations in their brain structure, function, and connectivity, some of which emerge as early as infancy in the context of prenatal maternal depression. For example, in a sample of 1-month-old infants,maternal prenatal depressive symptoms were associated with reduced right frontal white matter microstructure, particularly in female infants (Dean et al. 2018). In newborns with a genomic profile indicating elevated risk for MDD, maternal prenatal depressive symptoms were positively associated with larger right hippocampal volume and shape, right amygdala shape, and thicker orbitofrontal and ventromedial prefrontal cortices (Qiu et al. 2017); these findings suggest that MDD-relevant genes moderate the effects of prenatal maternal depressive symptoms on fetal development of brain regions implicated in cognitive-emotional functioning. Borchers et al. (2021) found that prenatal maternal depressive symptoms were associated with poorer white matter integrity in infancy.Prenatal maternal depressive symptoms have also been found to predict reduced cortical thickness in child offspring (Sandman et al. 2015). Interestingly, reductions in cortical thickness have been found to mediate the link between prenatal maternal depression and children’s externalizing problems (Sandman et al. 2015), which in turn have been demonstrated to increase risk for internalizing problems during childhood (Oh et al. 2020) and depression during adolescence (Nilsen et al. 2013). Longitudinal findings suggest that the effects of perinatal maternal depression on neuro- development in offspring are long-lasting and that chronic exposure to maternal depression is particularly impactful. For example, in a large birth cohort study, Zou et al. (2019) found that consistently high levels of perinatal maternal depression were associated with reduced gray and white matter volume in offspring at age 10. Finally, using resting-state functional magnetic reso- nance imaging, researchers have documented altered functional connectivity in infants exposed to prenatal depression, including greater positive functional connectivity of the amygdala with the left insula, bilateral anterior cingulate, and ventromedial prefrontal cortices (Qiu et al. 2015), and greater negative functional connectivity of the amygdala with dorsal prefrontal cortices (Posner et al. 2016). Thus, the effects of perinatal depression on structural and functional brain alterations in in- fancy, some of which may persist through childhood and adolescence, may contribute to the intergenerational transmission of risk for depression. Biological Characteristics of Offspring of Depressed Parents Over the last 20 years researchers have made significant advances in our understanding of biolog- ical anomalies in offspring of depressed parents, involving progress in studying fetal development, neuroimaging, stress biology, inflammation, and biological aging. Many of the observed biological alterations have been implicated in the development of psychopathology, including depression. Thus, findings of studies in this area may provide information about mechanisms that underlie the intergenerational transmission of risk for this disorder. Recent reviews have provided com- prehensive summaries of the literature organized by various biological methods and measures focused on elucidating specific mechanisms underlying familial risk for the development of psy- chopathology (e.g., Burkhouse & Kujawa 2022). In this section we build on these advances to describe differences in biological functioning between offspring of depressed versus nondepressed parents. Epigenetic alterations. Maternal depression occurring during the gestational period may alter the development of the fetus, setting offspring on a long-term course of vulnerability to the onset of depression. The Developmental Origins of Health and Disease hypothesis posits that early life experiences, including the prenatal environment, can have lifelong implications for mental and physical health (Gluckman et al. 2007). Researchers examining neonatal tissues (e.g., placenta, umbilical cord blood) have begun to elucidate the ways in which maternal depression during preg- nancy affects the functioning of offspring. For example, Conradt et al. (2013) found that higher levels of methylation of the glucocorticoid receptor gene NR3C1 in the placenta of mothers with depression predicted lower self-regulation scores in their newborn infants. Similarly, Zhang et al. (2018) found that maternal prenatal depressive symptoms were associated with increased infant negative affect, but only in infants with decreased HSD11B2, NR3C1, and NR3C2 (HPA-axis regu- latory genes) placental gene expression. Further, Liu et al. (2012) found that depressive symptoms during pregnancy were associated with higher methylation at the MEG3 differentially methyl- ated region, which has been implicated in altered fetal development (Heijmans et al. 2009) and internalizing symptoms in infancy (Fuemmeler et al. 2016). Collectively, these findings point to a likely cascade of epigenetic alterations related to maternal depression that contribute to risk for the development of depression in offspring, although additional research on the epigenome and replication of relevant findings are clearly needed to gain a more comprehensive understanding of the major pathways that are affected by prenatal depression. www.annualreviews.org • Functioning of Offspring of Depressed Parents 379 Brain structure, function, and connectivity. 380 Gotlib • Buthmann • Miller Biological Characteristics of Offspring of Depressed Parents This perspective is consistent with the De- velopmental Origins of Health and Disease hypothesis that early life is characterized by rapid neurobiological development and, thus, is a sensitive period of increased plasticity and open- ness to the effects of maternal depression (Gluckman et al. 2007). As Goodman & Gotlib (1999) posited over two decades ago, an altered prenatal environment related to maternal depression, which might include increased fetal exposure to inflammation and cortisol, may shape offspring neurodevelopment in ways that increase risk for the development of depression (Wang et al. 2022). Several researchers have also examined relations between parental depression and neural re- sponsivity of offspring to various tasks in the scanner. Given that disruptions in individuals’ responses to rewarding experiences have been implicated in diminished positive affect in depres- sion (Forbes & Dahl 2012), many of these efforts have focused on reward processing. As is the case with depressed adults (Fox & Lobo 2019), offspring of depressed parents show altered neural function when processing rewarding stimuli. Blunted reward-related neural responses have also been observed in children of depressed mothers at as young as 5 years of age (Wiggins et al. 2017); similarly, Kujawa et al. (2014) found reduced neural differentiation between the receipt of gains and losses in 9-year-old children of depressed mothers. Because adolescence is a period of both heightened risk for the onset of depression and heightened striatal responses to the receipt of re- wards (Luking et al. 2016), much of the research examining the brain basis of reward processing with offspring of depressed parents has focused on adolescents and young adults. For example, Gotlib • Buthmann • Miller 380 in an early study Gotlib et al. (2010) found that never-depressed daughters of depressed mothers exhibited diminished activation in the putamen and left insula and heightened activation in the right insula when anticipating monetary gains. Adolescents and young adult offspring of depressed parents have also been found to exhibit decreased activation in the orbitofrontal cortex (McCabe et al. 2012) and the nucleus accumbens (Monk et al. 2008) in response to reward. More recently, using electroencephalography, Freeman et al. (2022) found that a maternal history of depression was related to reduced neural responses to the receipt of social rewards in adolescent girls. Biological Characteristics of Offspring of Depressed Parents Col- lectively, therefore, this literature indicates that offspring of depressed parents are characterized by a blunted neural response in reward contexts, in both cortical and striatal regions. Finally, researchers have posited that amygdala hyperactivity in response to negative stimuli is a useful biomarker of risk for the development of depression (Mattson et al. 2016). Indeed, inves- tigators have found higher amygdala response to negative facial expressions in 6- to 9-year-old children of mothers who had prenatal depression (van der Knaap et al. 2018), in 8- to 14-year-old children of depressed parents (Chai et al. 2015), and in adolescents of depressed parents (Monk et al. 2008). Joormann et al. (2012) found stronger amygdala activation during a sad mood induc- tion task in 9- to 14-year-old daughters of remitted depressed than of never-depressed mothers. In a larger sample of adolescents with a depressed parent or grandparent, left amygdala response to negative facial expressions increased over a 2-year interval compared with those without a fam- ily history of depression (Swartz et al. 2015). Given that amygdala activation has been shown to predict response to antidepressant treatment (Williams et al. 2015), it may be an important target for assessment and intervention in offspring of depressed parents. Stress biology. Findings of studies examining the functioning of the HPA axis and autonomic ner- vous system indicate that offspring of depressed parents are characterized by dysregulated stress biology. Infants (Feldman et al. 2009), children (Apter-Levi et al. 2016), and adolescents (Gotlib et al. 2015) of depressed parents have all been found to have elevated levels of basal cortisol as well as potentiated increases in cortisol in response to stressors (Klimes-Dougan et al. 2022). For exam- ple, compared with adolescent daughters of mothers without a history of depression, daughters at familial risk for depression have elevated diurnal cortisol and cortisol reactivity to stress (Foland- Ross et al. 2014, Gotlib et al. 2015). Further, offspring of depressed parents exhibit patterns of autonomic activity that have been posited to underlie decreased or ineffective self-regulation. For instance, infants of depressed mothers have been found to exhibit elevated heart rate and de- creased vagal tone both at rest and during maternal interactions (Propper & Holochwost 2013). Similar findings have been reported in older offspring: Relative to adolescent offspring of nonde- pressed mothers, adolescent offspring of depressed mothers are characterized by elevated resting heart rate (Nelson et al. www.annualreviews.org • Functioning of Offspring of Depressed Parents Biological Characteristics of Offspring of Depressed Parents 2021) and increased autonomic reactivity to sad faces (Burkhouse et al. 2014). Thus, as early as infancy, offspring of depressed mothers show signs of increased arousal and hyperactivity in stress biology systems. Inflammation. Perhaps not surprisingly given that inflammation has been implicated in the pathophysiology of depression (Toenders et al. 2022), researchers have documented altered levels of inflammation in offspring of depressed parents. For example, toddlers of depressed mothers have been found to have heightened inflammation, indexed by a composite measure of proinflammatory cytokines (Measelle & Ablow 2018). Similarly, Wolf et al. (2008) found that parental depressive symptoms (primarily in mothers) were linked with increases in their children’s inflammatory markers (e.g., interleukin-4) over a 6-month period. These findings were extended both by O’Connor et al. (2020), who found that maternal depressive symptoms in childhood predicted elevated levels of C-reactive protein in their offspring almost a decade later, and by Plant et al. (2016), who demonstrated that prenatal maternal depression predicted elevated levels www.annualreviews.org • Functioning of Offspring of Depressed Parents 381 of C-reactive protein in offspring 25 years later. It is noteworthy that maternal depression and depressive symptoms have also been linked in offspring to physical health problems that have a strong inflammatory component. For example, prenatal maternal depressive symptoms have been found to be associated with infant infections (Schuez-Havupalo et al. 2018); similarly, autoimmune disorders, such as arthritis, psoriasis, and type 1 diabetes, are more common in children of parents with depression than in children of parents with no history of psychopathology (Nevriana et al. 2022). Thus, exposure to parental depression appears to alter the development of the immune system in offspring in ways that contribute to the expression of a maladaptive proinflammatory phenotype that may increase risk for the development of depression. Biological aging. Offspring of depressed parents have been found to show signs of accelerated biological aging, conceptualized as a more rapid decline in the integrity and function of biological systems than occurs solely with increasing chronological age. Biological aging has been studied using a variety of measures across multiple domains (Colich et al. 2020), although it appears that different measures capture different aging or developmental processes (Miller et al. 2020). Biological Characteristics of Offspring of Depressed Parents At the cellular level, researchers have perhaps most frequently assessed telomeres—protein caps at the end of chromosomes that shorten with cell division (Blackburn 2000); shorter telomere length has been used as a cellular measure of more rapid biological aging. In this context, adolescent offspring of depressed mothers have been found to have shorter telomeres than do their peers with nondepressed mothers (Gotlib et al. 2015, Nelson et al. 2021). Shorter telomere length has also been reported in children of mothers with elevated levels of depressive symptoms (Nelson et al. 2018). Although it is tempting to posit on the basis of these findings that shorter telomere length is a risk factor for the development of depression, it is important to recognize that shorter telomere length might instead be a consequence of other factors involved in the intergenerational transmission of risk for depression. For example, dysregulated stress biology, including increased HPA-axis and sympathetic nervous system activation, are putative mechanisms of processes, such as inflammation and oxidative stress, that contribute to telomere shortening ( Jiang et al. 2019). Indeed, changes in maternal depressive symptoms have been indirectly linked to shorter telo- mere length in toddlers via altered infant HPA-axis functioning (Nelson et al. 2018). Importantly, findings from longitudinal research suggest that symptoms of psychopathology in adolescents predict telomere shortening over time, but not the reverse (Humphreys et al. 2020, Wade et al. 2020). Thus, shorter telomere length in children and adolescents of depressed parents may reflect a consequence of other psychobiological processes involved in familial risk for depression. These diverse findings involving telomere length in the context of other biological processes highlight the complexity of the construct of biological aging and underscore the need for more research in this area and replication of relevant findings. Gotlib • Buthmann • Miller Mediators, Moderators, and Interacting Variables For example, harsh parent–child interactions at 2–3 years of age were found to partially mediate the association between early postnatal maternal depressive symptoms and child executive func- tioning at 4 years of age (Gueron-Sela et al. 2018). Similarly, maternal parenting at 18 months of age mediated the relation between maternal depressive symptoms at 9 months postpartum and child externalizing symptoms at 2 years of age (Taraban et al. 2019). Notably, Kuckertz et al. (2018) found that although parenting behaviors mediated the association of maternal depression with in- ternalizing symptoms at 9 years of age, the relation between maternal and child symptoms was bidirectional. Maternal depression in the first year of life has been found to be associated with decreased mother–child synchrony during play and child oxytocin levels at 6 and 10 years of age, both of which mediated the relation between maternal depression and subsequent child internal- izing and externalizing symptoms (Priel et al. 2019). Psychogiou et al. (2020) found that poorer parent–child relationships reported by 16-year-old offspring of depressed parents mediated the associations of parental depressive symptoms with child academic performance and mental health. Sex differences. Perhaps not surprisingly given the well-documented sex differences in the preva- lence of depression, some (but not all) researchers have also found sex differences in the magnitude of the association between parent depression and offspring outcomes. For example, researchers have found that maternal prenatal symptoms of depression were related to smaller amygdala volume in newborn males, but not females (Lehtola et al. 2020), and to poorer white matter mi- crostructure in 1-month-old females, but not males (Dean et al. 2018). Similarly, Letourneau et al. (2019) found that maternal postnatal depressive symptoms mediated the association between ma- ternal early life stress and internalizing and externalizing symptoms for 2-year-old boys, but not girls, and Swales et al. (2018) found that higher levels of prenatal maternal depressive symptoms were related to greater child emotional reactivity in preschool-aged girls, but not boys. The timing of exposure to depressive symptoms may also be an important consideration in un- derstanding sex differences in vulnerability to depression due to familial risk. In this context, Nolvi et al. Mediators, Moderators, and Interacting Variables Although offspring of depressed parents are clearly at elevated risk for the development of de- pression, the mechanisms by which this risk is transmitted intergenerationally are still unclear. We describe above alterations in the functioning of offspring of depressed parents in several diverse domains, any of which may represent the mechanisms underlying the relation between family his- tory and the development of depression. It is almost certain, however, that there is not a single mechanism underlying the intergenerational transmission of risk for depression but rather a con- stellation of contributing and likely interacting factors. Most researchers have focused on a single domain of risk for depression, such as anomalous neural characteristics, negative cognitive biases, or altered HPA-axis functioning. While the results of these investigations are informative, it is almost certain that many of these characteristics co-occur, and it is becoming increasingly clear 382 that the strongest advances in the understanding of risk for depression will come from studies that integrate assessments of psychobiological characteristics of offspring of depressed parents. Below, we briefly consider how the variables described above may interact with each other and with other factors to contribute to the development of depression in offspring with a depressed parent. Caregiving behaviors. Suboptimal caregiving behaviors may increase risk for depression in off- spring of depressed parents. Children of depressed parents have been found to report receiving fewer positive parenting behaviors from their parents (Loechner et al. 2020). Mechanistically, de- pression may lead parents to be less sensitive to their children’s needs and/or to engage in less warm parenting, perhaps attributable to the negative cognitive biases, social withdrawal and lack of warmth, or increased irritability that characterizes depressed individuals (Lovejoy et al. 2000), impairing their ability to be aware of and respond to their children’s needs. For example, moth- ers with prenatal depression have been found to be less responsive to their infants’ distress when experiencing a heel lance and to engage in less affectionate touch in response to infant crying (Mercuri et al.2023).Similarly,Salo et al.(2020) found that parents with high depressive symptoms exhibited less cognitive, emotional, and affective empathy toward their 1- to 3-year-old children. g p y y Importantly, researchers have consistently found that problematic caregiving behaviors and parent–child relationships mediate associations between parent symptoms and child functioning. www.annualreviews.org • Functioning of Offspring of Depressed Parents Mediators, Moderators, and Interacting Variables (2019) found that whereas maternal depression that occurred only during the prenatal period was associated with elevated fear in 6-month-old girls but not in boys, the pattern was reversed in infants whose mothers reported elevated symptoms during both the pre- and postnatal periods, with girls having lower levels of fear than did boys. In contrast, Braithwaite et al. (2020) found that the association between postnatal maternal depression and offspring’s emotional problems was 383 stronger in 3.5-year-old boys (but not in girls) whose mothers also had prenatal depression. Addi- tional research is needed to clarify the precise nature of the relation between parental depression and risk for psychopathology in male and female offspring. Sleep. Sleep disturbances have long been associated with psychological disorders and, indeed, are diagnostic symptoms of depressive disorders. Maternal depression, both prenatally and post- natally, has been found to affect offspring’s sleep behaviors and sleep quality. For example, in a large Finnish cohort, prenatal maternal symptoms of depression predicted more sleep problems and higher risk for developing a sleep disorder in 3.5-year-old offspring, an association that was mediated by concurrent maternal depressive symptoms (Toffol et al. 2019). Postnatal maternal de- pression symptoms have also been found to mediate the association of the goodness of fit between mother–child sleep at 8 months postpartum and mother–child attachment at 30 months postpar- tum (Newland et al. 2016). Finally, de Jong et al. (2016) found that maternal postnatal depressive symptoms were associated with increased variability in sleep onset time in 4.5-year-old offspring, although only for low- and not for high-socioeconomic-status dyads. Researchers have begun to focus explicitly on the temporal nature of the relation between sleep problems and depressive symptoms. While some research suggests that the association between these two constructs is bidirectional (Wang et al. 2020), other investigators have found disturbed sleep to precede and predict the development of depression (Lovato & Gradisar 2014), indicating that sleep difficulties may be a useful target for intervention in the study of children at familial risk for depression. Dysregulated emotion. Another mechanism by which parental mood may contribute to mal- adaptive functioning in offspring is through dysregulated emotion and stress reactivity. We describe above how offspring of depressed parents have altered regulatory abilities; impor- tantly, these difficulties have consistently been found to underlie behavioral differences associated with parental depression. Mediators, Moderators, and Interacting Variables For example, maternal prenatal depressive symptoms are related to lower levels of cortisol, which in turn are associated with more externalizing symptoms in 12-month-old children (Galbally et al. 2019). In a sample of adopted children, Laurent et al. (2013) found that child cortisol at 54 months of age moderated the association of parental depressive symptoms with child internalizing and externalizing symptoms. Maternal depression has been found to be associated with increased mother and child cortisol, which in turn predicted increased internalizing and externalizing symptoms in middle childhood (Ulmer-Yaniv et al. 2018). In 9- to 15-year-old participants, maternal depressive symptoms were associated with increased internal- izing and externalizing symptoms among adolescents using less adaptive stress coping strategies (Vreeland et al. 2019); this association was not evident in children who used more adaptive coping strategies. Further, in 8- to 17-year-old children, emotion regulation difficulties and negative life events partially mediated the relation between parent depressive symptoms and child depressive symptoms (Loechner et al. 2020). Thus, alterations in stress reactivity and emotion regulation may serve as mediating paths of intergenerational risk but may also magnify the relation between parental depressive symptoms and difficulties in offspring. 384 Gotlib • Buthmann • Miller Non-White Participants The associations between maternal mood and child outcomes have generally been assessed in White participants,to the exclusion of Black and Hispanic people in particular; there are important reasons to attend to racial and ethnic differences in this area. For example, Liu et al. (2016) found that concerns about financial and relational stressors were associated with depressed mood among Black and Hispanic pregnant people, but not White pregnant people, indicating that depres- sive symptoms may emerge and manifest differently in different racial or ethnic groups. Indeed, Gotlib • Buthmann • Miller 384 researchers have conducted studies in non-White populations to examine the generalizability of results found in White samples. Giurgescu et al. (2015) found that preterm birth was 16 times more likely and low birth weight 4 times more likely to occur in Black than in White women with depression. In a sample of low-income Black women, prenatal depression was associated with a more negative view of the child before, during, and after birth (Lee & Hans 2015). The relation between parental depression and offspring functioning has also been examined in countries other than the United States and in non-White participants. For example, newborns were more likely to be of lower birth weight in samples of Zambian and Nigerian women with prenatal depression (Sanchez et al. 2013, Wado et al. 2014). Similarly, Bangladeshi and Peruvian women with pre- natal depression gave birth to infants of younger gestational age (Nasreen et al. 2013, Sanchez et al. 2013). Maternal postpartum depression in Pakistani women has been found to be associated with a greater likelihood of delayed emotional, language, and motor development (Ali et al. 2013). Finally, among indigenous people in Canada, having a parent or grandparent who was forced to attend an Indian Residential School was associated with increased risk of suicide ideation and at- tempts (McQuaid et al. 2017). In general, studying the intergenerational transmission of risk for depression in ethnic minorities, in people of color, and in non-Western samples is particularly important given the historical underfunding of and lack of access to health care in these com- munities and the potential for members of these communities to have experienced racism and discrimination (Hankerson et al. 2022). COVID-19 The global COVID-19 pandemic has led to increased rates of depression, anxiety, and other psy- chopathological disorders (Gotlib et al. 2021, Racine et al. 2021); researchers are just beginning to explore the effects of the pandemic on the intergenerational transmission of risk for psychopathol- ogy. The pandemic appears to have increased levels of depressive symptoms in pregnant women (King et al. 2020, Racine et al. 2021), which has implications for the exposure of the developing fetus to prenatal depression and its associated factors.Buthmann et al.(2022) found that pandemic- related stress was associated with both increased prenatal depressive symptoms and infant negative affect. Comparing women who became mothers before versus during the pandemic, Chang et al. (2023) found that the association between postpartum depression and maternal anxiety about the child was stronger during than before the pandemic; further, parental bonding mediated the relation between postpartum depression and infant negative affect across both samples. The COVID-19 pandemic may have also exacerbated associations between parent depression and child mental health.For example,caregivers who reported having experienced greater pandemic-related stress were found to have more symptoms of depression and infants with higher levels of negative affect (Buthmann et al. 2022). Parental depression during the pandemic, in turn, has been asso- ciated with feeling less prepared to educate children in the home during school shutdowns (Lee et al. 2021) and with poorer caregiving quality (Roos et al. 2021). Non-White Participants Indeed, to this end, the World Health Organization has designated depression as a target of the Mental Health Gap Action Programme, which aims to increase prevention and treatment initiatives in low- and middle-income countries (World Health Organ. 2019). g. Guest (guest) IP: 195.242.1.103 On: Thu, 24 Oct 2024 04:44:48 ded from www.annualreviews.org. Guest (guest) IP: 195.242.1.103 On: Thu, 24 Oct 20 Altered Characteristics and Clinical Outcomes in Offspring of Depressed Parents One critical issue that has not yet been adequately examined concerns the nature of the relation between altered functional characteristics in offspring of depressed parents and the onset of de- pression or other forms of psychopathology in these individuals. Many studies in this area have assessed and reported altered psychobiological functioning in offspring with a family history of depression while they are not in a depressive episode. Thus, it seems reasonable to postulate that altered characteristics are not simply correlates of depression in the offspring but instead factors that might place offspring at risk for the development of disorder; however, it is still not clear whether and/or how these characteristics, individually or jointly, increase the likelihood that off- spring will develop psychopathology. That is, although there are exceptions in which researchers have tested explicitly whether specific alterations predict subsequent depression in offspring of depressed parents (e.g., Foland-Ross et al. 2015), investigators have not demonstrated reliably that offspring who are characterized by various forms of altered psychobiological functioning are more likely to develop depression than are their at-risk peers who do not exhibit these anoma- lies. In this context, not all researchers have examined and documented alterations in only those offspring who have not yet experienced an episode of depression (e.g., Dearing & Gotlib 2009, Foland-Ross et al. 2015, Joormann et al. 2007), raising the possibility that reported alterations are a consequence of having been depressed; still fewer studies have tested whether, within the same sample of offspring of depressed parents, those with altered characteristics are more likely to develop depression than are those without alterations. Elucidating these temporal differences in functioning within samples of offspring of depressed parents will further illuminate the nature of the relation between maladaptive psychobiological functioning and depression in offspring of depressed parents. Other important questions involve related aspects of the altered characteristics that have been documented in offspring of depressed parents. For example, are these characteristics transient, fading over time, or are they enduring aspects of these individuals’ functioning? Are they time- locked in a meaningful way to the onset or offset of a depressive episode? Does the development of depression in offspring exacerbate these characteristics and worsen the prognosis of the disor- der in depressed parents’ offspring who have them? Are different altered characteristics (or their interactions) related reliably to different symptoms or symptom profiles in offspring of depressed parents? CONCLUSIONS AND DIRECTIONS FOR FUTURE RESEARCH In this review we document that offspring of depressed parents are characterized not only by high rates of depression but also by alterations in diverse domains, including their processing of information; cognitive development; interpersonal relationships; neural function, structure, and connectivity; HPA-axis functioning; biological aging; inflammation; and sleep. The range of www.annualreviews.org • Functioning of Offspring of Depressed Parents 385 functioning across which we now know that offspring of depressed parents differ from their peers without a family history of disorder attests to the progress we have made over the last two decades in elucidating the extent of the difficulties experienced and exhibited by these offspring. In partic- ular, scientific advances during this period have permitted researchers to examine aspects of the functioning of offspring of depressed parents that were not previously possible or viable, allow- ing us to obtain a more complete characterization of these individuals. Nevertheless, there are important issues that are still unresolved. In this section we raise and discuss questions that we believe must be addressed if we are to continue to make significant progress in understanding the nature of the relation between parental depression and altered psychobiological functioning in their offspring. Issues of Specificity In this review we describe alterations in a range of psychobiological and social domains in offspring of depressed parents; it is not clear, however, whether these difficulties and anomalies are specific to parental depression. Indeed, given the high comorbidity of depression with other psychiatric and physical disorders (ter Meulen et al. 2021), it is likely that offspring of parents with diagnoses and difficulties other than depression also exhibit at least some of these anomalous characteristics. Similarly, it is not clear that depression is the only (or even the most prevalent) psychiatric dis- order diagnosed in offspring of depressed parents. Finally, frequent correlates of depression, such as poverty and environmental toxins, marital difficulties, and psychosocial stressors, both alone and in interaction with a parental diagnosis, are also likely to be important in understanding the intergenerational transmission of risk for psychopathology. Specificity to parental depression. With respect to parental diagnosis, early studies of offspring at familial risk for psychopathology, most of which focused on children of schizophrenic parents, typically included control groups of children of parents with a psychiatric diagnosis other than schizophrenia. For example, in the Stony Brook High Risk project, Neale, Weintraub, and their colleagues compared the functioning of children of parents diagnosed with schizophrenia with that of offspring of parents diagnosed with unipolar or bipolar depression in addition to children with no family history of any psychiatric disorder. In a number of publications from this project, children of unipolar depressed parents obtained lower scores than did children of healthy par- ents on measures of attention and cognitive functioning (Winters et al. 1981) and on teacher (Weintraub et al. 1975) and peer (Weintraub et al. 1978) ratings of behavior; often, however, children of depressed parents were indistinguishable from children of parents with schizophre- nia, raising questions early on about the specificity of these difficulties to offspring of depressed parents. Other studies have included offspring of medically ill patients as controls in studies of off- spring of depressed parents. For example, Hirsch et al. (1985) compared adolescent offspring of unipolar depressed parents with offspring of arthritic and healthy parents and found no difference between adolescents of depressed and of arthritic parents in the number of depressive symp- toms they endorsed. Similarly, Hammen et al. (1987) compared children of unipolar depressed mothers with children of bipolar depressed, medically ill, and healthy mothers. Altered Characteristics and Clinical Outcomes in Offspring of Depressed Parents And is the presentation of depression in offspring with a family history different than the presentation of depression in individuals without such a history? That is, are specific symptoms of depression more prevalent in offspring of depressed parents than they are in same-age depressed individuals without a family history of the disorder? In general, despite the progress we have made thus far, we need much more information concerning the nature of the relation between altered functioning and subsequent disorder in offspring of depressed parents. Gotlib • Buthmann • Miller 386 www.annualreviews.org • Functioning of Offspring of Depressed Parents 3 Issues of Specificity There were few differences in symptoms among children in the three experimental groups, and most of those dif- ferences were attenuated after controlling for current psychosocial stressors. Gotlib & Lee (1989) compared the functioning of children of depressed psychiatric patients, nondepressed psychiatric patients, nondepressed medical patients, and healthy controls and also found few differences be- tween children of depressed and nondepressed patients. Hirshfeld-Becker et al. (2012) assessed the functioning of offspring of parents diagnosed with depression and/or panic disorder, in addi- tion to offspring of healthy controls, and found that both forms of parental psychopathology were associated with elevated rates of diagnosed psychopathology in the offspring. Rasic et al. (2014) conducted a meta-analysis examining diagnoses in offspring of parents with depression, bipolar disorder, schizophrenia, and anxiety disorders. They found that although all forms of parental psychopathology were associated with elevated rates of diagnosable disorder in offspring, there was little evidence of specificity of difficulties to parental depression. Finally, Kerr et al. (2018) reported that comorbidity of personality disorders with parental depression increased offspring’s risk for psychopathology. Specificity of depression as an outcome in offspring. It appears, therefore, that there is lit- tle specificity to parental depression as a cause of adverse outcomes in offspring; offspring of 387 depressed parents are often indistinguishable from offspring of nondepressed psychiatric and med- ical patients with respect to their clinical and psychosocial functioning.A related issue concerns the specificity of a diagnosis of depression in offspring as a consequence of depression in the parents. Most studies that have examined diagnostic consequences of parental depression for their off- spring have focused on depression as the outcome. Importantly, studies that have included other diagnostic outcomes have reported that offspring of depressed parents have elevated rates not only of depression but also of anxiety disorders and substance dependence (Weissman et al. 2021), as well as higher rates of physical illness (Weissman et al. 2006). For example, Côté et al. (2018) found in a large sample of youth that individuals who were exposed to elevated symptoms of maternal depression during their first five years of life had higher rates of symptoms of major depression, generalized anxiety,and social phobia in adolescence.Similarly,Weissman et al.(2006,2021) found that offspring of depressed parents had significantly higher rates of bipolar disorder, anxiety disor- ders, and substance use disorders than did offspring of nondepressed parents. Issues of Specificity Thus, it is clear from our review that not only are more offspring of depressed than of nondepressed parents diagnosed with a significant psychiatric disorder (including, but not limited to, depression) but also offspring of depressed parents exhibit alterations in a wide range of domains of function, extending well beyond a psychiatric diagnosis. In the context of these findings concerning both the lack of specificity to parental depression of difficulties in offspring and the wide range of altered behavior and functioning in offspring of depressed parents, we should note that the National Institute of Mental Health has formulated Research Domain Criteria (RDoC), a research framework to serve as a foundation from which to investigate issues involving mental disorders (see Morris & Cuthbert 2022). The broad aim of RDoC is to understand the nature of mental health and illness not by psychiatric diagnosis but in terms of varying degrees of dysfunction in fundamental psychological and biological sys- tems, similar in important ways to the alterations documented in offspring of depressed parents. Because behavioral and biological aspects of functioning change throughout childhood and ado- lescence and across the life span, RDoC entails an explicit focus on developmental research. In fact, one can begin to map the maladaptive functioning of these offspring (and, indeed, of their depressed parents) onto the domains and constructs of the RDoC framework, including anomalies in neural function, structure, and connectivity, inflammation, sleep, and behavioral and cognitive difficulties. RDoC provides guidelines on assessing these alterations at different levels of analysis (in RDoC terms, units of analysis). Investigators have begun to consider how to best integrate RDoC constructs across different units of analysis and use computational methods to identify psychobiological targets for treatment (Sanislow et al. 2019). We believe that this approach has considerable potential to elucidate the broad effects of parental depression on the functioning of their offspring. Accompanying factors. The final aspect of the specificity of adverse consequences of parental depression to offspring concerns factors that often accompany depression, such as poverty and ethnic minority status (Smith & Mazure 2021), marital difficulties (Mead 2002), and psychosocial stressors (Gilman et al. 2013). These factors, both alone and in interaction with a parental diagno- sis, almost certainly contribute to the intergenerational transmission of risk for psychopathology, leading to findings of adverse consequences for offspring that are not due directly to the par- ent’s psychopathology. 388 Gotlib • Buthmann • Miller ACKNOWLEDGMENTS Preparation of this chapter was supported by National Institutes of Health grant R37MH101495 to I.H.G. DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. SUMMARY In this article we review the current literature examining the intergenerational effects of parental depression, describing maladaptive characteristics of offspring of depressed parents in a diverse range of domains,including clinical,cognitive,and biological functioning.Researchers have drawn on scientific advances over the past two decades to present a broader and more comprehensive picture of the functioning of offspring of depressed parents. Nevertheless, there are unresolved questions that warrant further investigation. We discuss several of these issues and offer directions for future research that we believe will move the field forward in elucidating the nature of the relation between parental depression and altered psychobiological functioning in their offspring. ded from www.annualreviews.org. Guest (guest) IP: 195.242.1.103 On: Thu, 24 Oct 20 Issues of Specificity For example, poverty, marital distress, and psychosocial stress all have been found to adversely affect children’s development (e.g., Knopp et al. 2017). In this context, in one of the most comprehensive studies in this area conducted to date, Barker et al. (2012) analyzed data from the Avon Longitudinal Study of Parents and Children, a population-based study of the effects of factors influencing children’s health and development. 388 Gotlib • Buthmann • Miller 388 They examined the effects of maternal depressive symptoms and of an index of cumulative risk, created by summing participants’ scores on measures of socioeconomic status, living conditions, and family risk factors (e.g., being a single caretaker, experiencing partner cruelty, lack of affection and support), on children’s psychiatric diagnoses. Importantly, Barker et al. (2012) found that ma- ternal depression was associated with 10 of the 11 individual risk factors that they included in their study and, further, that both maternal depression and cumulative risk factors increased children’s odds of experiencing internalizing and externalizing disorders. Thus, studies that do not assess and control for these factors are likely overestimating the impact of parents’ depression on their offspring’s functioning. org. Guest (guest) IP: 195.242.1.103 On: Thu, 24 Oct 2024 04:44:48 LITERATURE CITED Ali NS, Mahmud S, Khan A, Ali BS. 2013. Impact of postpartum anxiety and depression on child’s mental development from two peri-urban communities of Karachi, Pakistan: a quasi-experimental study. BMC Psychiatry 13(1):274. https://doi.org/10.1186/1471-244X-13-274 Am. Psychiatr. Assoc. 2013. Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Arlington, VA: Am. Psychiatr. Assoc. Apter-Levy Y, Feldman M, Vakart A, Ebstein RP, Feldman R. 2013. Impact of maternal depression across the first 6 years of life on the child’s mental health, social engagement, and empathy: the moderating role of oxytocin. Am. J. Psychiatry 170(10):1161–68. https://doi.org/10.1176/appi.ajp.2013.12121597 Apter-Levi Y, Pratt M, Vakart A, Feldman M, Zagoory-Sharon O, Feldman R. 2016. Maternal depression across the first years of life compromises child psychosocial adjustment; relations to child HPA-axis functioning. Psychoneuroendocrinology 64:47–56. https://doi.org/10.1016/j.psyneuen.2015.11.006 Barker ED, Copeland W, Maughan B, Jaffee SR, Uher R. 2012. Relative impact of maternal depression and associated risk factors on offspring psychopathology. Br. J. Psychiatry 200(2):124–29. https://doi.org/10. 1192/bjp.bp.111.092346 Barnes J, Theule J. 2019. Maternal depression and infant attachment security: a meta-analysis. Infant Ment. Health J. 40(6):817–34. https://doi.org/10.1002/imhj.21812 Barnes J, Theule J. 2019. Maternal depression and infant attachment security: a meta-analysis. Infant Ment. Health J. 40(6):817–34. https://doi.org/10.1002/imhj.21812 Blackburn EH. 2000. Telomere states and cell fates. Nature 408(6808):53–56. https://doi.org/10.1038/ 35040500 Blackburn EH. 2000. Telomere states and cell fates. Nature 408(6808):53–56. https://doi.org/10.1038/ 35040500 www.annualreviews.org • Functioning of Offspring of Depressed Parents 389 Borchers LR, Dennis EL, King LS, Humphreys KL, Gotlib IH. 2021. Prenatal and postnatal depres- sive symptoms, infant white matter, and toddler behavioral problems. J. Affect. Disord. 282:465–71. https://doi.org/10.1016/j.jad.2020.12.075 Braithwaite EC, Pickles A, Wright N, Sharp H, Hill J. 2020. Sex differences in foetal origins of child emo- tional symptoms: a test of evolutionary hypotheses in a large, general population cohort. J. Child Psychol. Psychiatry 61(11):1194–202. https://doi.org/10.1111/jcpp.13229 Burkhouse KL, Kujawa A. 2022. Annual research review: emotion processing in offspring of mothers with depression diagnoses – a systematic review of neural and physiological research. J. Child Psychol. Psychiatry 64(4):583–607. https://doi.org/10.1111/jcpp.13734 Burkhouse KL, Siegle GJ, Gibb BE. 2014. Pupillary reactivity to emotional stimuli in children of depressed and anxious mothers. J. Child Psychol. Psychiatry 55(9):1009–16. https://doi.org/10.1111/jcpp.12225 Buthmann JL, Miller JG, Gotlib IH. 2022. Maternal–prenatal stress and depression predict in- fant temperament during the COVID-19 pandemic. Dev. Psychopathol. https://doi.org/10.1017/ S0954579422001055 Cent. Dis. Control. 2023. Youth risk behavior survey, data summary & trends report: 2011–2021. Rep., Cent. Dis. LITERATURE CITED Control, Atlanta, GA Chai XJ, Hirshfeld-Becker D, Biederman J, Uchida M, Doehrmann O, et al. 2015. Functional and structural brain correlates of risk for major depression in children with familial depression. NeuroImage Clin. 8:398– 407. https://doi.org/10.1016/j.nicl.2015.05.004 Chang O, Huh K, Savoy CD, Krzeczkowski JE, Lieshout RJV. 2023. Associations between maternal post- partum depression and infant temperament in treatment-seeking mothers prior to and during the COVID-19 pandemic. Dev. Psychopathol. https://doi.org/10.1017/S0954579422001353 Colich NL, Rosen ML, Williams ES, McLaughlin KA. 2020. Biological aging in childhood and adolescence following experiences of threat and deprivation: a systematic review and meta-analysis. Psychol. Bull. 146(9):721–64. https://doi.org/10.1037/bul0000270 Conradt E, Lester BM, Appleton AA, Armstrong DA, Marsit CJ. 2013. The roles of DNA methylation of NR3C1 and 11β-HSD2 and exposure to maternal mood disorder in utero on newborn neurobehavior. Epigenetics 8(12):1321–29. https://doi.org/10.4161/epi.26634 Côté SM, Ahun MN, Herba CM, Brendgen M, Geoffroy MC, et al. 2018. Why is maternal depression re- lated to adolescent internalizing problems? A 15-year population-based study. J. Am. Acad. Child Adolesc. Psychiatry 57(12):916–24. https://doi.org/10.1016/j.jaac.2018.04.024 Davidovich S, Collishaw S, Thapar AK, Harold G, Thapar A, Rice F. 2016. Do better executive functions buffer the effect of current parental depression on adolescent depressive symptoms? J. Affect. Disord. 199:54–64. https://doi.org/10.1016/j.jad.2016.03.049 de Jong DM, Cremone A, Kurdziel LBF, Desrochers P, LeBourgeois MK, et al. 2016. Maternal depressive symptoms and household income in relation to sleep in early childhood. J. Pediatr. Psychol. 41(9):961–70. https://doi.org/10.1093/jpepsy/jsw006 Dean DC, Planalp EM, Wooten W, Kecskemeti SR, Adluru N, et al. 2018. Association of prenatal maternal depression and anxiety symptoms with infant white matter microstructure. JAMA Pediatr. 172(10):973– 81. https://doi.org/10.1001/jamapediatrics.2018.2132 Dearing KF, Gotlib IH. 2009. Interpretation of ambiguous information in girls at risk for depression. J. Abnorm. Child Psychol. 37(1):79–91. https://doi.org/10.1007/s10802-008-9259-z Ertel KA, Rich-Edwards JW, Koenen KC. 2011. Maternal depression in the United States: nationally representative rates and risks. J. Women’s Health 20(11):1609–17. https://doi.org/10.1089/jwh.2010. 2657 Feldman R, Granat A, Pariente C, Kanety H, Kuint J, Gilboa-Schechtman E. 2009. Maternal depression and anxiety across the postpartum year and infant social engagement, fear regulation, and stress reactivity. J. Am. Acad. Child Adolesc. Psychiatry 48(9):919–27. https://doi.org/10.1097/CHI.0b013e3181b21651 Foland-Ross LC, Kircanski K, Gotlib IH. 2014. Coping with having a depressed mother: the role of stress and coping in hypothalamic–pituitary–adrenal axis dysfunction in girls at familial risk for major depression. Dev. Psychopathol. 26(4 Part 2):1401–9. LITERATURE CITED https://doi.org/10.1017/S0954579414001102 Gotlib • Buthmann • Miller 390 Foland-Ross LC, Sacchet MD, Prasad G, Gilbert B, Thompson PM, Gotlib IH. 2015. Cortical thickness predicts the first onset of major depression in adolescence. Int. J. Dev. Neurosci. 46:125–31. https://doi. org/10.1016/j.ijdevneu.2015.07.007 Forbes EE, Dahl RE. 2012. Research review: altered reward function in adolescent depression: what, when and how? J. Child Psychol. Psychiatry 53(1):3–15. https://doi.org/10.1111/j.1469-7610.2011.02477.x Fox ME, Lobo MK. 2019. The molecular and cellular mechanisms of depression: a focus on reward circuitry. Forbes EE, Dahl RE. 2012. Research review: altered reward function in adolescent depression: what, when and how? J. Child Psychol. Psychiatry 53(1):3–15. https://doi.org/10.1111/j.1469-7610.2011.02477.x Forbes EE, Dahl RE. 2012. Research review: altered reward function in adolescent depression: what, when and how? J. Child Psychol. Psychiatry 53(1):3–15. https://doi.org/10.1111/j.1469-7610.2011.02477.x Fox ME, Lobo MK. 2019. The molecular and cellular mechanisms of depression: a focus on reward circuitry. Mol. Psychiatry 24(12):1798–815. https://doi.org/10.1038/s41380-019-0415-3 Fox ME, Lobo MK. 2019. The molecular and cellular mechanisms of depression: a focus on reward circuitry. Mol. Psychiatry 24(12):1798–815. https://doi.org/10.1038/s41380-019-0415-3 Freeman C, Ethridge P, Banica I, Sandre A, Dirks MA, et al. 2022. Neural response to rewarding social feed- back in never-depressed adolescent girls and their mothers with remitted depression: associations with multiple risk indices. J. Psychopathol. Clin. Sci. 131:141–51. https://doi.org/10.1037/abn0000728 Fuemmeler BF, Lee CT, Soubry A, Iversen ES, Huang Z, et al. 2016. DNA methylation of regulatory regions of imprinted genes at birth and its relation to infant temperament. Genet. Epigenet. 8. https://doi.org/ 10.4137/GEG.S40538 Galbally M, van Rossum EFC, Watson SJ, de Kloet ER, Lewis AJ. 2019. Trans-generational stress regulation: mother-infant cortisol and maternal mental health across the perinatal period. Psychoneuroendocrinology 109:104374. https://doi.org/10.1016/j.psyneuen.2019.104374 Gilman SE, Trinh NH, Smoller JW, Fava M, Murphy JM, Breslau J. 2013. Psychosocial stressors and the prognosis of major depression: a test of Axis IV. Psychol. Med. 43(2):303–16. https://doi.org/10.1017/ S0033291712001080 Giurgescu C, Misra DP, Sealy-Jefferson S, Caldwell CH, Templin TN, et al. 2015. The impact of neighbor- hood quality, perceived stress, and social support on depressive symptoms during pregnancy in African American women. Soc. Sci. Med. 130:172–80. https://doi.org/10.1016/j.socscimed.2015.02.006 Gluckman PD, Hanson MA, Beedle AS. 2007. Early life events and their consequences for later disease: a life history and evolutionary perspective.Am.J.Hum.Biol.19(1):1–19.https://doi.org/10.1002/ajhb.20590 Gluckman PD, Hanson MA, Beedle AS. 2007. Early life events and their consequences for later disease: a life history and evolutionary perspective.Am.J.Hum.Biol.19(1):1–19.https://doi.org/10.1002/ajhb.20590 Goodman SH, Garber J. 2017. Evidence-based interventions for depressed mothers and their young children. org/10.1176/appi.ajp.21101000 Heijmans BT, Tobi EW, Lumey LH, Slagboom PE. 2009. The epigenome: archive of the prenatal environment. Epigenetics 4(8):526–31. https://doi.org/10.4161/epi.4.8.10265 Henry LM, Steele EH, Watson KH, Bettis AH, Gruhn M, et al. 2020. Stress exposure and maternal depres- sion as risk factors for symptoms of anxiety and depression in adolescents. Child Psychiatry Hum. Dev. 51(4):572–84. https://doi.org/10.1007/s10578-019-00940-2 Hill J, Pickles A, Rollinson L, Davies R, Byatt M. 2004. Juvenile- versus adult-onset depression: mul- tiple differences imply different pathways. Psychol. Med. 34(8):1483–93. https://doi.org/10.1017/ S0033291704002843 Hirsch BJ, Moos RH, Reischl TM. 1985. Psychosocial adjustment of adolescent children of a depressed, arthritic, or normal parent. J. Abnorm. Psychol. 94:154–64. https://doi.org/10.1037/0021-843X.94.2. 154 Hirshfeld-Becker DR, Micco JA, Henin A, Petty C, Faraone SV, et al. 2012. Psychopathology in adolescent offspring of parents with panic disorder, major depression, or both: a 10-year follow-up. Am. J. Psychiatry 169(11):1175–84. https://doi.org/10.1176/appi.ajp.2012.11101514 Humphreys KL, Sisk LM, Manczak EM, Lin J, Gotlib IH. 2020. Depressive symptoms predict change in telomere length and mitochondrial DNA copy number across adolescence. J. Am. Acad. Child Adolesc. Psychiatry 59(12):1364–70. https://doi.org/10.1016/j.jaac.2019.09.031 Jiang Y, Da W, Qiao S, Zhang Q, Li X, et al. 2019. Basal cortisol, cortisol reactivity, and telomere length: a systematic review and meta-analysis. Psychoneuroendocrinology 103:163–72. https://doi.org/10.1016/j. psyneuen.2019.01.022 Joormann J, Cooney RE, Henry ML, Gotlib IH. 2012. Neural correlates of automatic mood regulation in girls at high risk for depression. J. Abnorm. Psychol. 121(1):61–72. https://doi.org/10.1037/a0025294 Joormann J, Gilbert K, Gotlib IH. 2010. Emotion identification in girls at high risk for depression. J. Child Psychol. Psychiatry 51(5):575–82. https://doi.org/10.1111/j.1469-7610.2009.02175.x Joormann J, Talbot L, Gotlib IH. 2007. Biased processing of emotional information in girls at risk for depression. J. Abnorm. Psychol. 116:135–43. https://doi.org/10.1037/0021-843X.116.1.135 Josefsson A, Vikström J, Bladh M, Sydsjö G. 2019. Major depressive disorder in women and risk for future generations: population-based three-generation study. BJPsych Open 5(1):E8. https://doi.org/10.1192/ bjo.2018.83 Kerr S, Dalrymple K, Chelminski I, Zimmerman M. 2018. Depression and substance use disorders in the off- spring of depressed parents as a function of the parent’s borderline personality disorder symptomatology. Ann. Clin. Psychiatry 30:207–14 Kessler RC, Wang PS. 2008. The descriptive epidemiology of commonly occurring mental disorders in the United States. Annu. Rev. Public Health 29:115–29. https://doi.org/10.1146/annurev.publhealth.29. 020907.090847 King L, Feddoes DE, Kirshenbaum JS, Humphreys K, Gotlib IH. 2020. Pregnancy during the pandemic: the impact of COVID-19-related stress on risk for prenatal depression. Psychol. Med. 53(1):170–80. https:// doi.org/10.1017/S003329172100132X Klein DN, Lewinsohn PM, Rohde P, Seeley JR, Olino TM. LITERATURE CITED Goodman SH, Garber J. 2017. Evidence-based interventions for depressed mothers and their young children. Child Dev. 88(2):368–77. https://doi.org/10.1111/cdev.12732 Goodman SH, Gotlib IH. 1999. Risk for psychopathology in the children of depressed mothers: a develop- mental model for understanding mechanisms of transmission. Psychol. Rev. 106(3):458–90. https://doi. org/10.1037/0033-295x.106.3.458 Gotlib IH, Borchers LR, Chahal R, Gifuni J, Teresi GI, Ho TC. 2021. Early life stress predicts depressive symptoms in adolescents during the COVID-19 pandemic: the mediating role of perceived stress. Front. Psychol. 11:3864. https://doi.org/10.3389/fpsyg.2020.603748 Gotlib IH, Goodman SH, Humphreys KL. 2020. Studying the intergenerational transmission of risk for de- pression: current status and future directions. Curr. Dir. Psychol. Sci. 29(2):174–79. https://doi.org/10. 1177/0963721420901590 Gotlib IH, Hamilton JP, Cooney RE, Singh MK, Henry ML, Joormann J. 2010. Neural processing of reward and loss in girls at risk for major depression. Arch. Gen. Psychiatry 67(4):380–87. https://doi.org/10. 1001/archgenpsychiatry.2010.13 Gotlib IH, Lee CM. 1989. The social functioning of depressed patients: a longitudinal assessment. J. Soc. Clin. Psychol. 8(3):223–37. https://doi.org/10.1521/jscp.1989.8.3.223 Gotlib IH, LeMoult J, Colich NL, Foland-Ross LC, Hallmayer J, et al. 2015. Telomere length and cortisol reactivity in children of depressed mothers. Mol. Psychiatry 20(5):615–20. https://doi.org/10.1038/mp. 2014.119 Gronemann FH, Jacobsen RK, Wium-Andersen MK, Jørgensen MB, Osler M, Jørgensen TSH. 2023. As- sociation of familial aggregation of major depression with risk of major depression. JAMA Psychiatry 80(4):350–59. https://doi.org/10.1001/jamapsychiatry.2022.4965 Gueron-Sela N, Camerota M, Willoughby MT, Vernon-Feagans L, Cox MJ, Fam. Life Proj. Key Investig. 2018. Maternal depressive symptoms, mother-child interactions, and children’s executive function. Dev. Psychol. 54(1):71–82. https://doi.org/10.1037/dev0000389 Gutierrez-Galve L, Stein A, Hanington L, Heron J, Lewis G, O’Farrelly C, Ramchandani PG. 2019. Associa- tion of maternal and paternal depression in the postnatal period with offspring depression at age 18 years. JAMA Psychiatry 76(3):290–96. https://doi.org/10.1001/jamapsychiatry.2018.3667 www.annualreviews.org • Functioning of Offspring of Depressed Parents 391 Hammen C. 2018. Risk factors for depression: an autobiographical review. Annu. Rev. Clin. Psychol. 14:1–28. https://doi.org/10.1146/annurev-clinpsy-050817-084811 Hammen CL, Gordon D, Burge D, Adrian C, Jaenicke C, Hiroto D. 1987. Maternal affective disorders, illness, and stress: risk for children’s psychopathology. Am. J. Psychiatry 144:736–41. https://doi.org/10.1176/ ajp.144.6.736 j Hankerson SH, Moise N, Wilson D, Waller BY, Arnold KT, et al. 2022. The intergenerational impact of structural racism and cumulative trauma on depression. Am. J. Psychiatry 179(6):434–40. https://doi. org/10 1176/appi ajp 21101000 org/10.1176/appi.ajp.21101000 2005. Psychopathology in the adolescent and young adult offspring of a community sample of mothers and fathers with major depression. Psychol. Med. 35(3):353–65. https://doi.org/10.1017/S0033291704003587 Klimes-Dougan B, Papke V, Carosella KA, Wiglesworth A, Mirza SA, et al. 2022. Basal and reactive cortisol: a systematic literature review of offspring of parents with depressive and bipolar disorders. Neurosci. Biobehav. Rev. 135:104528. https://doi.org/10.1016/j.neubiorev.2022.104528 Gotlib • Buthmann • Miller 392 Knopp K, Rhoades GK, Allen ES, Parsons A, Ritchie LL, Markman HJ, Stanley SM. 2017. Within- and between-family associations of marital functioning and child well-being. J. Marriage Fam. 79(2):451–61. https://doi.org/10.1111/jomf.12373 Koutra K, Roumeliotaki T, Kyriklaki A, Kampouri M, Sarri K, et. al. 2017. Maternal depression and person- ality traits in association with child neuropsychological and behavioral development in preschool years: mother-child cohort (Rhea Study) in Crete, Greece. J. Affect. Disord. 217:89–98. https://doi.org/10. 1016/j.jad.2017.04.002 Kuckertz JM, Mitchell C, Wiggins JL. 2018. Parenting mediates the impact of maternal depression on child internalizing symptoms. Depress. Anxiety 35(1):89–97. https://doi.org/10.1002/da.22688 Kujawa A, Arfer KB, Finsaas MC, Kessel EM, Mumper E, Klein DN. 2020. Effects of maternal depression and mother-child relationship quality in early childhood on neural reactivity to rejection and peer stress in adolescence: a 9-year longitudinal study. Clin. Psychol. Sci. 8(4):657–72. https://doi.org/10.1177/ 2167702620902463 Kujawa A, Proudfit GH, Klein DN. 2014. Neural reactivity to rewards and losses in offspring of mothers and fathers with histories of depressive and anxiety disorders. J. Abnorm. Psychol. 123(2):287–97. https://doi. org/10.1037/a0036285 Laurent HK, Leve LD, Neiderhiser JM, Natsuaki MN, Shaw DS, et al. 2013. Effects of parental depres- sive symptoms on child adjustment moderated by hypothalamic pituitary adrenal activity: within- and between-family risk. Child Dev. 84(2):528–42. https://doi.org/10.1111/j.1467-8624.2012.01859.x Lebrun-Harris LA, Ghandour RM, Kogan MD, Warren MD. 2022. Five-year trends in US children’s health and well-being, 2016-2020. JAMA Pediatr. 176(7):e220056. https://doi.org/10.1001/jamapediatrics. 2022.0056 Lee HY, Hans SL. 2015. Prenatal depression and young low-income mothers’ perception of their chil- dren from pregnancy through early childhood. Infant Behav. Dev. 40:183–92. https://doi.org/10.1016/ j.infbeh.2015.06.008 Lee SJ, Ward KP, Chang OD, Downing KM. 2021. Parenting activities and the transition to home-based education during the COVID-19 pandemic. Child. Youth. Serv. Rev. 122:105585. https://doi.org/10. 1016/j.childyouth.2020.105585 Lehtola SJ, Tuulari JJ, Scheinin NM, Karlsson L, Parkkola R, et al. 2020. Newborn amygdalar volumes are associated with maternal prenatal psychological distress in a sex-dependent way. NeuroImage Clin. 28:102380. org/10.1176/appi.ajp.21101000 Marital distress, co-occurring depression, and marital therapy: a review. J. Marital Fam. Ther. 28(3):299–314. https://doi.org/10.1111/j.1752-0606.2002.tb01188.xl Measelle JR, Ablow JC. 2018. Contributions of early adversity to pro-inflammatory phenotype in infancy: the buffer provided by attachment security. Attach. Hum. Dev. 20(1):1–23. https://doi.org/10.1080/ 14616734.2017.1362657 Mercuri M, Stack DM, Mantis I, Moszkowski R, Field TM. 2023. Maternal and infant touching behaviours during perturbed interactions: associations with maternal depressive symptomatology and infant crying. Infant Behav. Dev. 71:101821. https://doi.org/10.1016/j.infbeh.2023.101821 Miller JG, Ho TC, Humphreys KL, King LS, Foland-Ross LC, et al. 2020. Early life stress, frontoamygdala connectivity, and biological aging in adolescence: a longitudinal investigation. Cereb. Cortex 30(7):4269– 80. https://doi.org/10.1093/cercor/bhaa057 g Monk CS, Klein RG, Telzer EH, Schroth EA, Mannuzza S, et al. 2008. Amygdala and nucleus accumbens activation to emotional facial expressions in children and adolescents at risk for major depression. Am. J. Psychiatry 165(1):90–98. https://doi.org/10.1176/appi.ajp.2007.06111917 Montagner R, Mogg K, Bradley BP, Pine DS, Czykiel MS, et al. 2016. Attentional bias to threat in children at-risk for emotional disorders: role of gender and type of maternal emotional disorder. Eur. Child Adolesc. Psychiatry 25(7):735–42. https://doi.org/10.1007/s00787-015-0792-3 y y p g Morris SE, Cuthbert BN. 2022. Research domain criteria: cognitive systems, neural circuits, and dimensions of behavior. Dialogues Clin. Neurosci. 14(1):29–37. https://doi.org/10.31887/DCNS.2012.14.1/smorris Morris SE, Cuthbert BN. 2022. Research domain criteria: cognitive systems, neural circuits, and dimensions of behavior Dialogues Clin Neurosci 14(1) 29 37 https://doi org/10 31887/DCNS 2012 14 1/smorris behavior. Dialogues Clin. Neurosci. 14(1):29 37. https://doi.org/10.31887/DCNS.2012.14.1/smorris Nasreen HE, Nahar Kabir Z, Forsell Y, Edhborg M. 2013. Impact of maternal depressive symptoms and infant temperament on early infant growth and motor development: results from a population based study in B l d h J Aff t Di d 146(2) 254 61 htt //d i /10 1016/j j d 2012 09 013 Nasreen HE, Nahar Kabir Z, Forsell Y, Edhborg M. 2013. Impact of maternal depressive symptoms and infant temperament on early infant growth and motor development: results from a population based study in Bangladesh. J. Affect. Disord. 146(2):254–61. https://doi.org/10.1016/j.jad.2012.09.013 Nelson BW, Allen NB, Laurent H. 2018. Infant HPA axis as a potential mechanism linking maternal men- tal health and infant telomere length. Psychoneuroendocrinology 88:38–46. https://doi.org/10.1016/j. psyneuen.2017.11.008 Nelson BW, Sheeber L, Pfeifer J, Allen NB. 2021. Psychobiological markers of allostatic load in depressed and nondepressed mothers and their adolescent offspring. J. Child Psychol. Psychiatry 62(2):199–211. org/10.1176/appi.ajp.21101000 https://doi.org/10.1016/j.nicl.2020.102380 Letourneau N,Dewey D,Kaplan BJ,Ntanda H,Novick J,et al.2019.Intergenerational transmission of adverse childhood experiences via maternal depression and anxiety and moderation by child sex. J. Dev. Orig. Health Dis. 10(1):88–99. https://doi.org/10.1017/S2040174418000648 Lewinsohn PM, Olino TM, Klein DN. 2005. Psychosocial impairment in offspring of depressed parents. Psychol. Med. 35(10):1493–503. https://doi.org/10.1017/S0033291705005350 Liu CH, Giallo R, Doan SN, Seidman LJ, Tronick E. 2016. Racial and ethnic differences in prenatal life stress and postpartum depression symptoms. Arch. Psychiatr. Nurs. 30(1):7–12. https://doi.org/10.1016/ j.apnu.2015.11.002 Liu Y, Murphy SK, Murtha AP, Fuemmeler BF, Schildkraut J, et al. 2012. Depression in pregnancy, infant birth weight and DNA methylation of imprint regulatory elements. Epigenetics 7(7):735–46. https://doi. org/10.4161/epi.20734 Loechner J, Sfärlea A, Starman K, Oort F, Thomsen LA, et al. 2020. Risk of depression in the offspring of parents with depression: the role of emotion regulation, cognitive style, parenting and life events. Child Psychiatry Hum. Dev. 51(2):294–309. https://doi.org/10.1007/s10578-019-00930-4 Lovato N, Gradisar M. 2014. A meta-analysis and model of the relationship between sleep and depression in adolescents: recommendations for future research and clinical practice. Sleep Med. Rev. 18(6):521–29. https://doi.org/10.1016/j.smrv.2014.03.006 g j Lovejoy MC, Graczyk PA, O’Hare E, Neuman G. 2000. Maternal depression and parenting behavior: a meta- analytic review. Clin. Psychol. Rev. 20(5):561–92. https://doi.org/10.1016/S0272-7358(98)00100-7 Luking KR, Pagliaccio D, Luby JL, Barch DM. 2016. Reward processing and risk for depression across development. Trends Cogn. Sci. 20(6):456–68. https://doi.org/10.1016/j.tics.2016.04.002 www.annualreviews.org • Functioning of Offspring of Depressed Parents 393 Luppino FS, de Wit LM, Bouvy PF, Stijnen T, Cuijpers P, et al. 2010. Overweight, obesity, and depression: a systematic review and meta-analysis of longitudinal studies. Arch. Gen. Psychiatry 67(3):220–29. https:// doi.org/10.1001/archgenpsychiatry.2010.2 Martin SE, Williamson LR, Kurtz-Nelson EC, Boekamp JR. 2015. Emotion understanding (and misun- derstanding) in clinically referred preschoolers: the role of child language and maternal depressive symptoms. J. Child Fam. Stud. 24(1):24–37. https://doi.org/10.1007/s10826-013-9810-6 Mattson WI, Hyde LW, Shaw DS, Forbes EE, Monk CS. 2016. Clinical neuroprediction: amygdala reactivity predicts depressive symptoms 2 years later. Soc. Cogn. Affect. Neurosci. 11(6):892–98. https://doi.org/10. 1093/scan/nsw018 McCabe C,Woffindale C,Harmer CJ,Cowen PJ.2012.Neural processing of reward and punishment in young people at increased familial risk of depression. Biol. Psychiatry 72(7):588–94. https://doi.org/10.1016/j. biopsych.2012.04.034 McQuaid RJ, Bombay A, McInnis OA, Humeny C, Matheson K, Anisman H. 2017. Suicide ideation and attempts among First Nations peoples living on-reserve in Canada: the intergenerational and cumu- lative effects of Indian Residential Schools. Can. J. Psychiatry 62(6):422–30. https://doi.org/10.1177/ 0706743717702075 Mead DE. 2002. org/10.1176/appi.ajp.21101000 https:// d doi.org/10.1111/jcpp.13264 Nevriana A, Pierce M, Abel KM, Rossides M, Wicks S, et al. 2022. Association between parental mental ill- ness and autoimmune diseases in the offspring – a nationwide register-based cohort study in Sweden. J. Psychiatr. Res. 151:122–30. https://doi.org/10.1016/j.jpsychires.2022.04.017i Newland RP, Parade SH, Dickstein S, Seifer R. 2016. Goodness of fit between prenatal maternal sleep and infant sleep: associations with maternal depression and attachment security.Infant Behav.Dev.44:179–88. https://doi.org/10.1016/j.infbeh.2016.06.010 Nilsen W, Gustavson K, Røysamb E, Kjeldsen A, Karevold E. 2013. Pathways from maternal distress and child problem behavior to adolescent depressive symptoms: a prospective examination from early childhood to adolescence. J. Dev. Behav. Pediatr. 34(5):303–13. https://doi.org/10.1097/DBP.0b013e318293ab05 Gotlib • Buthmann • Miller 394 Nolvi S, Bridgett DJ, Korja R, Kataja EL, Junttila N, et al. 2019. Trajectories of maternal pre- and postnatal anxiety and depressive symptoms and infant fear: moderation by infant sex. J. Affect. Disord. 257:589–97. https://doi.org/10.1016/j.jad.2019.07.055 O’Connor TG, Willoughby MT, Moynihan JA, Messing S, Vallejo Sefair A, et al. 2020. Early childhood risk exposures and inflammation in early adolescence. Brain Behav. Immun. 86:22–29. https://doi.org/10. 1016/j.bbi.2019.05.001 Oh Y,Greenberg MT,Willoughby MT,Vernon-Feagans L,Greenberg MT,et al.2020.Examining longitudi- nal associations between externalizing and internalizing behavior problems at within- and between-child levels. J. Abnorm. Child Psychol. 48(4):467–80. https://doi.org/10.1007/s10802-019-00614-6 Plant DT, Pawlby S, Sharp D, Zunszain PA, Pariante CM. 2016. Prenatal maternal depression is associ- ated with offspring inflammation at 25 years: a prospective longitudinal cohort study. Transl. Psychiatry 6(11):e936. https://doi.org/10.1038/tp.2015.155 Posner J, Cha J, Roy AK, Peterson BS, Bansal R, et al. 2016. Alterations in amygdala-prefrontal circuits in infants exposed to prenatal maternal depression. Transl. Psychiatry 6(11):e935. https://doi.org/10.1038/ tp.2016.146 Priel A, Djalovski A, Zagoory-Sharon O, Feldman R. 2019. Maternal depression impacts child psychopathol- ogy across the first decade of life: oxytocin and synchrony as markers of resilience. J. Child Psychol. Psychiatry 60(1):30–42. https://doi.org/10.1111/jcpp.12880 Propper CB, Holochwost SJ. 2013. The influence of proximal risk on the early development of the autonomic nervous system. Dev. Rev. 33(3):151–67. https://doi.org/10.1016/j.dr.2013.05.001 Psychogiou L, Russell G, Owens M. 2020. Parents’ postnatal depressive symptoms and their children’s aca- demic attainment at 16 years: pathways of risk transmission. Br. J. Psychol. 111(1):1–16. https://doi.org/ 10.1111/bjop.12378 10.1111/bjop.12378 Clin. Psychol. 17:181–205. https://doi.org/10.1146/annurev-clinpsy-071219-022710 Swales DA, Winiarski DA, Smith AK, Stowe ZN, Newport DJ, Brennan PA. 2018. Maternal depression and cortisol in pregnancy predict offspring emotional reactivity in the preschool period. Dev. Psychobiol. 60(5):557–66. https://doi.org/10.1002/dev.21631 Swartz JR, Williamson DE, Hariri AR. 2015. Developmental change in amygdala reactivity during adoles- cence: effects of family history of depression and stressful life events. Am. J. Psychiatry 172(3):276–83. https://doi.org/10.1176/appi.ajp.2014.14020195 Székely E, Lucassen N, Tiemeier H, Bakermans-Kranenburg MJ, Van IJzendoorn MH, et al. 2014. Maternal depressive symptoms and sensitivity are related to young children’s facial expression recognition: the Generation R Study. Dev. Psychopathol. 26(2):333–45. https://doi.org/10.1017/S0954579413001028 Taraban L, Shaw DS, Leve LD, Natsuaki MN, Ganiban JM, et al. 2019. Parental depression, overreac- tive parenting, and early childhood externalizing problems: moderation by social support. Child Dev. 90(4):e468–85. https://doi.org/10.1111/cdev.13027 ter Meulen WG, Draisma S, van Hemert AM, Schoevers RA, Kupka RW, et al. 2021. Depressive and anxiety disorders in concert–a synthesis of findings on comorbidity in the NESDA study. J.Affect.Disord.284:85– 97. https://doi.org/10.1016/j.jad.2021.02.004l Toenders YJ, Laskaris L, Davey CG, Berk M, Milaneschi Y, et al. 2022. Inflammation and depression in young people: a systematic review and proposed inflammatory pathways. Mol. Psychiatry 27(1):315–27. https:// doi.org/10.1038/s41380-021-01306-8 Toffol E, Lahti-Pulkkinen M, Lahti J, Lipsanen J, Heinonen K, et al. 2019. Maternal depressive symptoms during and after pregnancy are associated with poorer sleep quantity and quality and sleep disorders in 3.5-year-old offspring. Sleep Med. 56:201–10. https://doi.org/10.1016/j.sleep.2018.10.042 Tsypes A, Gibb BE. 2015. Peer victimization mediates the impact of maternal depression on risk for suicidal ideation in girls but not boys: a prospective study. J. Abnorm. Child Psychol. 43(8):1439–45. https://doi. org/10.1007/s10802-015-0025-8 Ulmer-Yaniv A, Djalovski A, Priel A, Zagoory-Sharon O, Feldman R. 2018. Maternal depression alters stress and immune biomarkers in mother and child.Depress.Anxiety 35(12):1145–57.https://doi.org/10.1002/ da.22818 van der Knaap NJF, Klumpers F, El Marroun H, Mous S, Schubert D, et al. 2018. Maternal depressive symp- toms during pregnancy are associated with amygdala hyperresponsivity in children. Eur. Child Adolesc. Psychiatry 27(1):57–64. https://doi.org/10.1007/s00787-017-1015-x van Dijk MT, Murphy E, Posner JE, Talati A, Weissman MM. 2021. Association of multigenerational family history of depression with lifetime depressive and other psychiatric disorders in children: results from the Adolescent Brain Cognitive Development (ABCD) Study. JAMA Psychiatry 78(7):778–87. https:// doi.org/10.1001/jamapsychiatry.2021.0350 Vreeland A, Bettis AH, Reising MM, Dunbar JP, Watson KH, et al. 2019. Coping and stress reactivity as moderators of maternal depressive symptoms and youth’s internalizing and externalizing symptoms. 10.1111/bjop.12378 Qiu A, Anh TT, Li Y, Chen H, Rifkin-Graboi A, et al. 2015. Prenatal maternal depression alters amygdala functional connectivity in 6-month-old infants. Transl. Psychiatry 5(2):e508. https://doi.org/10.1038/tp. 2015.3 Qiu A, Shen M, Buss C, Chong YS, Kwek K, et al. 2017. Effects of antenatal maternal depressive symptoms and socio-economic status on neonatal brain development are modulated by genetic risk. Cereb. Cortex 27(5):3080–92. https://doi.org/10.1093/cercor/bhx065 Racine N, Hetherington E, McArthur BA, McDonald S, Edwards S, et al. 2021. Maternal depressive and anxiety symptoms before and during the COVID-19 pandemic in Canada: a longitudinal analysis. Lancet Psychiatry 8(5):405–15. https://doi.org/10.1016/S2215-0366(21)00074-2 Rasic D, Hajek T, Alda M, Uher R. 2014. Risk of mental illness in offspring of parents with schizophrenia, bipolar disorder, and major depressive disorder: a meta-analysis of family high-risk studies. Schizophr. Bull. 40(1):28–38. https://doi.org/10.1093/schbul/sbt114 Roos LE, Salisbury M, Penner-Goeke L, Cameron EE, Protudjer JLP, et al. 2021. Supporting families to protect child health: parenting quality and household needs during the COVID-19 pandemic. PLOS ONE 16(5):e0251720. https://doi.org/10.1371/journal.pone.0251720 Salo VC, Schunck SJ, Humphreys KL. 2020. Depressive symptoms in parents are associated with reduced empathy toward their young children. PLOS ONE 15(3):e0230636. https://doi.org/10.1371/journal. pone.0230636 Sanchez SE, Puente GC, Atencio G, Qiu C, Yanez D, et al. 2013. Risk of spontaneous preterm birth in relation to maternal depressive, anxiety and stress symptoms. J. Reprod. Med. 58:25–33 Sandman CA, Buss C, Head K, Davis EP. 2015. Fetal exposure to maternal depressive symptoms is associ- ated with cortical thickness in late childhood. Biol. Psychiatry 77(4):324–34. https://doi.org/10.1016/j. biopsych.2014.06.025 y Sanislow CA, Ferrante M, Pacheco J, Rudorfer MV, Morris SE. 2019. Advancing translational research using NIMH Research Domain Criteria and computational methods. Neuron 101(5):779–82. https://doi.org/ 10.1016/j.neuron.2019.02.024 Schuez-Havupalo L,Lahti E,Junttila N,Toivonen L,Aromaa M,et al.2018.Parents’ depression and loneliness during pregnancy and respiratory infections in the offspring: a prospective birth cohort study.PLOS ONE 13(9):e0203650. https://doi.org/10.1371/journal.pone.0203650 www.annualreviews.org • Functioning of Offspring of Depressed Parents 395 Sfärlea A, Löchner J, Neumüller J, Asperud Thomsen L, Starman K, et al. 2019. Passing on the half-empty glass: a transgenerational study of interpretation biases in children at risk for depression and their parents with depression. J. Abnorm. Psychol. 128(2):151–61. https://doi.org/10.1037/abn0000401 y g Smith MV, Mazure CM. 2021. Mental health and wealth: depression, gender, poverty, and parenting. Annu. R Cli P h l 17 181 205 h //d i /10 1146/ li 071219 022710 Smith MV, Mazure CM. 2021. Mental health and wealth: depression, gender, poverty, and parenting. Annu. Rev. 10.1111/bjop.12378 J Youth Adolesc 48(8):1580–91 https://doi org/10 1007/s10964-019-01033-y Vreeland A, Bettis AH, Reising MM, Dunbar JP, Watson KH, et al. 2019. Coping and stress reactivity as moderators of maternal depressive symptoms and youth’s internalizing and externalizing symptoms. J. Youth Adolesc. 48(8):1580–91. https://doi.org/10.1007/s10964-019-01033-y Wade M, Fox NA, Zeanah CH, Nelson CA, Drury SS. 2020. Telomere length and psychopathology: speci- ficity and direction of effects within the Bucharest Early Intervention Project. J. Am. Acad. Child Adolesc. Psychiatry 59(1):140–48. https://doi.org/10.1016/j.jaac.2019.02.013 Wado YD, Afework MF, Hindin MJ. 2014. Effects of maternal pregnancy intention, depressive symptoms and social support on risk of low birth weight: a prospective study from Southwestern Ethiopia. PLOS ONE 9(5):e96304. https://doi.org/10.1371/journal.pone.0096304 Wang J, Zhang X, Simons SR, Sun J, Shao D, Cao F. 2020. Exploring the bi-directional relationship between sleep and resilience in adolescence. Sleep Med. 73:63–69. https://doi.org/10.1016/j.sleep.2020.04.018 Wang S, Ding C, Dou C, Zhu Z, Zhang D, et al. 2022. Associations between maternal prenatal depression and neonatal behavior and brain function – evidence from the functional near-infrared spectroscopy. Psychoneuroendocrinology 146:105896. https://doi.org/10.1016/j.psyneuen.2022.105896 396 Gotlib • Buthmann • Miller Weintraub S, Neale JM, Liebert DE. 1975. Teacher ratings of children vulnerable to psychopathology. Am. J. Orthopsychiatry 45:838–45. https://doi.org/10.1111/j.1939-0025.1975.tb01211.x Weintraub S, Prinz RJ, Neale JM. 1978. Peer evaluations of the competence of children vulnerable to psychopathology. J. Abnorm. Child Psychol. 6(4):461–73. https://doi.org/10.1007/BF00926056 Weissman MM, Kidd KK, Prusoff BA. 1982. Variability in rates of affective disorders in relatives of depressed and normal probands. Arch. Gen. Psychiatry 39(12):1397–403. https://doi.org/10.1001/archpsyc.1982. 04290120033006 Weissman MM, Talati A, Gameroff MJ, Pan L, Skipper J, et al. 2021. Enduring problems in the offspring of depressed parents followed up to 38 years. eClinicalMedicine 38:101000. https://doi.org/10.1016/j. eclinm.2021.101000 www.annualreviews.org • Functioning of Offspring of Depressed Parents 397 eclinm.2021.101000 Weissman MM,Wickramaratne P,Nomura Y,Warner V,Pilowsky D,Verdeli H.2006.Offspring of depressed parents: 20 years later. Am. J. Psychiatry 163(6):1001–8. https://doi.org/10.1176/ajp.2006.163.6.1001 Weissman MM, Wickramaratne P, Nomura Y, Warner V, Verdeli H, et al. 2005. Families at high and low risk for depression: a 3-generation study. Arch. Gen. Psychiatry 62(1):29–36. https://doi.org/10.1001/ archpsyc.62.1.29 Wiggins JL, Schwartz KTG, Kryza-Lacombe M, Spechler PA, Blankenship SL, Dougherty LR. 2017. Neural reactivity to reward in school-age offspring of depressed mothers. J. Affect. Disord. 214:81–88. https:// doi.org/10.1016/j.jad.2017.03.020 Williams LM, Korgaonkar MS, Song YC, Paton R, Eagles S, et al. 2015. Amygdala reactivity to emotional faces in the prediction of general and medication-specific responses to antidepressant treatment in the randomized iSPOT-D Trial. Neuropsychopharmacology 40(10):2398–408. https://doi.org/10.1038/npp. 2015.89 Winters KC, Stone AA, Weintraub S, Neale JM. 1981. Cognitive and attentional deficits in children vulnerable to psychopathology. J. Abnorm. Child Psychol. 9(4):435–53. https://doi.org/10.1007/BF00917794 Wolf JM, Miller GE, Chen E. 2008. Parent psychological states predict changes in inflammatory markers in children with asthma and healthy children. Brain Behav. Immun. 22(4):433–41. https://doi.org/10.1016/ j.bbi.2007.10.016 World Health Organ. 2017. Depression and other common mental disorders: global health estimates. Rep., World Health Organ., Geneva, Switz. World Health Organ. 2019. MhGAP Intervention Guide for Mental, Neurological and Substance Use Disorders in Non-Specialized Health Settings, Version 2.0. Geneva, Switz.: World Health Organ. Zhang W, Finik J, Dana K, Glover V, Ham J, Nomura Y. 2018. Prenatal depression and infant temperament: the moderating role of placental gene expression. Infancy 23(2):211–31. https://doi.org/10.1111/infa. 12215 Zou R, Tiemeier H, van der Ende J, Verhulst FC, Muetzel RL, et al. 2019. Exposure to maternal depressive symptoms in fetal life or childhood and offspring brain development: a population-based imaging study. Am. J. Psychiatry 176(9):702–10. https://doi.org/10.1176/appi.ajp.2019.18080970 www.annualreviews.org • Functioning of Offspring of Depressed Parents 397
https://openalex.org/W4378212651	https://www.researchsquare.com/article/rs-2082820/latest.pdf	English	null	Associations of LH and FSH with reproductive hormones depending on each stage of the menopausal transition	BMC women's health	2,023	cc-by	6,887	Associations of LH and FSH with reproductive hormones depending on each stage of the menopausal transition Takako Kawakita (  Kawakita.takako@tokushima-u.ac.jp ) Version of Record: A version of this preprint was published at BMC Women's Health on May 25th, 2023. See the published version at https://doi.org/10.1186/s12905-023-02438-5. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at BMC Women's Health on May 25th, 2023. See the published version at https://doi.org/10.1186/s12905-023-02438-5. Page 1/16 Abstract Introduction: Associations of luteinizing hormone (LH) with androgens during the menopausal transition and associations between follicle-stimulating hormone (FSH) levels and various diseases related to reproductive hormones in postmenopause have received much attention. LH and FSH are also known to be associated with activities of enzymes related to reproductive hormones. We examined the associations of LH and FSH with androgens and estrogens in each stage according to a classification from menopausal transition to postmenopause. Methods: We divided the 173 subjects into 6 groups according to menstrual regularity and follicle- stimulating hormone level: mid reproductive stage (Group A), late reproductive stage (Group B), early menopausal transition (Group C), late menopausal transition (Group D), very early postmenopause (Group E) and early postmenopause (Group F). Levels of LH, FSH, dehydroepiandrosterone sulfate (DHEAS), estradiol, estrone, testosterone (T), free T, androstenedione and androstenediol were measured. Results: In Group A, LH showed significant positive correlations with androstenedione and estrone. In Group D, LH was positively associated with T and free T and was negatively associated with estradiol. In Groups B, C, D and F, LH showed significant positive correlations with FSH, and there was a tendency for an association between LH and FSH in Group E. FSH was associated with estradiol but not with estrone in Groups C and D. Conclusion: The associations of LH and FSH with reproductive hormones were different according to the stage of the menopausal transition. The associations between gonadotrophine levels and androgen levels may be important for metabolism from menopausal transition to postmenopause. Introduction During the menopausal transition, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) produced in the pituitary increase to compensate for the declining estradiol levels due to a decrease in ovarian function. Based on changes in FSH trajectory accelerations and decelerations and rates of change, four menopausal transition stages bounding the final menstrual period and eight epochs in chronological aging from ages of 28 to 60 years have been defined [1]. Also, in women aged 42-52 years, three FSH trajectories over the menopausal transition have been identified [2]. Previous studies have shown that FSH receptors are distributed in various tissues including the bone [3, 4], liver [5] and vessels [6] as well as the ovary. It has been shown that FSH has extragonadal actions and that FSH levels are associated with various diseases and with metabolism in postmenopause [7, 8]. On the other hand, LH receptors are also distributed in not only the ovary but also the adrenal gland [9], brain [10], skin [11] and bladder [12]. It has been reported that an LH level of less than 41 U/L showed a positive correlation with dehydroepiandrosterone sulfate (DHEAS) level in postmenopausal women but not in women during the menopausal transition [13], suggesting that DHEAS production from the adrenal gland may be stimulated by highly elevated LH levels [13]. Although much attention has been focused on the delta-4 steroidogenic Page 2/16 Page 2/16 pathway that produces cortisol, androstendione and testosterone, longitudinal studies have suggested that the delta-5 steroidogenic pathway that produces DHEA, DHEAS and androstenediol may play a more important role in women’s healthy aging [14, 15]. It has been reported that gonadotropins such as LH and FSH change the activities of enzymes, including cytochrome P450 (CYP) 17A, 3β-hydroxysteroid dehydrogenase (HSD) and 17β-HSD, that act in the delta-4 and delta-5 steroidogenic pathways [16, 17, 18, 19]. During the menopausal transition, increases in LH and FSH levels change the activities of enzymes and might be associated with changes in reproductive hormones. In 2012, the Stage of Reproductive Aging Workshop (STRAW)+10 staging system was revised. The revised staging system improves the comparability of studies of midlife women and is widely considered as the gold standard for characterizing reproductive aging through menopause [20]. Introduction Based on the STRAW staging system, we found associations of androstenediol levels related to the delta-5 steroidogenic pathway with estrogen and androgen in women during the menopausal transition in a previous study by using liquid chromatography mass spectrometry (LC-MS/MS) with high sensitivity and high specificity [21]. Associations of LH with androgens during the menopausal transition have been shown, but there has been no report on the associations between FSH and androgens including androstenediol. The associations of LH and FSH with reproductive hormones related to the adrenal gland may differ according to each stage in the STRAW classification. To our knowledge, there are few data on the associations of gonadotropins such as LH and FSH with androgens and estrogens by classifying menopausal transition in detail. The aim of this study was to determine the associations of LH and FSH with reproductive hormones in each stage of the menopausal transition. In 2012, the Stage of Reproductive Aging Workshop (STRAW)+10 staging system was revised. The revised staging system improves the comparability of studies of midlife women and is widely considered as the gold standard for characterizing reproductive aging through menopause [20]. Based on the STRAW staging system, we found associations of androstenediol levels related to the delta-5 steroidogenic pathway with estrogen and androgen in women during the menopausal transition in a previous study by using liquid chromatography mass spectrometry (LC-MS/MS) with high sensitivity and high specificity [21]. Associations of LH with androgens during the menopausal transition have been shown, but there has been no report on the associations between FSH and androgens including androstenediol. The associations of LH and FSH with reproductive hormones related to the adrenal gland may differ according to each stage in the STRAW classification. To our knowledge, there are few data on the associations of gonadotropins such as LH and FSH with androgens and estrogens by classifying menopausal transition in detail. The aim of this study was to determine the associations of LH and FSH with reproductive hormones in each stage of the menopausal transition. Subjects We recruited 173 Japanese women for this cross-sectional study from the outpatient clinic of the Department of Obstetrics and Gynecology in Tokushima University Hospital. These women visited a specialized health care outpatient clinic for consultation and for management and treatment of various conditions including decrease in bone mineral density, dyslipidemia and menopausal symptoms. Women who had received hormone therapy in the past year were excluded. At the time of the visit to the outpatient clinic, we obtained information on menstrual frequency and flow or years since the final menstrual period (FMP) and we measured FSH level and determined the stage of menopausal transition. Based on the STRAW staging system [20], we divided the subjects into 6 stages by menstrual regularity and FSH level: 1) women with a regular menstrual cycle (25–35 days per cycle) and normal FSH level (mid reproductive stage, Group A, n=21), 2) women with a regular menstrual cycle and elevated FSH level (>10 mIU/ml) (late reproductive stage, Group B, n=22)，3) women with an irregular menstrual cycle and elevated FSH level (>10 mIU/ml) (early menopausal transition, Group C, n=23), 4) women who had an irregular menstrual cycle in which the interval of amenorrhea was more than 2 months and who had elevated FSH level (late menopausal transition, Group D, n=35), 5) women for whom less than 1 year had passed since menopause (very early postmenopause, Group E, n=30), and 6) women for whom more than Page 3/16 Page 3/16 1 year and less than 5 years had passed since menopause (early postmenopause, Group F, n=42). Informed consent for participation in this study was obtained from each woman. The Ethics Committee of Tokushima University Hospital approved the study. 1 year and less than 5 years had passed since menopause (early postmenopause, Group F, n=42). Informed consent for participation in this study was obtained from each woman. The Ethics Committee of Tokushima University Hospital approved the study. Measurements Blood in women with menstruation was drawn from 9:00 to 12:00 during a period of 3-7 days after the commencement of menstruation. Blood samples for measurements were obtained by venipuncture and drawn into tubes. They were frozen at -40℃ until used for analysis. Levels of LH, FSH, estradiol and testosterone (T) were measured by a chemiluminescent immunoassay. DHEAS level was measured by a chemiluminescent enzyme immunoassay. Levels of androstenedione and free T were measured by a radioimmunoassay. The intra- and inter-assay coefficients (CVs) for DHEAS, T, free T, estradiol, androstenedione, LH and FSH were less than 10%. Serum androstenediol and estrone concentrations were measured by using LC-MS/MS, and the measurements were described in our previous report (21). The intra- and inter-assay CVs for androstenedione measurements were 2.3-2.9% and 4.4-6.3%, respectively. The intra- and inter-assay CVs for estrone measurements were 2.0-2.3% and 3.0-3.2%, respectively. The sensitivity of the assay was 0.01 ng/ml for androstenediol and estrone. Statistical analysis All statistical analyses were performed by using SPSS statics version 20.0 (IBM, Armonk, New York). Data are presented as medians with 25th and 75th ranges and LH/FSH ratio is shown as a mean. The Kruskal- Wallis rank test was used to compare differences between different menopausal stages, and Bonferroni adjustment was used for a multiple comparison test. Correlations between variables were assessed by Spearman’s rank correlation analysis. Results Background characteristics of the subjects are shown in Table 1. Median body mass index (BMI) ranged from 20.1 to 22.5 kg/㎡, and there was no significant difference in BMI among the groups. BMI in one woman was more than 30.0 kg/㎡ (Table 1). Correlations of LH with reproductive hormones As can be seen in Table 2, in Group A, LH level showed significant and positive correlations with levels of androstenedione and estrone (r=0.747, p=0.003; r=0.782, p=0.038). In Group D, LH level was positively associated with levels of T and free T (r=0.356, p=0.036 and r=0.414, p=0.041, respectively) and was negatively associated with estradiol level (r=-0.484, p=0.003). Positive associations between LH and FSH were found in Groups B, C, D and F (r=0.713, p<0.001; r=0.776, p<0.001; r=0.692, p<0.001; r=0.688, p<0.001), and a tendency for a positive correlation between LH and FSH was found in Group E (r=0.358, p=0.052) (Table 2). Changes in LH and FSH levels during the menopausal transition Changes in LH and FSH levels during the menopausal transition are shown in Figure 1. Both LH and FSH levels were significantly increased in Groups D, E and F compared to those in Group A. In addition, LH level tended to be increased in Group C compared to that in Group A (p=0.062) FSH level continued to increase after Group D, but LH level reached a plateau at Group E (Fig.１A and B). There was no significant difference in the LH/FSH ratio among the 6 groups (Table 1) The box indicates values from the 25th percentile to the 75th percentile. The vertical line in the box indicates the median and the cross mark indicates the mean. Vertical lines represent minimum and Page 4/16 Page 4/16 maximum values. * p <0.05 vs Group A. ** p=0.06 vs Group A Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. Correlations of FSH with reproductive hormones In Groups C and D, FSH level showed a significant and negative association with estradiol level (r=-0.432, p=0.039 and r=-0.720, p<0.001, respectively) but not with estrone level. FSH level was not associated with testosterone, DHEAS, androstenediol or androstenedione (Table 3). Discussion In the present study, we found that the associations of LH and FSH with reproductive hormones differ according to stages of the menopausal transition. Women in Group A had a normal range of FSH levels. However, the ages of women in Group A ranged from 41.6 to 46.5 years and gradually approached the age for menopause. The fact that the delta-4 pathway for synthesis of androstenedione and estrone acts well along with actions of 17β-HSD and 3β- HSD in women in that age range is thought to be the reason for the significant correlations of LH level with estrone and androstenedione levels in Group A. In women in Group A, the delta-4 pathway in the ovary may have been maintained due to a balance between LH and FSH. In Group B, in which ovarian function had begun to decrease, there was no significant association of LH level with androstenedione or estrone level. Through an increase in FSH level, a relationship in which the balance between LH and FSH was maintained in Group A was attenuated, and the shift was beginning toward the delta-5 pathway. In addition, stimulation of DHEAS production by augmentation of CYP17A1 activity due to an increase in LH may be the reason for the disappearance of correlations of LH with androstenedione and estrone. A correlation between LH and FSH was found with increasing FSH level. In several studies, fibrosis in the stroma was observed in the ovary and associations between LH and androgens were found in women approaching menopause. Reproductive age-associated fibrosis was found in the stroma of ovaries in mice and humans [22, 23]. Matt et al. reported that alterations in Page 5/16 hypothalamic-pituitary function such as a prolonged interpulse interval of LH and increased LH pulse width were found in middle-aged women with a mean age of 42.6 years [24]. Thompson et al. found in a study using ovarian tumor cells that androgens are synthesized and secreted by the human ovary and that the primary source of androgens is the stromal cell compartment in the menopausal ovary [25]. A tripartile relationship among increase in LH, increase in androgens and fibrosis in the stroma in the ovary was also found in women with polycystic ovary syndrome (PCOS) [26]. Wickenheisser et al. reported that CYP17 gene expression increased for biosynthesis of androgens in theca interna cells in women with PCOS [17]. Moran et al. Discussion reported that women with PCOS who had an excess of adrenal androgen had significantly higher activity of CYP17 than that in women with PCOS who did not have an excess of adrenal androgen [16]. Thus, stimulation of DHEAS production by activation of CYP17A1 induces a hyperandrogenic state in women with PCOS [16, 17]. The results of those previous studies suggest that there are changes such as fibrosis in the ovarian stroma and transient increases in LH and androgens with aging. In Group C, production of androgen was started by the effect of increased LH level and a transition from the delta-4 pathway to the delta-5 pathway was proceeding. Negative correlations between FSH and estradiol were found to be significant in Group C and remarkable in Group D. When women enter into the periods of Group C and Group D, which indicate menopausal transition, the negative correlation between estradiol and FSH might become stronger due to the remarkable decline in estradiol level. Murayama et al. reported that theca cells pretreated with a high concentration of LH showed increased CYP17 gene expression [18]. Oktem et al. reported that FSH up-regulated the mRNA expression of 17β- HSD and 3β-HSD in granulosa cells [19]. Due to an increase in LH in the late menopausal transition, CYP17A1 activity increases via LH receptors in the adrenal gland and the conversion from pregnenolone to DHEAS is stimulated. DHEAS level tended to increase, but the difference was not statistically significant (p = 0.062), in the present study. However, in a previous study, a transient increase in DHEAS was found in the late menopausal transition [27]. In the present study, LH was significantly associated with total T and free T in Group D (late menopausal transition). As well as DHEAS production in response to LH stimulation, conversion to T by stimulation of 17β-HSD activity via FSH increase is considered to be involved in this significant correlation. The positive relationships between level of LH and levels of androgens such as T, free T and DHEAS may indicate that the delta-5 pathway is the main pathway in Group D. In the late menopausal transition, LH and FSH may act cooperatively on enzyme activities and stimulate reproductive hormone production. In Groups B, C, D and F, there were positive and significant associations between LH and FSH, but there was only a tendency for a correlation between LH and FSH in Group E. Discussion In the present study, in Group F, FSH level continued to increase, although LH level showed a plateau (Fig. 1). The time difference in hormonal changes in which FSH reached a plateau later than LH might be involved in the weak correlation between LH and FSH in Group E. In addition, in Group E, the correlations of LH with T and free T shown in Group D disappeared. The reason is conversion from T to estradiol by an increase in aromatase through continuation of the increase in FSH, and this phenomenon was maintained in Group Page 6/16 F. During the menopausal transition, FSH may regulate androgen levels for prevention of an excessive increase in androgen levels. In postmenopausal women, associations of FSH levels with the development of various diseases and with metabolism have been reported [28, 29, 30]. A high FSH level was shown to be associated with prevalence of vasomotor symptoms [31] and an increase in low-density lipoprotein cholesterol [5], and a low FSH level was shown to be associated with non-alcoholic fatty liver disease [32] and diabetes mellitus [30]. Serum FSH levels have been shown to be correlated with the rate of bone loss in perimenopausal women [33] and in older women [34]. However, the associations that were investigated in those studies were for FSH levels, not LH levels, in postmenopausal women. The associations of FSH levels with lipid metabolism, carbohydrate metabolism and vascular function in postmenopause may be affected by the increase in androgen levels due to an increase in LH during the menopausal transition. Combined studies on LH levels and FSH levels from menopausal transition to postmenopause may be valuable. Fibrosis in the stroma in the ovary and increases in LH and androstenedione occur in women with aging. Stimulation of 17β-HSD activity due to an increase in FSH might be regulated so as to prevent an excessive increase in androgen levels, particularly in postmenopausal women with high FSH levels. We did not examine the associations of LH and FSH with lipid metabolism and insulin resistance in each stage of the menopausal transition. Studies on associations of LH with the development of various diseases and with metabolism should be carried out not only for women in postmenopause but also for women during the menopausal transition. There are some limitations in this study. Conclusion The associations of LH and FSH with reproductive hormones are different depending on the stage of the menopausal transition. The associations between gonadotropin levels and androgen levels may be important for metabolism from menopausal transition to postmenopause. Discussion The sample size in the present study might be insufficient for a generalization for all Japanese women. Further study with a large sample size is needed. This study was a cross-sectional study. Thus, a causal relationship needs to be clarified in a longitudinal study. In addition, measurements of various enzymes related to the production of hormones may be important to clarify individual differences in enzyme activities. In the present study, total circulating reproductive hormones were measured, and we could not separate ovary-derived reproductive hormones and adrenal gland-derived reproductive hormones. Declarations Ethics approval and consent to participate Consent for publication The participant informed consent form included a statement related to the authorization for publishing study results. Funding All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Ethics approval and consent to participate Page 7/16 Informed consent was obtained from all participants before participating in the study. The study protocol was developed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Tokushima University Hospital. Authors' contributions Takako Kawakita participated in research conception and design, data acquisition, data analysis and interpretation, drafting, and critical revision of the critical intellectual content. Takako Kawakita participated in research conception and design, data acquisition, data analysis and interpretation, drafting, and critical revision of the critical intellectual content. Toshiyuki Yasui participated in the conception and design of the study, data acquisition, data analysis and interpretation, and critical revision of the intellectual content of the paper. Toshiyuki Yasui participated in the conception and design of the study, data acquisition, data analysis and interpretation, and critical revision of the intellectual content of the paper. Kanako Yoshida participated in data analysis and interpretation, as well as critical revision of key intellectual content. Kanako Yoshida participated in data analysis and interpretation, as well as critical revision of key intellectual content. Sumika Matsui participated in the conception and design of the study, data acquisition, data analysis and interpretation, and critical revision of the critical intellectual content. Sumika Matsui participated in the conception and design of the study, data acquisition, data analysis and interpretation, and critical revision of the critical intellectual content. Takeshi Iwasa participated in data analysis and interpretation, as well as critical revision of key intellectual content. Takeshi Iwasa participated in data analysis and interpretation, as well as critical revision of key intellectual content. All authors have seen and approved the final version, and no one else has made a substantive contribution to the paper. All authors have seen and approved the final version, and no one else has made a substantive contribution to the paper. Availability of data and materials All data and materials are available upon reasonable request from the corresponding author. Competing interests The authors declare that they have no competing interest. Acknowledgments Page 8/16 We would like to express our deep gratitude to the subjects who participated in this study. Toshiyuki Yasui The authors have no relevant financial or non-financial interests to disclos The study protocol was developed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Tokushima University Hospital. Informed consent was obtained from all individual participants included in the study. Consent to participate Informed consent was obtained from all individual participants included in the study. Authors' information Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, The University of Tokushima Graduate School, Japan Takako Kawakita , Kanako Yoshida, Takeshi Iwasa Department of Reproductive and Menopausal Medicine, Institute of Biomedical Sciences, The University of Tokushima Graduate School, Tokushima, Japan Department of Reproductive and Menopausal Medicine, Institute of Biomedical Sciences, The University of Tokushima Graduate School, Tokushima, Japan References Journal of Clinical Endocrinology and Metabolism 101:254-263. doi: 10.1210/jc.2015-2724. Epub 2015 Nov 19. 5. Song Y, Wang ES, Xing LL, Shi S, Qu F, Zhang D, Li JY, Shu J, Meng Y, Sheng JZ, Zhou JH, Huang HF (2016) Follicle-stimulating hormone induces postmenopausal dyslipidemia through inhibiting hepatic cholesterol metabolism. Journal of Clinical Endocrinology and Metabolism 101:254-263. doi: 10.1210/jc.2015-2724. Epub 2015 Nov 19. 6. Radu A, Pichon C, Camparo P, Antoine M, Allory Y, Couvelard A, Fromont G, Hai MT, Ghinea N (2010) Expression of follicle-stimulating hormone receptor in tumor blood vessels. The New England Journal of Medicine21:1621-1630. doi: 10.1056/NEJMoa1001283. 6. Radu A, Pichon C, Camparo P, Antoine M, Allory Y, Couvelard A, Fromont G, Hai MT, Ghinea N (2010) Expression of follicle-stimulating hormone receptor in tumor blood vessels. The New England Journal of Medicine21:1621-1630. doi: 10.1056/NEJMoa1001283. 7. Serviente C, Tuomainen TP, Virtanen J, Witkowski S, Niskanen L, Bertone-Johnson (2019) Follicle- stimulating hormone is associated with lipids in postmenopausal women. Menopause 26:540-545. doi: 10.1097/GME.0000000000001273. 7. Serviente C, Tuomainen TP, Virtanen J, Witkowski S, Niskanen L, Bertone-Johnson (2019) Follicle- stimulating hormone is associated with lipids in postmenopausal women. Menopause 26:540-545. doi: 10.1097/GME.0000000000001273. 8. Sowers M, Zheng H, Tomey K, Karvonen-Gutierrez C, Jannausch M, Li X, Yosef M, Symons J (2007) Changes in body composition in women over six years at midlife: ovarian and chronological aging. Journal of Clinical Endocrinology and Metabolism 92: 895-901. doi:10.1210/jc.2006-1393. Epub 2006 Dec 27. 9. Pabon JE, Li X, Lei ZM, Sanfilippo JS, Yussman MA, Rao CV (1996) Novel presence of luteinizing hormone/chorionic gonadotropin receptors in human adrenal glands. Journal of Clinical Endocrinology and Metabolism 81: 2397–2400. 10. Blair JA, Bhatta S, McGee H, Casadesus G (2015) Luteinizing hormone: Evidence for direct action in the CNS. Hormones and Behavior 76 : 57-62. doi: 10.1016/j.yhbeh.2015.06.020. Epub 2015 Jul 12. 10. Blair JA, Bhatta S, McGee H, Casadesus G (2015) Luteinizing hormone: Evidence for direct action in the CNS. Hormones and Behavior 76 : 57-62. doi: 10.1016/j.yhbeh.2015.06.020. Epub 2015 Jul 12. 11. Pabon JE, Bird JS, Li X, Huang ZH, Lei ZM, Sanfilippo JS, Yussman MA, Rao CV (1996) Human skin contains luteinizing hormone/chorionic gonadotropin receptors. Journal of Clinical Endocrinology and Metabolism 81:2738-2741. doi: 10.1210/jcem.81.7.8675605. 11. Pabon JE, Bird JS, Li X, Huang ZH, Lei ZM, Sanfilippo JS, Yussman MA, Rao CV (1996) Human skin contains luteinizing hormone/chorionic gonadotropin receptors. Journal of Clinical Endocrinology and Metabolism 81:2738-2741. doi: 10.1210/jcem.81.7.8675605. 12. References Page 9/16 1. Sowers MR, Zheng H, McConnell D, Nan B, Harlow S, Randolph JF (2008) Follicle stimulating hormone and its rate of change in defining menopause transition stages. Journal of Clinical Endocrinology and Metabolism 93:3958-3964. https://doi.org/10.1210/jc.2008-0482. 2. Tepper PG, Randolph JF, McConnell DS, Crawford SL, El Khoudary SR, Joffe H, Gold EB, Zheng H, Bromberger JT, Sutton-Tyrrell K (2012) Trajectory clustering of estradiol and follicle-stimulating 1. Sowers MR, Zheng H, McConnell D, Nan B, Harlow S, Randolph JF (2008) Follicle stimulating hormone and its rate of change in defining menopause transition stages. Journal of Clinical Endocrinology and Metabolism 93:3958-3964. https://doi.org/10.1210/jc.2008-0482. Page 9/16 hormone and its rate of change in defining menopause transition stages. Journal of Clinical Endocrinology and Metabolism 93:3958-3964. https://doi.org/10.1210/jc.2008-0482. 2. Tepper PG, Randolph JF, McConnell DS, Crawford SL, El Khoudary SR, Joffe H, Gold EB, Zheng H, Bromberger JT, Sutton-Tyrrell K (2012) Trajectory clustering of estradiol and follicle-stimulating Page 9/16 2. Tepper PG, Randolph JF, McConnell DS, Crawford SL, El Khoudary SR, Joffe H, Gold EB, Zheng H, Bromberger JT, Sutton-Tyrrell K (2012) Trajectory clustering of estradiol and follicle-stimulating Page 9/16 2. Tepper PG, Randolph JF, McConnell DS, Crawford SL, El Khoudary SR, Joffe H, Gold EB, Zheng H, Bromberger JT, Sutton-Tyrrell K (2012) Trajectory clustering of estradiol and follicle-stimulating hormones during the menopausal transition among women in the study of women’s health across the nation (SWAN). Journal of Clinical Endocrinology and Metabolism 97:2872-2880. doi: 10.1210/jc.2012-1422. hormones during the menopausal transition among women in the study of women’s health across the nation (SWAN). Journal of Clinical Endocrinology and Metabolism 97:2872-2880. doi: 10.1210/jc.2012-1422. 3. Sun L, Peng Y, Sharrow AC, Iqbal J, Zhang Z, Papachristou DJ, Zaidi S, Zhu LL, Yaroslavskiy BB, Zhou H, Zallone A, Sairam MR, Kumar TR, Bo W, Braun J, Cardoso-Landa L, Schaffler MB, Moonga BS, Blair HC, Zaidi M (2006) FSH directly regulates bone mass. Cell :247-260. doi:10.1016/j.cell.2006.01.051. 4. Robinson LJ, Tourkova I, Wang Y, Sharrow AC, Landau MS, Yaroslavskiy BB, Sun L, Zaidi M, Blair HC (2010) FSH-receptor isoforms and FSH-dependent gene transcription in human monocytes and osteoclasts. Biochemical and Biophysical Research Communications 26:12-17. doi: 10.1016/j.bbrc.2010.02.112. Epub 2010 Feb 19. 5. Song Y, Wang ES, Xing LL, Shi S, Qu F, Zhang D, Li JY, Shu J, Meng Y, Sheng JZ, Zhou JH, Huang HF (2016) Follicle-stimulating hormone induces postmenopausal dyslipidemia through inhibiting hepatic cholesterol metabolism. doi: 10.1530/eje.1.02165. doi: 10.1530/eje.1.02165. 14. Santoro N, Torrens J, Crawford S, Allsworth JE, Finkelstein JS, Gold EB, Korenman S, Lasley WL, Luborsky JL, McConnell D, Sowers MF, Weiss G (2005) Correlates of circulating androgens in mid-life women: The Study of Women’s Health Across the Nation. Journal of Clinical Endocrinology and Metabolism 90: 4836-4845. doi: 10.1210/jc.2004-2063. Epub 2005 Apr 19. 15. Torrens JI, Sutton-Tyrrell K, Zhao X, Matthews K, Brockwell S, Sowers M, Santoro N(2009) Relative androgen excess during the menopausal transition predicts incident metabolic syndrome in midlife women: Study of Women’s Health Across the Nation. Menopause 16: 257-264. doi: 10.1097/gme.0b013e318185e249. 16. Moran C, Reyna R, Boots LS, Azziz R (2004) Adrenocortical hyperresponsiveness to corticotropin in polycystic ovary syndrome patients with adrenal androgen excess. Fertility and Sterility 81:126-131. doi: 10.1016/j.fertnstert.2003.07.008. 17. Wickenheisser JK, Nelson-Degrave VL, McAllister JM (2005) Dysregulation of cytochrome P450 17alpha-hydroxylase messenger ribonucleic acid stability in theca cells isolated from women with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 90: 1720-1727. doi: 10.1210/jc.2004-1860. Epub 2004 Dec 14. 18. Murayama C, Miyazaki H, Miyamoto A, Shimizu T (2012) Luteinizing hormone (LH) regulates production of androstenedione and progesterone via control of histone acetylation of StAR and CYP17 promoters in ovarian theca cells. Molecular and Cellular Endocrinology 5:1-9. doi: 10.1016/j.mce.2011.11.014. Epub 2011 Dec 2. 18. Murayama C, Miyazaki H, Miyamoto A, Shimizu T (2012) Luteinizing hormone (LH) regulates production of androstenedione and progesterone via control of histone acetylation of StAR and CYP17 promoters in ovarian theca cells. Molecular and Cellular Endocrinology 5:1-9. doi: 10.1016/j.mce.2011.11.014. Epub 2011 Dec 2. 19. Oktem O, Akin N, Bildik G, Yakin K, Alper E, Balaban B, Urman B (2017) FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization. Human Reproduction 32: 643-652. doi: 10.1093/humrep/dex010. 19. Oktem O, Akin N, Bildik G, Yakin K, Alper E, Balaban B, Urman B (2017) FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization. Human Reproduction 32: 643-652. doi: 10.1093/humrep/dex010. 20. Harlow SD, Gass M, Hall JE, Lobo R, Maki P, Reber RW, Sherman S, Sluss PM, de Villiers TJ, for the STRAW+10 Collaborative Group (2012) Executive summary of the stages of reproductive aging workshop +10: addressing the unfinished agenda of staging reproductive aging. Journal of Clinical Endocrinology and Metabolism 97: 1159-1168. doi: 10.1210/jc.2011-3362. Epub 2012 Feb 16. 20. References Tao YX, Heit M, Lei ZM, Rao CV(1998) The urinary bladder of a woman is a novel site of luteinizing hormone-human chorionic gonadotropin receptor gene expression. American Journal of Obstetrics and Gynecology179 :1026-1031. doi: 10.1016/s0002-9378 98 70222-4. 12. Tao YX, Heit M, Lei ZM, Rao CV(1998) The urinary bladder of a woman is a novel site of luteinizing hormone-human chorionic gonadotropin receptor gene expression. American Journal of Obstetrics and Gynecology179 :1026-1031. doi: 10.1016/s0002-9378 98 70222-4. 13. Alevizaki M, Saltiki K, Mantzou E, Anastasiou E, Huhtaniemi I (2006) The adrenal gland may be a target of LH action in postmenopausal women. European Journal of Endocrinology 154 875-881. 13. Alevizaki M, Saltiki K, Mantzou E, Anastasiou E, Huhtaniemi I (2006) The adrenal gland may be a target of LH action in postmenopausal women. European Journal of Endocrinology 154 875-881. 13. Alevizaki M, Saltiki K, Mantzou E, Anastasiou E, Huhtaniemi I (2006) The adrenal gland may be a target of LH action in postmenopausal women. European Journal of Endocrinology 154 875-881. Page 10/16 Page 10/16 doi: 10.1530/eje.1.02165. Harlow SD, Gass M, Hall JE, Lobo R, Maki P, Reber RW, Sherman S, Sluss PM, de Villiers TJ, for the STRAW+10 Collaborative Group (2012) Executive summary of the stages of reproductive aging workshop +10: addressing the unfinished agenda of staging reproductive aging. Journal of Clinical Endocrinology and Metabolism 97: 1159-1168. doi: 10.1210/jc.2011-3362. Epub 2012 Feb 16. 21. Kawakita T, Yasui T, Yoshida K, Matsui S, Iwasa T (2021) Correlations of androstenediol with reproductive hormones and cortisol according to stages during the menopausal transition in Japanese women. Journal of Steroid Biochemistry and molecular biology 214:106009. doi: 10.1016/j.jsbmb.2021.106009. Epub 2021 Sep 25. 21. Kawakita T, Yasui T, Yoshida K, Matsui S, Iwasa T (2021) Correlations of androstenediol with reproductive hormones and cortisol according to stages during the menopausal transition in Japanese women. Journal of Steroid Biochemistry and molecular biology 214:106009. doi: 10.1016/j.jsbmb.2021.106009. Epub 2021 Sep 25. 22. Briley SM, Jasti S, McCracken JM, Hornick JE, Fegley B, Pritchard MT, Duncan FE (2016) Reproductive age-associated fibrosis in the stroma of the mammalian ovary. Reproduction 152: 245- 260. doi: https://doi.org/10.1530/REP-16-0129 22. Briley SM, Jasti S, McCracken JM, Hornick JE, Fegley B, Pritchard MT, Duncan FE (2016) Reproductive age-associated fibrosis in the stroma of the mammalian ovary. Reproduction 152: 245- 260. doi: https://doi.org/10.1530/REP-16-0129 23. McCloskey CW, Cook DP, Kelly BS, Azzi F, Allen CH, Forsyth A, Upham J, Rayner KJ, Gray DA, Boyd RW, Murugkar S, Lo B, Trudel D, Senterman MK, Vanderhyden BC (2020) Metformin abrogates age- associated ovarian fibrosis. Clinical Cancer Research 26: 632- 642. https://doi.org/10.1158/1078- 0432.CCR-19-0603 23. McCloskey CW, Cook DP, Kelly BS, Azzi F, Allen CH, Forsyth A, Upham J, Rayner KJ, Gray DA, Boyd RW, Murugkar S, Lo B, Trudel D, Senterman MK, Vanderhyden BC (2020) Metformin abrogates age- associated ovarian fibrosis. Clinical Cancer Research 26: 632- 642. https://doi.org/10.1158/1078- 0432.CCR-19-0603 Page 11/16 24. Matt DW, Kauma SW, Pincus SM, Veldhuis JD, Evans WS (1998) Characteristics of luteinizing hormone secretion in younger versus older premenopausal women. American Journal of Obstetrics and Gynecology178: 504-510. doi: 10.1016/s0002-9378(98)70429-6. 25. Thompson MA, Adelson MD (1993) Aging and development of ovarian epithelial carcinoma: the relevance of changes in ovarian stromal androgen production. Advances in Experimental Medicine and Biology 330: 155-165. doi: 10.1007/978-1-4615-2926-2_12. 26. Schildkraut JM, Schwingl PJ, Bastos E, Evanoff A, Hughes C (1996) Epithelial ovarian cancer risk among women with polycystic ovary syndrome. Obstetrics & Gynecology 88:554-559. doi: 10.1016/0029-7844(96)00226-8. 27. doi: 10.1530/eje.1.02165. Matsui S, Yasui T, Tani A, Kato T, Kunimi K, Uemura H, Kuwahara A, Matsuzaki T, Irahara M (2012) Association of circulating adiponectin with testosterone in women during the menopausal transition. Maturitas. 73: 255-260. doi: 10.1016/j.maturitas.2012.08.003 28. Matthews KA, Chang Y, Brooks MM, Crawford SL, Janssen I, Joffe H, Kravitz HM, Thurston RC, El Khoudary SR (2020) Identifying women who share patterns of reproductive hormones, vasomotor symptoms, and sleep maintenance problems across the menopause transition: group-based multi- trajectory modeling in the Study of Women's Health Across the Nation. Menopause 5:126-134. doi: 10.1097/GME.0000000000001663. 29. Chu MC, Rath KM, Huie J, Taylor HS (2003) Elevated basal FSH in normal cycling women is associated with unfavourable lipid levels and increased cardiovascular risk. Human Reproduction 18:1570-1573. doi: 10.1093/humrep/deg330. 29. Chu MC, Rath KM, Huie J, Taylor HS (2003) Elevated basal FSH in normal cycling women is associated with unfavourable lipid levels and increased cardiovascular risk. Human Reproduction 18:1570-1573. doi: 10.1093/humrep/deg330. 30. Wang N, Kuang L, Han B, Li Q, Chen Y, Zhu C, Chen Y, Xia F, Cang Z, Zhu C, Lu M, Meng Y, Guo H, Chen C, Lin D, Lu Y (2016) Follicle-stimulating hormone associates with prediabetes and diabetes in postmenopausal women. Acta Diabetologica 53: 227-236. doi: 10.1007/s00592-015-0769-1. Epub 2015 May 12. 31. Randolph JF Jr, Sowers M, Bondarenko I, Gold EB, Greendale GA, Bromberger JT, Brockwell SE, Matthews KA (2005) The relationship of longitudinal change in reproductive hormones and vasomotor symptoms during the menopausal transition. Journal of Clinical Endocrinology and Metabolism 90: 6106-6112. doi: 10.1210/jc.2005-1374. Epub 2005 Sep 6. 32. Wang N, Li Q, Han B, Chen Y, Zhu C, Chen Y, Xia F, Lu M, Meng Y, Guo Y, Ye L, Sui C, Kuang L, Lin D, Lu Y (2016) Follicle-stimulating hormone is associated with non-alcoholic fatty liver disease in Chinese women over 55 years old. Journal of Gastroenterology and Hepatology 31:1196-1202. doi: 10.1111/jgh.13271. 33. Sowers MR, Finkelstrein JS, Ettinger B Bondarenko I, Neer RM, Cauley JA, Sherman S, Greendale GA; Study of Women's Health Across the Nation (2003) The association of endogenous hormone concentrations and bone mineral density measures in pre- and perimenopausal women of four ethnic groups: SWAN. Osteoporosis International 14: 44-52.doi:10.1007/s00198-002-1307-x. 34. doi: 10.1530/eje.1.02165. Veldhuis-Vlug AG, Woods GN, Sigurdsson S, Ewing SK, Le PT, Hue TF, Vittinghoff E, Xu K, Gudnason V, Sigurdsson G, Kado DM, Eiriksdottir G, Harris T, Schafer AL, Li X, Zaidi M, Rosen CJ, Schwartz AV 34. Veldhuis-Vlug AG, Woods GN, Sigurdsson S, Ewing SK, Le PT, Hue TF, Vittinghoff E, Xu K, Gudnason V, Sigurdsson G, Kado DM, Eiriksdottir G, Harris T, Schafer AL, Li X, Zaidi M, Rosen CJ, Schwartz AV 34. Veldhuis-Vlug AG, Woods GN, Sigurdsson S, Ewing SK, Le PT, Hue TF, Vittinghoff E, Xu K, Gudnason V, Sigurdsson G, Kado DM, Eiriksdottir G, Harris T, Schafer AL, Li X, Zaidi M, Rosen CJ, Schwartz AV Page 12/16 Page 12/16 (2021) Serum FSH is associated with BMD, bone marrow adiposity, and body composition in the AGES-Reykjavik Study of older adults. Journal of Clinical Endocrinology and Metabolism 106 e1156- e1169. doi: 10.1210/clinem/dgaa922. Tables Table 1. Clinical characteristics of the subjects Group P value A B C D E F Number 21 22 23 35 30 42 Age years 43.5 41.50- 46.50 47 44.50- 49.00 47* 46.00- 49.00 49* 46.50- 50.50 51* 49.50- 53.00 53* 51.75- 54.00 <0.001 Height cm 155.0 152.25- 157.00 158.0 154.00- 160.85 160.4 158.93- 163.00 158.7 156.63- 160.00 158.0 154.00- 160.00 156.5 155.00- 159.05 0.189 Weight kg 50.1 47.70- 56.25 55.9 50.5- 57.65 56.0 52.65- 58.53 59.0 52.00- 61.20 52.0 49.40- 57.40 53.1 50.00- 57.15 0.182 BMI kg/cm² 20.1 19.50- 21.50 21.4 20.00- 23.55 21.8 20.51- 23.13 22.5 20.31- 25.00 21.9 20.09- 22.79 21.7 20.30- 23.53 0.379 LH mIU/ml 3.7 2.83- 5.90 7.2 4.05- 13.28 8.8 7.7- 26.3 30.05* 17.48- 35.78 33.30* 20.80- 43.90 30.55* 23.5- 38.48 <0.001 FSH mIU/ml 5.6 4.80- 8.00 14.05 11.15- 35.18 23.9 13.5- 42.9 56.95* 30.55- 87.35 93.40* 70.70- 110.0 95.85* 71.43- 123.70 <0.001 Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. Table 1. Clinical characteristics of the subjects Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. Age, height, weight, BMI, LH and FSH are shown as medians. LH/FSH ratio is shown as a mean. LH: luteinizing hormone, FSH: follicle-stimulating hormone. Table 2. Tables Correlations of LH with reproductive hormones Page 13/16 Group A Group B Group C Group D Group E Group F testosterone R=0.223 P=0.331 R=－0.033 P=0.883 R=－0.037 P=0.867 R=0.356 P=0.036 R=－0.004 P=0.985 R=－0.004 P=0.978 Free testosterone R=0.233 P=0.309 R=0.216 P=0.335 R=－0.180 P=0.410 R=0.414 P=0.041 R=－0.205 P=0.277 R=－0.153 P=0.332 DHEAS R=0.235 P=0.304 R=－0.051 P=0.827 R=0.234 P=0.283 R=0.295 P=0.090 R=0.136 P=0.480 R=0.076 P=0.632 androstenedione R=0.747 P=0.003 R=0.000 P=1.000 R=0.473 P=0.142 R=－0.010 P=0.970 R=0.024 P=0.955 R=0.000 P=1.000 androstenediol R=－0.146 P=0.688 R=－0.071 P=0.867 R=0.052 P=0.813 R=0.250 P=0.516 R=－0.204 P=0.279 R=－0.018 P=0.907 estradiol R=0.427 P=0.053 R=0.166 P=0.459 R=0.300 P=0.433 R=－0.484 P=0.003 R=－0.214 P=0.645 R=0.533 P=0.139 estrone R=0.782 P=0.038 R=0.048 P=0.911 R=－0.217 P=0.576 R=－0.095 P=0.823 R=0.501 P=0.116 R=－0.009 P=0.979 FSH R=0.233 P=0.309 R=0.713 P=0.000 R=0.776 P=0.000 R=0.692 P=0.000 R=0.358 P=0.052 R=0.688 P=0.000 Cortisol R=0.408 P=0.067 R=－0.311 P=0.170 R=－0.057 P=0.797 R=0.034 P=0.849 R=0.203 P=0.290 R=－0.040 P=0.804 arman correlation coefficients were used to evaluate the correlations of LH with reproductive Spearman correlation coefficients were used to evaluate the correlations of LH with reproductive hormones for each group. Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. DHEAS: dehydroepiandrosterone sulfate, T: testosterone FSH: follicle-stimulating hormone. Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. DHEAS: dehydroepiandrosterone sulfate, T: testosterone FSH: follicle-stimulating hormone. Table 3. Correlations of FSH with reproductive hormones DHEAS: dehydroepiandrosterone sulfate, T: testosterone FSH: follicle-stimulating hormone. Table 3. Tables Correlations of FSH with reproductive hormones Page 14/16 Stage Group A Group B Group C Group D Group E Group F testosterone R=－0.085 P=0.715 R=－0.163 P=0.469 R=－0.059 P=0.789 R=0.145 P=0.407 R=－0.159 P=0.402 R=－0.030 P=0.853 Free testosterone R=－0.021 P=0.929 R=0.120 P=0.595 R=－0.079 P=0.719 R=0.135 P=0.438 R=－0.334 P=0.071 R=－0.180 P=0.253 DHEAS R=0.067 P=0.772 R＝－0.065 P= 0.780 R=0.354 P=0.098 R=0.302 P=0.082 R=－0.159 P=0.411 R=0.076 P=0.632 androstenedione R=－0.307 P=0.308 R=0.013 P=0.965 R=0.227 P=0.502 R=－0.216 P=0.405 R=0.048 P=0.911 R=－0.393 P=0.383 androstenediol R=0.129 P=0.723 R＝－0.024 P=0.955 R=－0.275 P=0.509 R=－0.233 P=0.546 R=－0.500 P=0.253 R=0.000 P=1.000 estradiol R=－0.318 P=0.160 R=－0.222 P=0.321 R=－0.432 P=0.039 R=－0.720 P=0.000 R=－0.158 P=0.406 R=0.015 P=0.925 estrone R=－0.309 P=0.500 R=－0.167 P=0.693 R=－0.600 P=0.088 R=－0.452 P=0.260 R=0.209 P=0.537 R=0.300 P=0.370 LH R=0.233 P=0.309 R=0.713 P＝0.000 R=0.776 P=0.000 R=0.692 P=0.000 R=0.358 P=0.052 R=0.688 P=0.000 Cortisol R=0.026 P=0.911 R=－0.268 P=0.240 R=0.094 P=0.670 R=0.009 P=0.960 R=0.018 P=0.926 R=0.003 P=0.983 earman correlation coefficients were used to evaluate the correlations of FSH with reproductive mones and cortisol for each group Spearman correlation coefficients were used to evaluate the correlations of FSH with reproductive hormones and cortisol for each group. Group A: mid reproductive stage, Group B: late reproductive stage, Group C: early menopausal transition, Group D: late menopausal transition, Group E: very early post menopause, Group F: early postmenopause. DHEAS: dehydroepiandrosterone sulfate, T: testosterone, LH: luteinizing hormone. Figures Page 15/16 Figure 1 See image above for figure legend Page 16/16
https://openalex.org/W2138112715	https://europepmc.org/articles/pmc3410317?pdf=render	English	null	Retinal Damage in Multiple Sclerosis Disease Subtypes Measured by High-Resolution Optical Coherence Tomography	Multiple sclerosis international	2,012	cc-by	8,464	Timm Oberwahrenbrock,1 Sven Schippling,2, 3 Marius Ringelstein,4 Falko Kaufhold,1 Hanna Zimmermann,1 Nazmiye Keser,4 Kim Lea Young,2 Jens Harmel,4 Hans-Peter Hartung,4 Roland Martin,2, 3 Friedemann Paul,1, 5 Orhan Aktas,4 and Alexander U. Brandt1 Timm Oberwahrenbrock,1 Sven Schippling,2, 3 Marius Ringelstein,4 Falko Kaufhold,1 Hanna Zimmermann,1 Nazmiye Keser,4 Kim Lea Young,2 Jens Harmel,4 Hans-Peter Hartung,4 Roland Martin,2, 3 Friedemann Paul,1, 5 Orhan Aktas,4 and Alexander U. Brandt1 1NeuroCure Clinical Research Center and Experimental and Clinical Research Center, Charit´e University Medicine Berlin/Max Delbr¨uck Center for Molecular Medicine, 10117 Berlin, Germany 2Institute for Neuroimmunology and Clinical Multiple Sclerosis Research (inims) f gy p ( University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany f gy p University Medical Center Hamburg-Eppendorf, 20251 Hamburg, Germany 3Department of Neuroimmunology and Clinical Multiple Sclerosis Research, Neurology Clinic, University Hospita 8091 Zurich, Switzerland Department of Neuroimmunology and Clinical Multiple Sclerosis Research, Neurology Clinic, University Hospital Zu 091 Zurich, Switzerland 4Department of Neurology, Medical Faculty, Heinrich-Heine-University D¨usseldorf, 40225 D¨usseldorf, Germany 5Clinical and Experimental Multiple Sclerosis Research Center, Charit´e University Medicine Berlin, 10117 Berlin, Germany 4Department of Neurology, Medical Faculty, Heinrich-Heine-University D¨usseldorf, 40225 D¨usseldorf, Germany 5Clinical and Experimental Multiple Sclerosis Research Center, Charit´e University Medicine Berlin, 10117 Berlin, Germany Correspondence should be addressed to Friedemann Paul, friedemann.paul@charite.de Received 24 February 2012; Revised 8 May 2012; Accepted 18 May 2012 Academic Editor: Mark S. Freedman Academic Editor: Mark S. Freedman Copyright © 2012 Timm Oberwahrenbrock et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Optical coherence tomography (OCT) has facilitated characterisation of retinal alterations in MS patients. Only scarce and in part conﬂicting data exists on diﬀerent MS subtypes. Objective. To analyse patterns of retinal changes in diﬀerent subtypes of MS with latest spectral-domain technology. Methods. In a three-centre cross-sectional study 414 MS patients and 94 healthy controls underwent spectral-domain OCT examination. Results. Eyes of MS patients without a previous optic neuritis showed a signiﬁcant reduction of both retinal nerve ﬁbre layer (RNFL) thickness and total macular volume (TMV) compared to healthy controls independent of the MS subtype (P < 0.001 for all subtypes). RNFL thickness was lower in secondary progressive MS (SPMS) eyes compared to relapsing-remitting MS (RRMS) eyes (P = 0.007), and TMV was reduced in SPMS and primary progressive MS (PPMS) eyes compared to RRMS eyes (SPMS: P = 0.039, PPMS: P = 0.005). Independent of the subtype a more pronounced RNFL thinning and TMV reduction were found in eyes with a previous optic neuritis compared to unaﬀected eyes. Conclusion. Timm Oberwahrenbrock,1 Sven Schippling,2, 3 Marius Ringelstein,4 Falko Kaufhold,1 Hanna Zimmermann,1 Nazmiye Keser,4 Kim Lea Young,2 Jens Harmel,4 Hans-Peter Hartung,4 Roland Martin,2, 3 Friedemann Paul,1, 5 Orhan Aktas,4 and Alexander U. Brandt1 Analysis of this large-scale cross-sectional dataset of MS patients studied with spectral-domain OCT conﬁrmed and allows to generalize previous ﬁndings. Furthermore it carves out distinct patterns in diﬀerent MS subtypes. Hindawi Publishing Corporation Multiple Sclerosis International Volume 2012, Article ID 530305, 10 pages doi:10.1155/2012/530305 Hindawi Publishing Corporation Multiple Sclerosis International Volume 2012, Article ID 530305, 10 pages doi:10.1155/2012/530305 1. Introduction Optical coherence tomography (OCT) is an increasingly recognized, noninvasive tool in MS imaging that allows cost- eﬀective investigation of the retina [2] in a disease in which pathology of the anterior visual system is common. Over the recent past, OCT has emerged as a potential marker of axonal retinal degeneration in MS patients [3]. Of note, an increasing number of studies have consistently shown an association between OCT measures of retinal atrophy and markers of degeneration derived from either structural mag- netic resonance imaging or MR spectroscopy (MRS) studies Multiple sclerosis is an inﬂammatory and neurodegenerative disorder of the central nervous system that leads to a progres- sive axonal loss and degeneration of neurons. Whereas a vast majority of MS patients present with a relapsing-remitting disease course (RRMS) that may subsequently transforms into secondary progressive MS (SPMS), a smaller portion of patients shows a progressive course (PPMS) from the very beginning of the disease [1]. 2 Multiple Sclerosis International 2 [4–11] and between OCT measures and functional visual parameters as well as physical disability and cognitive perfor- mance [12–17]. Retinal nerve ﬁbre layer (RNFL) thickness and total macular volume (TMV) are the most frequently investigated OCT parameters. They provide a unique oppor- tunity to quantify the integrity of nonmyelinated axonal tissue (RNFL) as well as all retinal layers (TMV), including cellular segments, by a noninvasive imaging technique. RNFL and TMV reduction can be detected in eyes with (MS- ON) or without a previous history of optic neuritis (MS- NON) applying diﬀerent OCT devices [15, 18–29]. Data on distinct diﬀerences in OCT ﬁndings between MS subtypes are scarce, and the results are at least in part conﬂicting. In general, cross-sectional studies show a more profound thinning of RNFL in progressive forms of MS compared to RRMS patients. However, it remains unresolved to date whether these diﬀerences are a genuine eﬀect of the disease subtype per se or rather a function of disease duration, the number of optic neuritis (ON) episodes in the patients’ history, or disease severity [30–34]. or any other acute relapse or steroid treatment less than six months prior to OCT assessment as well as any other neuro- logical disease with possible ocular manifestations. Disease duration was calculated as time since diagnosis in months. Participants were assessed in a clinical examination under supervision of board certiﬁed neurologists within 6 months of OCT. 1. Introduction For assessing the macular volume two diﬀerent scan protocols were used: the system built-in macula scan (25 B-scans, scanning angle = 15◦× 15◦, ART = 9) was performed at the Hamburg and D¨usseldorf sites, while for the macular volume determination at the NCRC a custom protocol (61 B-scans, scanning angle = 30◦× 25◦, ART = 13) was used. Irrespective of the macular scan type, the TMV was calculated using the device’s software. All scans were performed by trained operators and were reviewed for suﬃcient signal strength, correct centring, and beam placement as well as segmentation. Only eyes that passed the quality review were included in the subsequent analysis. 1. Introduction Extended disability status scale (EDSS) was calcu- lated according to the current guidelines [41]. Time since diagnosis was determined by reviewing patients’ medical records. Healthy control participants were recruited from the medical staﬀ, patients’ relatives, and other volunteers. A total of 937 eyes (754 MS eyes and 183 HC eyes) were ﬁnally included, 79 eyes were excluded from the analysis either due to poor scan quality or incomplete clinical data, in detail missing data on history of ON. 2.2. Ethics. The study protocol was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki (1964) in its currently applicable version, the Guidelines of the International Conference on Harmonization of Good Clinical Practice (ICH-GCP) and applicable German laws. All participants gave written in- formed consent. 2.2. Ethics. The study protocol was approved by the local ethics committee and was conducted in accordance with the Declaration of Helsinki (1964) in its currently applicable version, the Guidelines of the International Conference on Harmonization of Good Clinical Practice (ICH-GCP) and applicable German laws. All participants gave written in- formed consent. Most OCT studies previously performed in MS applied time-domain technology. Only very recently spectral-do- main technology became available which enables imaging at substantially higher resolution without an increase in scanning time [16, 22, 34–38]. Here we report results from the largest multicentre cohort of MS patients and healthy controls (HC) thus far, studied in three dedicated MS centres in Germany, applying latest high-resolution spectral-domain OCT (SD-OCT) technology. In this large cohort, we reliably identiﬁed patterns of RNFL thinning and TMV reduction among diﬀerent MS subtypes both with and without a history of ON when controlling for disease duration and severity, age, and gender. 2.3. Optical Coherence Tomography. Participants underwent SD-OCT examination using the Heidelberg Engineering Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany). For both eyes of each participant, RNFL thickness around the optic disc was acquired using the 3.4 mm circle scan with the eye tracker system (TrueTrack) activated and the maximum number of averaging frames in ART-MEAN mode were tried to achieve. 3. Results 3.1. Cohort Description. An overview of the demographic and basic clinical data including MS subtypes is given in Table 1. Healthy controls showed no signiﬁcant gender dif- ferences to RRMS (Chi-square: P = 0.452) and SPMS (Chi- square: P = 0.186) patients, while gender composition of PPMS patients diﬀered compared to HC (Chi-square: P = 0.010). Mean age of HC was signiﬁcantly lower compared to all MS patients and all subtypes (Mann-Whitney U tests, P < 0.001 for all subtypes). RRMS patients were signiﬁcantly younger than the progressive subtypes (Mann-Whitney U tests, P < 0.001 for SPMS and PPMS). As a consequence, age and gender were included as covariates in all GEE models. Time since diagnosis of RRMS and PPMS was signiﬁcantly shorter compared to SPMS (Mann-Whitney U tests, P < 0.001 for both). Therefore, GEE models for MS subtype comparison were additionally adjusted for disease duration. Disease severity estimated by the EDSS was heterogeneous between MS subtypes as evaluated by Mann-Whitney U tests (RRMS versus SPMS: P < 0.001; RRMS versus PPMS: P < 0.001; SPMS versus PPMS: P = 0.03). 3.3. Comparison of MS-ON Eyes with MS-NON Eyes and HC. Irrespective of the MS subtype, MS-ON eyes were signiﬁ- cantly diﬀerent from HC eyes for RNFL thickness and TMV (Figure 2) and showed a more pronounced RNFL thinning and TMV reduction when compared to MS-NON eyes (Table 3). RNFL thickness of RRMS-ON eyes and SPMS- ON eyes was signiﬁcantly thinner compared to RRMS-NON or SPMS-NON eyes, respectively. The same was true for the TMV of ON-aﬀected MS eyes, which was signiﬁcantly reduced when compared to HC and to unaﬀected eyes of the same MS subtype (Table 3, Figure 2). The extent of RNFL thinning as well as TMV reduction was comparable between RRMS-ON and SPMS-ON eyes (RNFL GEE: P = 0.369; TMV GEE: P = 0.124). RRMS-ON eyes showed a signiﬁcant inverse correlation between EDSS and RNFL (P = 0.012) while TMV did not reach signiﬁcance (P = 0.085). For SPMS-ON eyes no signiﬁcant correlation of EDSS with OCT parameters was found (RNFL: P = 0.169; TMV: P = 0.573). 3.2. RNFL and TMV in MS-NON Eyes of Diﬀerent MS Sub- types Compared to HC. For MS-NON eyes, diﬀerences in RNFL thickness and TMV compared to HC are given in Table 2. 2. Materials and Methods MS patients are subdivided in the subtypes relapsing-remitting MS (RRMS), secondary-progressive MS (SPMS), and primary-progressive MS (PPMS). Table 1: Demographic and clinical data of multiple sclerosis patients (MS) and healthy controls (HC). MS patients are subdivided in the subtypes relapsing-remitting MS (RRMS), secondary-progressive MS (SPMS), and primary-progressive MS (PPMS). HC MS RRMS SPMS PPMS All MS No. of Subjects Total 94 308 65 41 414 Berlin 63 95 22 8 125 Hamburg 0 158 23 22 203 D¨usseldorf 31 55 20 11 86 No. of Eyes (eyes with ON) Total 183 561 (156) 116 (27) 77 (0) 754 (183) Berlin 122 187 (77) 44 (15) 16 (0) 247 (92) Hamburg 0 270 (54) 35 (2) 41 (0) 346 (56) D¨usseldorf 61 104 (25) 37 (10) 20 (0) 161 (35) Gender Male (%) 31 (33) 87 (28) 29 (45) 24 (59) 140 (34) Female (%) 63 (67) 221 (72) 36 (55) 24 (41) 274 (66) Age (in years) Mean (SD) 34.47 (10.25) 39.10 (9.50) 48.23 (6.11) 46.90 (7.10) 41.31 (9.59) Range 19–56 19–58 33–59 32–59 19–59 Disease duration (in months) Mean (SD) NA 91.05 (80.26) 186.15 (87.94) 100.02 (93.33) 106.87 (89.51) Range NA 0–384 39–403 4–426 0–426 EDSS Median NA 2.0 5.5 4.0 2.5 Range NA 0-7 3-8 2-8 0-8 Likewise, TMV was reduced for the pooled cohort of all MS patients’ eyes and in all MS subtypes when compared to HC (Figure 1(b)). All alterations of RNFL and TMV in MS-NON eyes compared to control eyes showed strong statistical signiﬁcance based on GEE models (Table 2). EDSS was inversely correlated with RNFL in case of all MS subtypes without a history of prior ON (RRMS-NON: P = 0.007; SPMS-NON: P = 0.034; PPMS-NON: P = 0.006). In contrast, the TMV was only signiﬁcantly correlated with the EDSS in RRMS-NON eyes (P = 0.003), but not in SPMS- NON (P = 0.321) or PPMS-NON (P = 0.085). models OCT measurements were included as the dependent variable. All statistical analyses were performed with R (R Version 2.12.2) including the geepack package for GEE models. Statistical signiﬁcance was established at P < 0.05. 2. Materials and Methods 2.1. Patients and Controls. 414 patients and 94 healthy con- trols were recruited in three dedicated MS units in the respec- tive outpatient clinics at the Charit´e University Medicine Berlin (NeuroCure Clinical Research Center (NCRC)), at the Department of Neurology of the Heinrich-Heine Uni- versity D¨usseldorf (UKD) and in Hamburg (Institute for Neuroimmunology and Clinical MS Research (inims)). Data from a subgroup has previously been reported in Brandt et al. [23]. Inclusion criteria were age between 18 and 60 years and a deﬁnite diagnosis of MS according to the revised 2005 McDonald criteria [39]. MS subtype classiﬁcation in RRMS, SPMS, or PPMS was based on the clinical course as assessed by the treating physician using Lublin criteria [40]. A history of ON had to be clearly determinable either by existing medical records, a VEP suggestive of optic neuritis, or by patient self-reports. Only eyes in which a history of ON could either be conﬁrmed by clinical records or ruled out were subsequently included in the analysis. Patients with a refractive error of ≥5.0 diopters or with a history of eye disease that may impact signiﬁcantly on OCT measures (e.g., glaucoma, retinal disease, retinal surgery, and diabetes) were excluded. Other exclusion criteria were acute optic neuritis 2.4. Statistical Analysis. Group comparisons of demographic factors were analysed using Mann-Whitney U test (for age and EDSS) and Pearson’s Chi-square test (for gender, alpha = 0.05). Within the MS cohort Spearman’s Rho analysis was used for correlation of EDSS and disease duration. 2.4. Statistical Analysis. Group comparisons of demographic factors were analysed using Mann-Whitney U test (for age and EDSS) and Pearson’s Chi-square test (for gender, alpha = 0.05). Within the MS cohort Spearman’s Rho analysis was used for correlation of EDSS and disease duration. Generalized estimation equation (GEE) models account- ing for within-subject intereye correlations were applied to test for diﬀerences of RNFL thickness and TMV between the study cohorts. GEE models were corrected for age and gender for the comparison of MS patients with HC and additionally for disease duration for MS subtype analysis. Associations of OCT with EDSS and regression analysis of disease duration with RNFL thickness and TMV were investigated with GEE models in a similar fashion. In all GEE Multiple Sclerosis International 3 Multiple Sclerosis International Table 1: Demographic and clinical data of multiple sclerosis patients (MS) and healthy controls (HC). 3. Results However, correlations of RNFL and TMV with disease duration were not signiﬁcant for PPMS- NON eyes. adjusting GEE models for age, gender, and disease duration, the only signiﬁcant diﬀerence based on the mean total RNFL thickness was found between RRMS and SPMS patients, while patients with PPMS did not diﬀer from either RRMS or SPMS (Table 4, Figure 1). As opposed to RNFL thickness a diﬀerent pattern was obtained for TMV measures, in which a signiﬁcant reduction was found for SPMS and PPMS when compared to RRMS patients. GEE models in which we additionally corrected for EDSS to account for diﬀerent stages of disease severity did not show diﬀerences in RNFL thickness or TMV between MS subtypes (data not shown). 3.5. Association with Disease Duration and Yearly Atrophy Estimate. GEE models were used to test for an association of disease duration and RNFL thickness or TMV in MS eyes. MS-NON eyes showed an association of RNFL thickness and TMV with disease duration in the pooled cohort of all MS subtypes (Table 5, Figure 3). This association was retained in RRMS and SPMS eyes only for RNFL thickness and only in RRMS eyes for TMV. In all MS subtypes the correlation between RNFL thickness and TMV and disease duration was lost in ON eyes (data not shown). 3. Results In summary, average peripapillary RNFL thickness was reduced in the pooled cohort of all MS patients’ eyes and in all MS subtypes compared to HC (Figure 1(a)). 3.4. Diﬀerences between Subtypes in MS-NON Eyes. Among MS subtypes, RRMS patients showed less RNFL thinning when compared to progressive subtypes (Table 2). When 4 Multiple Sclerosis International Table 2: OCT results for the subtypes of MS patients without a history of optic neuritis (NON). Total retinal nerve ﬁber layer (RNFL) thickness (in µm) and total macular volume (TMV in mm3) are given as mean values with standard deviation (SD). Generalized estimation equation (GEE) models were used to compare the MS cohorts to healthy controls. GEE models estimate the eﬀect size with standard error (SE) and the respective P value. GEE models comparing MS OCT parameters to the healthy control cohort RNFL thickness TMV Total RNFL thickness mean (SD) [µm] TMV mean (SD) [mm3] Eﬀect Eﬀect size SE P value Eﬀect size SE P value Group −9.571 1.177 <0.001 −0.235 0.043 <0.001 MS-NON (n = 571) 90.15 (12.27) 8.48 (0.43) Age −0.132 0.053 0.013 −0.006 0.002 0.002 Gender 2.272 1.116 0.042 −0.075 0.041 0.064 Group −8.470 1.188 <0.001 −0.197 0.044 <0.001 RRMS-NON (n = 405) 92.03 (11.91) 8.54 (0.42) Age −0.080 0.061 0.189 −0.003 0.002 0.094 Gender 2.933 1.224 0.017 −0.107 0.045 0.019 Group −9.951 1.084 <0.001 −0.248 0.039 <0.001 SPMS-NON (n = 89) 83.14 (12.07) 8.32 (0.41) Age 0.139 0.098 0.158 0.003 0.003 0.267 Gender −1.807 1.788 0.312 −0.150 0.062 0.015 Group −4.253 0.792 <0.001 −0.141 0.027 <0.001 PPMS-NON (n = 77) 88.35 (11.31) 8.34 (0.42) Age 0.037 0.093 0.691 −0.001 0.003 0.654 Gender 0.802 1.796 0.655 −0.044 0.064 0.492 Group N/A N/A N/A N/A N/A N/A HC (n = 183) 100.60 (9.41) 8.75 (0.34) Age N/A N/A N/A N/A N/A N/A Gender N/A N/A N/A N/A N/A N/A thinning and TMV reduction per year of ongoing disease in MS-NON eyes only (Table 5). RRMS-NON eyes showed the strongest and highly signiﬁcant yearly changes for RNFL thickness (−0.495 µm/year) and TMV (−0.0155 mm3/year). Interestingly, the signiﬁcant yearly RNFL thinning in SPMS-NON eyes (−0.464 µm/year) was not concomitantly associated with a signiﬁcant correlation in TMV change (−0.0016 mm3/year, P = 0.838). In contrast, PPMS-NON eyes showed a less pronounced yearly RNFL thinning (−0.105 µm/year) but in relation a distinct reduction of TMV (−0.0111 mm3/year). 4. Discussion Here we present the largest ever performed cross-sectional study on retinal atrophy measures in MS subtypes applying latest SD-OCT technology. Groups of disease subtypes in our study were suﬃciently large to contrast ﬁndings in ON versus ON-free eyes within subgroups. Hereby we show that both RNFL and TMV are reduced in MS-NON eyes versus HC when pooling all disease subtypes, but also when separately comparing disease subtypes (RRMS, SPMS, Based on the eﬀect size of the association of disease duration and RNFL thickness or TMV we estimated RNFL 5 Multiple Sclerosis International 120 110 100 90 80 70 60 HC RRMS NON SPMS NON PPMS NON ∗∗∗ ∗ n = 183 n = 405 n = 89 n = 77 Mean RNFL thickness (µm) (a) 9.5 9 8.5 8 7.5 7 ∗∗ ∗ ∗∗∗ HC RRMS NON SPMS NON PPMS NON n = 183 n = 405 n = 89 n = 77 Mean TMV (mm3) (b) Figure 1: Mean retinal nerve ﬁbre layer (RNFL) thickness (a) and mean total macular volume (TMV) (b) for the healthy controls (HC) and MS subtypes (RRMS, SPMS, and PPMS) without a history of optic neuritis (NON). Signiﬁcant diﬀerences between the groups are indicated with ∗(P < 0.05), ∗∗(P < 0.01), and ∗∗∗(P < 0.001), respectively. (a) (b) Figure 1: Mean retinal nerve ﬁbre layer (RNFL) thickness (a) and mean total macular volume (TMV) (b) for the healthy controls (HC) and MS subtypes (RRMS, SPMS, and PPMS) without a history of optic neuritis (NON). Signiﬁcant diﬀerences between the groups are indicated with ∗(P < 0.05), ∗∗(P < 0.01), and ∗∗∗(P < 0.001), respectively. 4. Discussion The large sample size of our study enabled a statistically robust comparison of various disease GEE models comparing OCT parameters between ON-aﬀected and unaﬀected eyes of the same subtype GEE models comparing OCT parameters between ON-aﬀected and unaﬀected eyes of the same subtype RNFL thickness TMV Total RNFL thickness mean (SD) [µm] TMV mean (SD) [mm3] Eﬀect Eﬀect size SE P value Eﬀect size SE P value Group −12.199 1.336 <0.001 −0.237 0.043 <0.001 MS-ON (n = 183) 77.88 (14.61) 8.24 (0.45) Age −0.147 0.056 0.008 −0.007 0.002 0.001 Gender 2.989 1.161 0.010 −0.041 0.040 0.299 Group −12.859 1.478 <0.001 −0.263 0.048 <0.001 RRMS-ON (n = 156) 78.69 (14.91) 8.28 (0.45) Age −0.087 0.066 0.185 −0.004 0.002 0.071 Gender 4.573 1.352 0.001 −0.048 0.046 0.295 Group −9.297 2.802 0.001 −0.252 0.100 0.012 SPMS-ON (n = 27) 73.19 (11.89) 8.05 (0.41) Age 0.419 0.216 0.052 0.009 0.008 0.234 Gender −7.310 2.591 0.005 −0.250 0.091 0.006 PPMS-ON (n = 0) N/A N/A N/A N/A N/A N/A N/A N/A N/A HC (n = 183) 100.60 (9.41) 8.75 (0.34) N/A N/A N/A N/A N/A N/A N/A Table 4: Diﬀerences between MS subtypes without a history of optic neuritis (NON) were analyzed with generalized estimation equations (GEE) models including age, disease duration, and gender as eﬀects. GEE models comparing OCT parameters between NON eyes of diﬀerent MS subtypes RNFL thickness TMV Eﬀect Eﬀect size SE P value Eﬀect size SE P value RRMS-NON versus SPMS-NON Group −5.144 1.921 0.007 −0.137 0.066 0.039 Age 0.062 0.077 0.419 −0.001 0.003 0.775 Duration −0.044 0.009 <0.001 −0.001 0.0003 <0.001 Gender 1.864 1.377 0.176 −0.139 0.051 0.007 RRMS-NON versus PPMS-NON Group −1.204 1.022 0.239 −0.104 0.037 0.005 Age −0.016 0.073 0.823 −0.002 0.003 0.371 Duration −0.034 0.008 <0.001 −0.001 0.0003 <0.001 Gender 2.785 1.393 0.045 −0.102 0.052 0.051 SPMS-NON versus PPMS-NON Group 2.634 2.494 0.291 −0.053 0.090 0.553 Age 0.339 0.175 0.053 0.002 0.006 0.729 Duration −0.031 0.011 0.007 −0.001 0.0004 0.224 Gender −3.932 2.287 0.086 −0.127 0.086 0.139 Table 4: Diﬀerences between MS subtypes without a history of optic neuritis (NON) were analyzed with generalized estimation equations (GEE) models including age, disease duration, and gender as eﬀects. and PPMS) to HC. Not surprisingly and in accordance with previous studies, MS-ON eyes exhibited more severe RNFL and TMV damage than MS-NON eyes. 4. Discussion 120 110 100 90 80 70 60 HC RRMS ON SPMS ON ∗∗∗ Mean RNFL thickness (µm) n = 183 n = 156 n = 27 (a) 9.5 9 8.5 8 7.5 7 Mean TMV (mm3) HC RRMS ON SPMS ON n = 183 n = 156 n = 27 ∗∗∗ (b) 120 110 100 90 80 70 60 HC RRMS ON SPMS ON ∗∗∗ Mean RNFL thickness (µm) n = 183 n = 156 n = 27 (a) 9.5 9 8.5 8 7.5 7 Mean TMV (mm3) HC RRMS ON SPMS ON n = 183 n = 156 n = 27 ∗∗∗ (b) Figure 2: Mean retinal nerve ﬁbre layer (RNFL) thickness (a) and mean total macular volume (TMV) (b) for the healthy controls (HC) and MS subtypes (RRMS, SPMS) with a history of optic neuritis (ON). Signiﬁcant diﬀerences between the groups are indicated with ∗(P < 0.05), ∗∗(P < 0.01), and ∗∗∗(P < 0.001), respectively. Mean RNFL thickness (µm) (a) (b) Figure 2: Mean retinal nerve ﬁbre layer (RNFL) thickness (a) and mean total macular volume (TMV) (b) for the healthy controls (HC) and MS subtypes (RRMS, SPMS) with a history of optic neuritis (ON). Signiﬁcant diﬀerences between the groups are indicated with ∗(P < 0.05), ∗∗(P < 0.01), and ∗∗∗(P < 0.001), respectively. 6 Multiple Sclerosis International Table 3: OCT results for the subtypes of MS patients with a history of optic neuritis (ON). Total retinal nerve ﬁber layer (RNFL) thickness (in µm) and total macular volume (TMV in mm3) are given as mean values with standard deviation (SD). ON eyes were compared to ON- non aﬀected eyes of the same MS subtype using generalized estimation equation (GEE) models. GEE models estimate the eﬀect size with standard error (SE) and the respective P value. standard error (SE) and the respective P value. 4. Discussion GEE models comparing OCT parameters between ON-aﬀected and unaﬀected eyes of the same subtype RNFL thickness TMV Total RNFL thickness mean (SD) [µm] TMV mean (SD) [mm3] Eﬀect Eﬀect size SE P value Eﬀect size SE P value Group −12.199 1.336 <0.001 −0.237 0.043 <0.001 MS-ON (n = 183) 77.88 (14.61) 8.24 (0.45) Age −0.147 0.056 0.008 −0.007 0.002 0.001 Gender 2.989 1.161 0.010 −0.041 0.040 0.299 Group −12.859 1.478 <0.001 −0.263 0.048 <0.001 RRMS-ON (n = 156) 78.69 (14.91) 8.28 (0.45) Age −0.087 0.066 0.185 −0.004 0.002 0.071 Gender 4.573 1.352 0.001 −0.048 0.046 0.295 Group −9.297 2.802 0.001 −0.252 0.100 0.012 SPMS-ON (n = 27) 73.19 (11.89) 8.05 (0.41) Age 0.419 0.216 0.052 0.009 0.008 0.234 Gender −7.310 2.591 0.005 −0.250 0.091 0.006 PPMS-ON (n = 0) N/A N/A N/A N/A N/A N/A N/A N/A N/A HC (n = 183) 100.60 (9.41) 8.75 (0.34) N/A N/A N/A N/A N/A N/A N/A Table 4: Diﬀerences between MS subtypes without a history of optic neuritis (NON) were analyzed with generalized estimation equations (GEE) models including age, disease duration, and gender as eﬀects. GEE models comparing OCT parameters between NON eyes of diﬀerent MS subtypes RNFL thickness TMV Eﬀect Eﬀect size SE P value Eﬀect size SE P value RRMS-NON versus SPMS-NON Group −5.144 1.921 0.007 −0.137 0.066 0.039 Age 0.062 0.077 0.419 −0.001 0.003 0.775 Duration −0.044 0.009 <0.001 −0.001 0.0003 <0.001 Gender 1.864 1.377 0.176 −0.139 0.051 0.007 RRMS-NON versus PPMS-NON Group −1.204 1.022 0.239 −0.104 0.037 0.005 Age −0.016 0.073 0.823 −0.002 0.003 0.371 Duration −0.034 0.008 <0.001 −0.001 0.0003 <0.001 Gender 2.785 1.393 0.045 −0.102 0.052 0.051 SPMS-NON versus PPMS-NON Group 2.634 2.494 0.291 −0.053 0.090 0.553 Age 0.339 0.175 0.053 0.002 0.006 0.729 Duration −0.031 0.011 0.007 −0.001 0.0004 0.224 Gender −3.932 2.287 0.086 −0.127 0.086 0.139 and PPMS) to HC. Not surprisingly and in accordance with previous studies, MS-ON eyes exhibited more severe RNFL and TMV damage than MS-NON eyes. Both ﬁndings have been previously described in a similar way by various methodological advances compared to previous works that have important implications for the interpretation of our and previous OCT ﬁndings. 4. Discussion RNFL thickness TMV Eﬀect size SE P value Change per year (µm) Eﬀect size SE P value Change per year (mm3) All MS-NON −0.0444 0.0068 <0.001 −0.533 −0.0012 0.0002 <0.001 −0.014 RRMS-NON −0.0413 0.0088 <0.001 −0.495 −0.0013 0.0003 <0.001 −0.016 SPMS-NON −0.0387 0.0174 0.026 −0.464 −0.0001 0.0006 0.838 −0.002 PPMS-NON −0.0088 0.0151 0.562 −0.105 −0.0009 0.0006 0.131 −0.011 PPMS 0 100 200 300 400 SPMS 0 100 200 300 400 Disease duration (months) 50 60 70 80 90 100 110 120 RRMS 0 100 200 300 400 RNFL thickness (µm) 50 60 70 80 90 100 110 120 RRMS 0 100 200 300 400 SPMS 0 100 200 300 400 PPMS 0 100 200 300 400 Disease duration (months) RNFL thickness (µm) 7 8 8.5 9 9.5 RRMS 0 100 200 300 400 TMV (mm3) SPMS 0 100 200 300 400 Disease duration (months) (b) PPMS 0 100 200 300 400 (b) Figure 3: Association of RNFL thickness (a) and TMV (b) with disease duration for RRMS, SPMS and PPMS subtypes in eyes without previous optic neuritis. The blue lines indicate the 95%-, 50%- and 5%-quantiles. models. In particular, we had larger numbers of patients in the progressive subgroups (65 SPMS, 41 PPMS) than any other previous study which allowed us to compare not only disease subtypes with HC but also with each other. This is of special interest against the background of the ongoing scientiﬁc debate on distinct pathogenetic mechanisms in, for example, PPMS versus RRMS. The subgroup comparisons revealed a signiﬁcant reduction of RNFL thickness in SPMS patients versus RRMS after correction for age, gender, and disease duration and a signiﬁcant reduction of TMV in both SPMS and PPMS patients versus RRMS. considerable diﬀerences in the statistical models—at least partly explain the inconsistent ﬁndings. Pulicken et al. (number of subjects: 135 RRMS, 16 SPMS, 12 PPMS, and 47 HC) found only trends towards lower RNFL thickness values in progressive disease versus RRMS and no diﬀerence in TMV in progressive MS versus RRMS [30]. Henderson et al. (number of subjects: 27 SPMS, 23 PPMS, and 20 HC) reported a signiﬁcant reduction of RNFL and TMV versus HC only in NON eyes from SPMS patients but not PPMS patients and no diﬀerence between PPMS and SPMS [31]. Siepman et al. 4. Discussion Both ﬁndings have been previously described in a similar way by various groups so that the nature of our study appears to be largely conﬁrmatory at ﬁrst glance. However, our study has some and PPMS) to HC. Not surprisingly and in accordance with previous studies, MS-ON eyes exhibited more severe RNFL and TMV damage than MS-NON eyes. Both ﬁndings have been previously described in a similar way by various groups so that the nature of our study appears to be largely conﬁrmatory at ﬁrst glance. However, our study has some methodological advances compared to previous works that have important implications for the interpretation of our and previous OCT ﬁndings. The large sample size of our study enabled a statistically robust comparison of various disease subgroups with the inclusion of possible confounding factors such as age, disease duration, and gender in the statistical Multiple Sclerosis International 7 Table 5: Generalized estimation equation (GEE) models correlating disease duration with RNFL thickness and TMV, respectively. The yearly change based on the eﬀect sizes of the respective GEE model. Table 5: Generalized estimation equation (GEE) models correlating disease duration with RNFL thickness and TMV, respectively. The yearly change based on the eﬀect sizes of the respective GEE model. Table 5: Generalized estimation equation (GEE) models correlating disease duration with RNFL thickness and TMV, respectively. The yearly change based on the eﬀect sizes of the respective GEE model. change based on the eﬀect sizes of the respective GEE model. RNFL thickness TMV Eﬀect size SE P value Change per year (µm) Eﬀect size SE P value Change per year (mm3) All MS-NON −0.0444 0.0068 <0.001 −0.533 −0.0012 0.0002 <0.001 −0.014 RRMS-NON −0.0413 0.0088 <0.001 −0.495 −0.0013 0.0003 <0.001 −0.016 SPMS-NON −0.0387 0.0174 0.026 −0.464 −0.0001 0.0006 0.838 −0.002 PPMS-NON −0.0088 0.0151 0.562 −0.105 −0.0009 0.0006 0.131 −0.011 50 60 70 80 90 100 110 120 RRMS 0 100 200 300 400 SPMS 0 100 200 300 400 PPMS 0 100 200 300 400 Disease duration (months) RNFL thickness (µm) (a) 7 8 8.5 9 9.5 RRMS 0 100 200 300 400 SPMS 0 100 200 300 400 PPMS 0 100 200 300 400 Disease duration (months) TMV (mm3) (b) Figure 3: Association of RNFL thickness (a) and TMV (b) with disease duration for RRMS, SPMS and PPMS subtypes in eyes without previous optic neuritis. The blue lines indicate the 95%-, 50%- and 5%-quantiles. 4. Discussion (number of subjects: 26 RRMS, 10 SPMS, and 29 PPMS) could not detect diﬀerences between PPMS- NON eyes and the pooled RRMS/SPMS-NON eyes [33]. In contrast, most previous works had only small sample sizes, especially for progressive subtypes which may—besides Multiple Sclerosis International 8 Serbecic et al. (number of subjects: 42 RRMS, 17 SPMS, and 59 HC) did not speciﬁcally address diﬀerences in OCT measures between disease subtypes [34] as did numerous other studies with highly heterogeneous patient populations (Gordon-Lipkin et al. [6], number of subjects: 20 RRMS, 15 SPMS, 5 PPMS, and 15 HC; Toledo et al. [12], number of subjects: 7 CIS, 36 RRMS, 3 SPMS, 3 PPMS, 4 progressive- relapsing, and 18 HC; Fisher et al. [15], number of subjects: 90 MS, 76 of whom RRMS, and 36 HC; Sepulcre et al. [7], number of subjects: 22 CIS, 28 RRMS, 5 SPMS, 6 PPMS, and 29 HC), either because of insuﬃcient subgroup sample sizes or owing to the fact that the study had goals other than comparing disease subtypes. greatly increase the value of macular scans over the cur- rently preferred peripapillary ring scans. In addition, spatial scans allow for correction of positioning errors after scan acquisition by limiting the analysed area to a subset of the actual scan. For example, the Cirrus SD-OCT uses a spatial scan for the peripapillary ring scan, allowing for subsequent correction of alignment errors, whereas the Heidelberg Engineering Spectralis facilitates an eye tracker function to correct for eye movements. In TD-OCT, incorrect placement of peripapillary ring scans accounts to a signiﬁcant extent for a weaker inter-measurement reliability and cannot be corrected after the scan has been acquired [48]. Next to the ability to analyse all intraretinal layers, improved test/retest- reliability distinguishes SD-OCT from TD-OCT and makes it an ideal instrument for the use in a longitudinal setting where inter-measurement reliability is crucial [49]. p g yp In line with several previous studies [16, 30, 31, 42, 43] we found higher RNFL measures in PPMS as compared to SPMS (88.4 µm versus 83.1 µm), which is in striking accordance with another study that also reported a diﬀerence of approximately 5 µm between PPMS and SPMS-NON eyes (93.9 µm versus 88.4 µm) [31]. 4. Discussion Although these diﬀerences were not signiﬁcant in both studies, these ﬁndings may point to a more severe RNFL damage in SPMS as compared to PPMS, which is in line with the clinical features of PPMS with a lower proportion of visual loss, less frequent ON attacks, a predominant clinical involvement of the spinal cord, and smaller brain lesions as compared to SPMS [31, 44, 45]. However, in contrast to Henderson et al. we found like in SPMS a signiﬁcant reduction of TMV in NON eyes of PPMS patients versus RRMS and HC, which may display the neurodegenerative component of PPMS concomitantly reﬂected through brain atrophy measures [46]. y The time course of RNFL thinning and TMV reduction by atrophy of diﬀerent retinal layers—be it in the context of ON or independent thereof—is an essential characteristic in rating the usefulness of OCT as a potential marker of axonal loss in longitudinal clinical trials. For MS-ON eyes it has previously been shown that RNFL thinning occurs within the ﬁrst 6 months after the ON attack [21, 50]. Overall little is known about temporal dynamics of retinal thinning in MS-NON eyes. Based on published data from cross-sectional studies in MS patients with diﬀerent disease duration a rough estimate of the yearly atrophy rate appears to be around 1 µm/year, which is ten times as much as what can be expected from normal ageing [3]. In previous cross- sectional studies signiﬁcant inverse correlations of RNFL thickness and disease duration could be established by some authors [11, 15, 24], while others did not ﬁnd a signiﬁcant correlation [20, 31]. In PPMS, an MS subtype in which frequency of clinical attacks of ON is probably lowest, Henderson et al. [31] estimated an RNFL thinning of approximately 0.12 µm (TMV reduction: 0.01 mm3) per year of disease, which is in good agreement with our results in PPMS eyes (RNFL thinning −0.105 µm/year; TMV reduction: −0.011 mm3/year). Correlations of OCT measures of retinal atrophy and disease duration were not signiﬁcant for PPMS patients in both studies. In case of RRMS and SPMS patients without ON we estimated higher yearly RNFL changes than for PPMS (nearly 0.5 µm/year). It is, however, important to note that yearly atrophy rates are considerably lower than the optimized axial resolution of SD-OCT devices, which is approximately 4–6 µm [51, 52]. References [1] A. Compston and A. Coles, “Multiple sclerosis,” The Lancet, vol. 372, no. 9648, pp. 1502–1517, 2008. [18] M. Bock, A. U. Brandt, J. D¨orr et al., “Patterns of retinal nerve ﬁber layer loss in multiple sclerosis patients with or without optic neuritis and glaucoma patients,” Clinical Neurology and Neurosurgery, vol. 112, no. 8, pp. 647–652, 2010. [2] D. Huang, E. A. Swanson, C. P. Lin et al., “Optical coherence tomography,” Science, vol. 254, no. 5035, pp. 1178–1181, 1991. [3] A. Petzold, J. F. de Boer, S. Schippling et al., “Optical coherence tomography in multiple sclerosis: a systematic review and meta-analysis,” The Lancet Neurology, vol. 9, no. 9, pp. 921– 932, 2010. [19] F. Costello, S. Coupland, W. Hodge et al., “Quantifying axonal loss after optic neuritis with optical coherence tomography,” Annals of Neurology, vol. 59, no. 6, pp. 963–969, 2006. [4] C. F. Pfueller, A. U. Brandt, F. Schubert et al., “Metabolic changes in the visual cortex are linked to retinal nerve ﬁber layer thinning in multiple sclerosis,” PLoS One, vol. 6, no. 4, Article ID e18019, 2011. [20] A. Klistorner, H. Arvind, T. Nguyen et al., “Axonal loss and myelin in early on loss in postacute optic neuritis,” Annals of Neurology, vol. 64, no. 3, pp. 325–331, 2008. [5] J. D¨orr, K. D. Wernecke, M. Bock et al., “Association of retinal and macular damage with brain atrophy in multiple sclerosis,” PLoS One, vol. 6, no. 4, Article ID e18132, 2011. [21] S. A. Trip, P. G. Schlottmann, S. J. Jones et al., “Retinal nerve ﬁber layer axonal loss and visual dysfunction in optic neuritis,” Annals of Neurology, vol. 58, no. 3, pp. 383–391, 2005. PLoS One, vol. 6, no. 4, Article ID e18132, 2011. [6] E. Gordon-Lipkin, B. Chodkowski, D. S. Reich et al., “Retinal nerve ﬁber layer is associated with brain atrophy in multiple sclerosis,” Neurology, vol. 69, no. 16, pp. 1603–1609, 2007. [22] S. Saidha, S. B. Syc, M. A. Ibrahim et al., “Primary retinal pathology in multiple sclerosis as detected by optical coher- ence tomography,” Brain, vol. 134, no. 2, pp. 518–533, 2011. [7] J. Sepulcre, M. Murie-Fernandez, A. Salinas-Alaman, A. Gar- c´ıa-Layana, B. Bejarano, and P. Villoslada, “Diagnostic accu- racy of retinal abnormalities in predicting disease activity in MS,” Neurology, vol. 68, no. 18, pp. 1488–1494, 2007. [23] A. U. Brandt, T. Oberwahrenbrock, M. 4. Discussion This is of relevance in case OCT endpoints are taken into consideration for future clinical trials, for example, in proof- of-concept trials for neuroprotective agents. Depending on the disease subtypes, model timelines and sample sizes have to be planned accordingly. Regarding the comparison of RNFL measures in RRMS and SPMS patients, we made another interesting observa- tion: as described previously by Costello et al. [32], dif- ferences between SPMS-NON and RRMS-NON eyes were about twice that of diﬀerences between SPMS-ON and RRMS-ON eyes (∼20 µm versus ∼10 µm in Costello’s study, ∼10 µm versus ∼5 µm in our study). Costello et al. suggested that the impact of prior ON may outweigh the eﬀects of disease subtype. yp Further limitation of most of the previous studies is the utilization of time-domain OCT devices (TD-OCT) that only allow for 2-dimensional retinal imaging, limiting its use especially in the demanding macular investigations. The newer high-resolution spectral-domain OCT allows spatial imaging of the retina thus potentially greatly increasing the accuracy and value of OCT in MS [35, 36, 47]. First studies have already applied SD-OCT with intraretinal segmentation [16, 22, 26, 29]. Interestingly, the recent work by Saidha et al. supports the ﬁnding of a more severe neuroaxonal retinal damage in SPMS as compared to PPMS; a separate analysis of the combined ganglion cell layer and inner plexiform layer measured by Cirrus SD-OCT in diﬀerent MS subtypes showed lowest values in SPMS [16]. In contrast, another study by Albrecht et al. [29] applying manual segmentation on Spectralis SD-OCT showed reduced measures in the deeper inner nuclear layer of PPMS but not SPMS patients versus HC. We presume that the ability of SD-OCT to measure spatial scans (earlier TD-OCT had to interpolate macular volume by using 6 radial linear scans) will in future In a ﬁrst longitudinal OCT study by Talman et al. [53] a thorough examination of the time course of RNFL thinning in a mixed cohort of diﬀerent MS subtypes was performed with TD-OCT (Stratus) revealing a yearly loss of approxi- mately 2 µm in MS-NON eyes (GEE: P < 0.001). 4. Discussion The study included a preliminary sample size calculation (supplemen- tary data of [53]) for future clinical trials that aimed to detect a 30% reduction in the proportion of eyes with an RNFL 9 Multiple Sclerosis International thinning greater than the test-retest variability of the Stratus OCT (6.6 µm) over a follow-up period of 2-3 years. With a power of 80–90% and a type 1 error of 0.05, the authors’ sample size calculation estimated roughly a number of 400– 600 patients per group. The yearly loss of 2 µm reported by Talman et al. from Stratus OCT is considerably higher than the yearly reduction of approximately 0.5 µm calculated from our dataset. Discrepancies may derive from the diﬀerences in the devices applied (TD-OCT versus SD-OCT) and the fact that our calculation is based on cross-sectional data only. [10] E. Grazioli, R. Zivadinov, B. Weinstock-Guttman et al., “Reti- nal nerve ﬁber layer thickness is associated with brain MRI outcomes in multiple sclerosis,” Journal of the Neurological Sciences, vol. 268, no. 1-2, pp. 12–17, 2008. [11] M. Siger, K. Dzie¸gielewski, L. Jasek et al., “Optical coherence tomography in multiple sclerosis: thickness of the retinal nerve ﬁber layer as a potential measure of axonal loss and brain atrophy,” Journal of Neurology, vol. 255, no. 10, pp. 1555–1560, 2008. [12] J. Toledo, J. Sepulcre, A. Salinas-Alaman et al., “Retinal nerve ﬁber layer atrophy is associated with physical and cognitive disability in multiple sclerosis,” Multiple Sclerosis, vol. 14, no. 7, pp. 906–912, 2008. In sum, this study, based on a large SD-OCT data set, conﬁrms previous data on neuroaxonal retinal damage in MS subtypes. At the same time, it extends previous ﬁndings by providing new insights into diﬀerences between MS-ON and MS-NON eyes in the various subgroups and—in addition— allowing for reliable correction for non-disease-related fac- tors such as age, gender disease duration, and severity. [13] B. M. Burkholder, B. Osborne, M. J. Loguidice et al., “Macular volume determined by optical coherence tomography as a measure of neuronal loss in multiple sclerosis,” Archives of Neurology, vol. 66, no. 11, pp. 1366–1372, 2009. [14] M. Bock, A. U. Brandt, J. Kuchenbecker et al., “Impairment of contrast visual acuity as a functional correlate of retinal nerve ﬁbre layer thinning and total macular volume reduction in multiple sclerosis,” British Journal of Ophthalmology, vol. 96, no. 1, pp. 62–67, 2011. Acknowledgments [16] S. Saidha, S. B. Syc, M. K. Durbin et al., “Visual dysfunction in multiple sclerosis correlates better with optical coherence tomography derived estimates of macular ganglion cell layer thickness than peripapillary retinal nerve ﬁber layer thick- ness,” Multiple Sclerosis, vol. 17, no. 12, pp. 1449–1463, 2011. This study was supported by DFG Exc Grant 257 and BMWi Grant ZIM KF2286101FO9. The inims is supported by an This study was supported by DFG Exc Grant 257 and BMWi Grant ZIM KF2286101FO9. The inims is supported by an unrestricted grant of the “Gemeinn¨utzige Hertie-Stiftung”. This study was supported by DFG Exc Grant 257 and BMWi Grant ZIM KF2286101FO9. The inims is supported by an unrestricted grant of the “Gemeinn¨utzige Hertie-Stiftung”. [17] S. Noval, I. Contreras, S. Mu˜noz, C. Oreja-Guevara, B. Manzano, and G. Rebolleda, “Optical coherence tomography in multiple sclerosis and neuromyelitis optica: an update,” Multiple Sclerosis International, vol. 2011, Article ID 472790, 11 pages, 2011. Authors’ Contribution T. Oberwahrenbrock, S. Schippling, and M. Ringelstein contributed equally to this work. [15] J. B. Fisher, D. A. Jacobs, C. E. Markowitz et al., “Relation of visual function to retinal nerve ﬁber layer thickness in multiple sclerosis,” Ophthalmology, vol. 113, no. 2, pp. 324–332, 2006. References Mesec, “Clinical, MRI, CSF and electrophys- iological ﬁndings in diﬀerent stages of multiple sclerosis,” Clinical Neurology and Neurosurgery, vol. 108, no. 3, pp. 271– 274, 2006. [30] M. Pulicken, E. Gordon-Lipkin, L. J. Balcer, E. Frohman, G. Cutter, and P. A. Calabresi, “Optical coherence tomography and disease subtype in multiple sclerosis,” Neurology, vol. 69, no. 22, pp. 2085–2092, 2007. [46] N. De Stefano, A. Giorgio, M. Battaglini et al., “Assessing brain atrophy rates in a large population of untreated multiple sclerosis subtypes,” Neurology, vol. 74, no. 23, pp. 1868–1876, 2010. [31] A. P. D. Henderson, S. A. Trip, P. G. Schlottmann et al., “An investigation of the retinal nerve ﬁbre layer in progressive multiple sclerosis using optical coherence tomography,” Brain, vol. 131, no. 1, pp. 277–287, 2008. [47] E. T´atrai, M. Sim´o, A. Iljicsov, J. N´emeth, D. C. DeBuc, and G. M. Somfai, “In vivo evaluation of retinal neurodegeneration in patients with multiple sclerosis,” PLoS One, vol. 7, no. 1, Article ID e30922, 2012. [32] F. Costello, W. Hodge, Y. I. Pan, M. Freedman, and C. DeMeulemeester, “Diﬀerences in retinal nerve ﬁber layer atro- phy between multiple sclerosis subtypes,” Journal of the Neu- rological Sciences, vol. 281, no. 1-2, pp. 74–79, 2009. [48] P. Tewarie, L. Balk, F. Costello et al., “The OSCAR-IB consen- sus criteria for retinal OCT quality assessment,” PLoS One, vol. 7, no. 4, Article ID e34823, 2012. [49] N. Serbecic, S. C. Beutelspacher, F. C. Aboul-Enein, K. Kircher, A. Reitner, and U. Schmidt-Erfurth, “Reproducibility of high- resolution optical coherence tomography measurements of the nerve ﬁbre layer with the new Heidelberg Spectralis optical coherence tomography,” British Journal of Ophthalmology, vol. 95, no. 6, pp. 804–810, 2011. [33] T. A. Siepman, M. W. Bettink-Remeijer, and R. Q. Hintzen, “Retinal nerve ﬁber layer thickness in subgroups of multiple sclerosis, measured by optical coherence tomography and scanning laser polarimetry,” Journal of neurology, vol. 257, no. 10, pp. 1654–1660, 2010. [34] N. Serbecic, F. Aboul-Enein, S. C. Beutelspacher et al., “Het- erogeneous pattern of retinal nerve ﬁber layer in multiple scle- rosis. high resolution optical coherence tomography: potential and limitations,” PLoS One, vol. 5, no. 11, Article ID e13877, 2010. [50] F. Costello, W. Hodge, Y. I. Pan, E. Eggenberger, S. Coupland, and R. H. Kardon, “Tracking retinal nerve ﬁber layer loss after optic neuritis: a prospective study using optical coherence tomography,” Multiple Sclerosis, vol. 14, no. 7, pp. 893–905, 2008. References Ringelstein et al., “Primary retinal pathology in multiple sclerosis as detected by optical coherence tomography,” Brain, vol. 134, no. 11, article e193, 2011. [8] F. Costello, “Evaluating the use of optical coherence tomog- raphy in optic neuritis,” Multiple Sclerosis International, vol. 2011, Article ID 148394, 9 pages, 2011. [24] V. Pueyo, J. Martin, J. Fernandez et al., “Axonal loss in the retinal nerve ﬁber layer in patients with multiple sclerosis,” Multiple Sclerosis, vol. 14, no. 5, pp. 609–614, 2008. [9] E. M. Frohman, M. G. Dwyer, T. Frohman et al., “Relationship of optic nerve and brain conventional and non-conventional MRI measures and retinal nerve ﬁber layer thickness, as as- sessed by OCT and GDx: a pilot study,” Journal of the Neu- rological Sciences, vol. 282, no. 1-2, pp. 96–105, 2009. [25] M. S. Zaveri, A. Conger, A. Salter et al., “Retinal imaging by laser polarimetry and optical coherence tomography evidence of axonal degeneration in multiple sclerosis,” Archives of Neu- rology, vol. 65, no. 7, pp. 924–928, 2008. Multiple Sclerosis International 10 multiple sclerosis patients,” Canadian Journal of Ophthalmol- ogy, vol. 45, no. 5, pp. 520–526, 2010. [26] S. B. Syc, S. Saidha, S. D. Newsome et al., “Optical coherence tomography segmentation reveals ganglion cell layer pathol- ogy after optic neuritis,” Brain, vol. 135, no. 2, pp. 521–533, 2012. [43] A. P. D. Henderson, S. A. Trip, P. G. Schlottmann et al., “A preliminary longitudinal study of the retinal nerve ﬁber layer in progressive multiple sclerosis,” Journal of Neurology, vol. 257, no. 7, pp. 1083–1091, 2010. [27] A. P. D. Henderson, D. R. Altmann, S. A. Trip et al., “Early factors associated with axonal loss after optic neuritis,” Annals of Neurology, vol. 70, no. 6, pp. 955–963, 2011. [44] G. V. McDonnell and S. A. Hawkins, “Clinical study of pri- mary progressive multiple sclerosis in Northern Ireland, UK,” Journal of Neurology Neurosurgery and Psychiatry, vol. 64, no. 4, pp. 451–454, 1998. [28] E. Garcia-Martin, V. Pueyo, J. R. Ara et al., “Eﬀect of optic neuritis on progressive axonal damage in multiple sclerosis patients,” Multiple Sclerosis, vol. 17, no. 7, pp. 830–837, 2011. [29] P. Albrecht, M. Ringelstein, A.-M. M¨uller et al., “Degeneration of retinal layers in multiple sclerosis subtypes quantiﬁed by optical coherence tomography,” Multiple Sclerosis Journal. In press. [45] U. Rot and A. References [35] M. Bock, A. U. Brandt, J. D¨orr et al., “Time domain and spectral domain optical coherence tomography in multiple sclerosis: a comparative cross-sectional study,” Multiple Scle- rosis, vol. 16, no. 7, pp. 893–896, 2010. [51] U. E. K. Wolf-Schnurrbusch, L. Ceklic, C. K. Brinkmann et al., “Macular thickness measurements in healthy eyes using six diﬀerent optical coherence tomography instruments,” Investigative Ophthalmology and Visual Science, vol. 50, no. 7, pp. 3432–3437, 2009. [36] S. B. Syc, C. V. Warner, G. S. Hiremath et al., “Reproducibility of high-resolution optical coherence tomography in multiple sclerosis,” Multiple Sclerosis, vol. 16, no. 7, pp. 829–839, 2010. [52] B. B. Tan, M. Natividad, K.-C. Chua, and L. W. Yip, “Com- parison of retinal nerve ﬁber layer measurement between 2 spectral domain oct instruments,” Journal of Glaucoma, vol. 21, no. 4, pp. 266–273, 2012. [37] N. Serbecic, F. Aboul-Enein, S. C. Beutelspacher et al., “High resolution spectral domain optical coherence tomography (SD-OCT) in multiple sclerosis: the ﬁrst follow up study over two years,” PLoS One, vol. 6, no. 5, Article ID e19843, 2011. [53] L. S. Talman, E. R. Bisker, D. J. Sackel et al., “Longitudinal study of vision and retinal nerve ﬁber layer thickness in multi- ple sclerosis,” Annals of Neurology, vol. 67, no. 6, pp. 749–760, 2010. [38] C. V. Warner, S. B. Syc, A. M. Stankiewicz et al., “The impact of utilizing diﬀerent optical coherence tomography devices for clinical purposes and in multiple sclerosis trials,” PLoS One, vol. 6, no. 8, Article ID e22947, 2011. [39] C. H. Polman, S. C. Reingold, G. Edan et al., “Diagnostic cri- teria for multiple sclerosis: 2005 Revisions to the ’McDonald Criteria’,” Annals of Neurology, vol. 58, no. 6, pp. 840–846, 2005. [40] F. D. Lublin and S. C. Reingold, “Deﬁning the clinical course of multiple sclerosis: results of an international survey,” Neurology, vol. 46, no. 4, pp. 907–911, 1996. [41] J. F. Kurtzke, “Rating neurologic impairment in multiple scle- rosis: an expanded disability status scale (EDSS),” Neurology, vol. 33, no. 11, pp. 1444–1452, 1983. [42] F. Costello, W. Hodge, Y. I. Pan, E. Eggenberger, and M. S. Freedman, “Using retinal architecture to help characterize
https://openalex.org/W4281639438	https://rbc.inca.gov.br/index.php/revista/article/download/1961/1636	Portuguese	null	Linfoma de Burkitt Primário de Mama: Relato de Caso	Revista Brasileira de Cancerologia	2,022	cc-by	3,388	ABSTRACT d Introducción: El linfoma primario de mama (LMP) representa aproximadamente el 0,5% de los cánceres de mama, siendo considerado un tipo de tumor poco común. Algunos de los tipos de LPM, a su vez, tienen una conexión intensa con el embarazo y el posparto debido a la estimulación hormonal. El objetivo de este estudio es reportar un caso de LMP con rara presentación de linfoma de Burkitt, considerando propuestas terapéuticas efectivas para el seguimiento. Relato del caso: Paciente, 23 años, con un tumor periareolar en la mama derecha con aspecto de piel de naranja y rápido crecimiento durante un mes, quejas flogísticas en el sitio de la lesión, buen estado general y ningún otro. síntomas asociados. La condición reportada comenzó siete meses después de la expulsión fetal con un feto muerto. El paciente fue sometido a biopsia de la lesión y fue derivado sin éxito a seguimiento ambulatorio, requiriendo el retorno al entorno hospitalario por empeoramiento de la clínica y extensión del tumor. Hubo un diagnóstico de linfoma de Burkitt, con tratamiento multidisciplinario. Se sometió al protocolo de quimioterapia CODOX-M y murió a los 22 días de seguimiento hospitalario. Conclusión: Este informe demuestra una situación poco común en una paciente joven, enfatizando la importancia de investigar de manera efectiva los cambios en los senos para un diagnóstico temprano correcto y un tratamiento adecuado en todos los grupos de edad. Palabras clave: linfoma de Burkitt/tratamiento farmacológico; linfoma de Burkitt/radioterapia; neoplasias de la mama/tratamiento farmacológico; neoplasias de la mama/radioterapia; informes de casos. Introduction: Primary breast lymphoma (PML) represents about 0.5% of breast cancers, being considered a rare type of tumor. Some of the types of PML, in turn, have an intense connection with the pregnancy and postpartum period due to hormonal stimulation. The aim of this study is to report a case of PML with a rare presentation of Burkitt’s lymphoma, considering effective therapeutic proposals for follow-up. Case report: A 23-year-old female patient with a peri-areolar tumor in the right breast with orange peel aspect and rapid growth for one month, phlogistic complaints at the lesion site, good general condition and no other associated symptoms. The reported condition started seven months after fetal expulsion with a dead fetus. The patient underwent lesion biopsy and was referred to an outpatient follow-up with unsuccessful outcome, requiring return to the hospital due to worsening of clinical conditions and extension of the tumor. RESUMO Introdução: O linfoma primário de mama (LPM) representa cerca de 0,5% das neoplasias mamárias, sendo considerado um tipo raro de tumor. Alguns dos tipos de LPM, por sua vez, possuem ligação intensa com o período gravídico e pós-parto em virtude do estímulo hormonal. O objetivo deste estudo é relatar um caso de LPM com apresentação rara de linfoma de Burkitt, considerando propostas terapêuticas eficazes para o seguimento. Relato do caso: Paciente do sexo feminino, 23 anos, portadora de tumoração periareolar em mama direita com aspecto de casca de laranja e crescimento rápido há um mês, queixas flogísticas no local da lesão, bom estado geral e sem outros sintomas associados. O quadro relatado iniciou-se sete meses após a expulsão fetal com feto morto. A paciente foi submetida à biópsia da lesão e encaminhada para seguimento ambulatorial sem sucesso, necessitando de retorno ao ambiente hospitalar por piora das condições clínicas e extensão da tumoração. Houve diagnóstico de linfoma de Burkitt, com tratamento multidisciplinar, sendo submetida a protocolo CODOX-M de quimioterapia, com óbito após 22 dias de acompanhamento hospitalar. Conclusão: Este relato demonstra uma situação rara em uma paciente jovem, ressaltando a importância de investigar as alterações mamárias, de maneira eficaz, para um diagnóstico precoce correto e um tratamento adequado, em todas as faixas etárias. Palavras-chave: linfoma de Burkitt/tratamento farmacológico; linfoma de Burkitt/radioterapia; neoplasias da mama/tratamento farmacológico; neoplasias da mama/radioterapia; relatos de casos. 1Universidade Iguaçu (Unig). Itaperuna (RJ), Brasil. 1-6Hospital São José do Avaí. Itaperuna (RJ), Brasil. 1E-mail: lais.gomees@hotmail.com. Orcid iD: https://orcid.org/0000-0003-4084-4040 2E-mail: isabela_nagime8@hotmail.com. Orcid iD: https://orcid.org/0000-0003-3352-9982 3E-mail: brunoacsoare@hotmail.com. Orcid iD: https://orcid.org/0000-0003-4085-3254 4E-mail: camilafncaldas@gmail.com. Orcid iD: https://orcid.org/0000-0002-9645-0393 5E-mail: ximenes_luciana@hotmail.com. Orcid iD: https://orcid.org/0000-0001-9066-8124 6E-mail: rmartinsc@uol.com.br. Orcid iD: https://orcid.org/0000-0001-9828-159X Endereço para correspondência: Laís Gomes Ferreira. Rua Padre Humberto Lindelauf, 374/202 - Cidade Nova. Itaperuna (RJ), Brasil. CEP 28300-000. E-mail: lais.gomees@hotmail.com Linfoma de Burkitt Primário de Mama: Relato de Caso doi: https://doi.org/10.32635/2176-9745.RBC.2022v68n2.1961 Primary Breast Burkitt Lymphoma: Case Report Linfoma de Burkitt Primario de Mama: Relato de Caso Laís Gomes Ferreira1; Isabela Nagime Barros Gomes2; Bruno de Almeida Castro Soares3; Camila Fleckner Navarro Rodrigues Caldas4; Luciana Ximenes Bonani Alvim Brito5; Rogério Martins de Castro6 Laís Gomes Ferreira1; Isabela Nagime Barros Gomes2; Bruno de Almeida Castro Soares3; Camila Fleckner Navarro Rodrigues Caldas4; Luciana Ximenes Bonani Alvim Brito5; Rogério Martins de Castro6 de Mama: Relato RELATO DE CASO de Mama: Relato RELATO DE CASO INTRODUÇÃO principalmente região periareolar de mama direita, com um mês de evolução e aumento progressivo (Figura 1). De acordo com a história pregressa, a paciente relatava óbito fetal há sete meses, sendo internada e submetida à biópsia da lesão mamária com inserção de dreno de penrose em um procedimento sem intercorrências, com alta médica subsequente e ferida cirúrgica em bom aspecto (Figura 2). A paciente deu seguimento ambulatorial com antibioticoterapia para acompanhamento e realização de exames ginecológicos, bem como para aguardar o resultado anatomopatológico da biópsia mamária. O câncer de mama é uma neoplasia com alta morbimortalidade no sexo feminino. Consiste na neoplasia maligna mais comum entre as mulheres no Brasil e no mundo – depois do câncer de pele não melanoma – e responde por cerca de 25% dos novos casos de câncer a cada ano. A doença também pode afetar homens, em uma condição rara, sendo apenas 1% do total de casos da doença1. Entre os tipos histológicos dessa neoplasia, os linfomas compreendem um pequeno número de casos1,2. O linfoma definido como foco primário de mama, ou seja, doença de mama como manifestação inicial, por sua vez, apresenta raridade ainda maior, podendo chegar a 0,5% de todos os cânceres de mama2-5. Figura 1. Tumoração mamária direita Na patologia em questão, o acometimento de linfonodos axilares ipsilaterais não é regra, podendo estar presente ou não5. Na maioria dos casos, o subtipo histológico dos linfomas primários de mama (LPM) é não Hodgkin2,4,5. O linfoma de Burkitt é um outro subtipo tumoral altamente agressivo e apresenta de 10,3% de todos os LPM6-8. A origem neoplásica dos LPM é nos tecidos linfoides periductal e perilobular da mama, podendo estar associado com o tecido linfoide junto às mucosas1,6,9. O gatilho para que isso ocorra pode ser um estímulo hormonal, especialmente em período gravídico ou puerperal2,3,6,8. Em razão da raridade desses tipos tumorais, não há uma padronização terapêutica descrita. Contudo, a quimioterapia específica para o tipo histológico e a radioterapia podem ser consideradas como métodos de bom resultado. Cirurgias terapêuticas têm descrição divergentes na literatura, ao contrário das cirurgias diagnósticas para biópsia que são amplamente utilizadas1,5,7. Figura 1. Tumoração mamária direita Figura 1. Tumoração mamária direita Figura 2. Mama após biópsia de lesão suspeita O objetivo deste estudo foi descrever um caso de LPM com apresentação rara de linfoma de Burkitt. ABSTRACT d There was a diagnosis of Burkitt’s lymphoma, with multidisciplinary treatment. She underwent the CODOX-M chemotherapy protocol, and died after 22 days of hospital follow-up. Conclusion: This report demonstrates a rare situation in a young patient, emphasizing the importance of effective investigation of breast changes so that correct early diagnosis and appropriate treatment can be made for all age groups. Key words: Burkitt lymphoma/drug therapy; Burkitt lymphoma/ radiotherapy; breast neoplasms/drug therapy; breast neoplasms/ radiotherapy; case reports. 1Universidade Iguaçu (Unig). Itaperuna (RJ), Brasil. 1-6Hospital São José do Avaí. Itaperuna (RJ), Brasil. 1E-mail: lais.gomees@hotmail.com. Orcid iD: https://orcid.org/0000-0003-4084-4040 2E-mail: isabela_nagime8@hotmail.com. Orcid iD: https://orcid.org/0000-0003-3352-9982 3E-mail: brunoacsoare@hotmail.com. Orcid iD: https://orcid.org/0000-0003-4085-3254 4E-mail: camilafncaldas@gmail.com. Orcid iD: https://orcid.org/0000-0002-9645-0393 5E-mail: ximenes_luciana@hotmail.com. Orcid iD: https://orcid.org/0000-0001-9066-8124 6E-mail: rmartinsc@uol.com.br. Orcid iD: https://orcid.org/0000-0001-9828-159X Endereço para correspondência: Laís Gomes Ferreira. Rua Padre Humberto Lindelauf, 374/202 - Cidade Nova. Itaperuna (RJ), Brasil. CEP 28300-000. E-mail: lais.gomees@hotmail.com Este é um artigo publicado em acesso aberto (Open Access) sob a licença Creative Commons Attribution, que permite uso, distribuição e reprodução em qualquer meio, sem restrições, desde que o trabalho original seja corretamente citado. Revista Brasileira de Cancerologia 2022; 68(2): e-151961 Ferreira LG, Gomes INB, Soares BAC, Caldas CFNR, Brito LXBA, Castro RM INTRODUÇÃO Além disso, relatar o seguimento clínico da paciente, incluindo propostas terapêuticas, na intenção de aprimorar o conhecimento sobre o câncer de mama e suas formas menos comuns e agressivas, principalmente em momentos de maior sobrecarga hormonal, como no período gravídico-puerperal. É O estudo foi aprovado pelo Comitê de Ética em Pesquisa do Hospital São José do Avaí, em Itaperuna/RJ (CAAE 47226121.8.0000.5288) em 15/4/2021. Revista Brasileira de Cancerologia 2022; 68(2): e-151961 DISCUSSÃO O linfoma maligno da mama é um diagnóstico raro, podendo se manifestar de forma primária ou secundária como parte de um processo metastático3. O caso discutido neste estudo chama a atenção por se tratar de uma paciente apresentando tumoração mamária após estímulo hormonal intenso decorrente do período gravídico-puerperal e sem alterações no restante da avaliação clínica8. Isso remete à hipótese de uma patologia primária. A idade apresentada no estudo difere da média comum de apresentação dos casos de LPM, que, em sua grande maioria, acometem pacientes em idades mais avançadas1. Contudo, está tipicamente representado por uma mulher jovem, sendo compatível com o padrão do linfoma de Burkitt4. Sete dias após admissão, foi realizada videolaparoscopia diagnóstica evidenciando liquido livre em cavidade com coleta para análise. Pela hematologia, diagnosticou-se linfoma de Burkitt, com solicitação de exames laboratoriais para estadiamento, avaliação de liquor e biópsia de medula óssea. Iniciou-se tratamento quimioterápico após 15 dias da admissão atual com o protocolo CODOX-M, envolvendo uso de doxorrubicina e vincristina, ciclofosfamida, metotrexate, leucovorin e fator estimulador de colônias de granulócitos (G-CSF). Além disso, foi proposta quimioterapia intratecal com citarabina, metotrexate e dexametasona. Esse protocolo foi iniciado mesmo na ausência dos resultados dos exames solicitados previamente por causa da piora clínica da paciente. Os critérios diagnósticos propostos para LPM incluem: 1) a mama deve ser o sítio inicial de apresentação da neoplasia; 2) ausência de história pregressa de linfoma; 3) ausência de doença neoplásica disseminada ao diagnóstico; 4) associação do linfoma com o tecido mamário na análise histopatológica; 5) possibilidade de acometimento de linfonodos axilares, desde que de forma simultânea aos mamários1,4,10,11. Após início de quimioterapia, a paciente se apresentou sonolenta e taquipneica, mantendo alteração no hemograma, porém com melhora da leucocitose (3.700). Evidenciaram-se valores extremamente elevados de potássio (6,5) e baixos de cálcio (0,95). A paciente evoluiu com quadro de acidose metabólica crítica e massa palpável em hemiabdome direito. No quarto dia de protocolo, foi observada melhora da tumoração mamária, com controle das intercorrências previamente expostas e quadro de pancitopenia e síndrome da lise tumoral. No quinto dia, evoluiu com quadro grave de neutropenia, plaquetopenia e uremia, expressando encefalopatia urêmica/séptica, crise convulsiva, hipocalcemia, sepse de foco urinário e sangramento gengival. Por esses motivos, houve suspensão do esquema quimioterápico até realização de diálise, melhora clínica e reavaliação da nefrostomia por serviço de hemodinâmica. Diante do exposto, o diagnóstico clínico presuntivo foi de neoplasia mamária. Linfoma de Burkitt Primário de Mama: Relato de Caso Linfoma de Burkitt Primário de Mama: Relato de Caso Linfoma de Burkitt Primário de Mama: Relato de Caso Linfoma de Burkitt Primário de Mama: Relato de Caso Em janeiro de 2020, retornou ao ambiente hospitalar com laudo de neoplasia indeterminada de mama e neoplasia do colo uterino avançado, apresentando piora do estado geral e exames laboratoriais alterados, enfatizando uma leucocitose (19.200) com presença de bastões e metamielócitos, além de anemia e função renal prejudicada. Ademais, a paciente apresentou injúria renal aguda (IRA), consequente a uma compressão ureteral pela tumoração pélvica. Tal mecanismo desencadeou hidronefrose e dilatação pielocalicial, sendo indicada nefrostomia esquerda pelo serviço de cirurgia geral. A paciente mantinha estabilidade hemodinâmica e apresentou hemograma alterado. Depois de 22 dias da admissão atual e três dias após a suspensão de quimioterapia, a paciente encontrava-se em estado grave com múltiplas disfunções orgânicas, choque séptico pulmonar, instabilidade hemodinâmica com aminas em doses elevadas, coagulopatia, IRA, acidose mista refratária à ventilação mecânica e bicarbonato (HCO3), sangramento oral e de esclera, evoluindo a óbito após três episódios de parada cardiorrespiratória por hipoxemia e acidose mista. RELATO DO CASO Paciente do sexo feminino, negra, 23 anos, admitida no Serviço de Ginecologia Oncológica do Hospital São José do Avaí – referência da Região Noroeste Fluminense – situado no município de Itaperuna/RJ, em dezembro de 2019, em virtude de uma tumoração de grande volume, apresentando aspecto de casca de laranja, envolvendo Figura 2. Mama após biópsia de lesão suspeita Revista Brasileira de Cancerologia 2022; 68(2): e-151961 2 Revista Brasileira de Cancerologia 2022; 68(2): e-151961 DISCUSSÃO Isso se deve à semelhança entre a maioria das malignidades de mama1. A clínica específica de linfoma de Burkitt possui particularidades como massa dolorosa, geralmente no quadrante superior lateral com inflamação local e evolução rápida6. Para confirmação diagnóstica, orientou-se a investigação por meio de biópsia mamária e a condução de uma avaliação ginecológica complementar, a fim de avaliar outros órgãos passiveis de acometimentos secundários2,8. Isso tudo auxilia no diagnóstico histopatológico e no estabelecimento de prognóstico das pacientes acometidas. No caso relatado, à colpocitologia, foi evidenciada neoplasia do colo uterino avançada, provavelmente secundária ao processo inicial, além de um envolvimento sistêmico destacado pelos exames laboratoriais. A paciente evoluiu para instabilidade hemodinâmica com necessidade de intubação orotraqueal por hipoxemia e administração de aminas endovenosas. Apesar do quadro hemodinâmico, apresentou melhora progressiva da tumoração mamária, abdome com edema de parede à direita, petéquias no tronco e má perfusão de extremidades. À radiografia de tórax, houve hipótese de síndrome da angústia respiratória do adulto (SARA). A avaliação complementar inicial pode incluir imagem por mamografia, ressonância nuclear magnética, ultrassonografia e tomografia computadorizada por 3 Revista Brasileira de Cancerologia 2022; 68(2): e-151961 Ferreira LG, Gomes INB, Soares BAC, Caldas CFNR, Brito LXBA, Castro RM Ferreira LG, Gomes INB, Soares BAC, Caldas CFNR, Brito LXBA, Castro RM emissão de pósitrons (PET-TC)6. Contudo, não há presença de achados patognomônicos, e as alterações encontradas variam de massas e alterações vasculares a comprometimento ganglionar6,11. Em razão da inespecificidade, todos os achados devem possuir correlação clinicorradiológica11. terapêutica. Porém, o mais preconizado pelas literaturas atuais inclui quimioterapia individualizada e específica para o tipo histológico, podendo ser associado à radioterapia da mama acometida. Inclui-se ainda a profilaxia do SNC na terapêutica de formas agressivas de LPM, mesmo que em estágios iniciais, pois isso pode melhorar o resultado e reduzir o risco de recaída do SNC. Neste estudo, notou-se, à ultrassonografia abdominal, que a neoplasia metastática do colo do útero promoveu uma compressão ureteral e desencadeou hidronefrose e posterior IRA. Mesmo que sem relação direta, as repercussões sistêmicas do LPM puderam ser esclarecidas por uma abordagem holística. A avaliação para a necessidade cirúrgica é controversa, uma vez que se discute não haver benefícios da mastectomia em relação ao risco de sobrevivência e recorrência do LPM. Conclui-se, portanto, que o linfoma de Burkitt é um subtipo tumoral de LPM com comprometimento altamente agressivo e consequentemente apresenta um prognóstico mais reservado. DISCUSSÃO O seguimento para essas pacientes exige abordagem mais invasiva e conforme as repercussões sistêmicas apresentadas pela neoplasia. Após diagnosticar a patologia e começar a pensar no manejo terapêutico, deve-se entender que o tratamento de LPM é variável na literatura11. A terapia combinada com quimioterapia e radioterapia é o tratamento mais difundido e com resultados positivos. A cirurgia terapêutica como a mastectomia não oferece benefícios em relação à sobrevivência e/ou recorrência da doença4. A única conduta que possui relação com status axilar da paciente é a indicação da quimioterapia. Este caso demonstra uma situação rara em uma paciente jovem, ressaltando a importância de investigar as alterações mamárias de maneira eficaz para que seja realizado um diagnóstico precoce correto e um tratamento adequado, em todas as faixas etárias. Todavia, ao se tratar de formas agressivas, como no linfoma de Burkitt, a quimioprofilaxia de sistema nervoso central (SNC) é a indicação majoritária mesmo que em estágios iniciais. Tal conduta indica melhor resultado e reduz significativamente o risco de recaída do SNC2. O estudo foi de grande relevância para os acadêmicos e profissionais médicos dessa instituição, pois proporcionou aprimorar o conhecimento sobre o câncer de mama e suas formas menos comuns e agressivas, incentivar a prática do autoexame das mamas e rastreamento, sobretudo nos momentos oportunos de maior sobrecarga hormonal como no período gravídico-puerperal, conscientizando as mulheres sobre o diagnóstico precoce e melhor desfecho. Foi também muito importante para o ensino e a pesquisa, pois trouxe subsídios para que outros pesquisadores possam aprofundar mais o tema proposto, sugerindo terapêuticas consolidadas e totalmente eficazes para melhora do prognóstico. i Para a avaliação do prognóstico dos linfomas de Burkitt, relacionou-se o tipo histológico ao grau de comprometimento linfonodal e aos fatores de risco apresentados por cada paciente7. Na paciente em questão, a proposta foi iniciar quimioterapia sistêmica associada à quimioterapia intratecal, com o objetivo de melhorar a sobrevida da paciente, inibir o crescimento tumoral, reduzir o risco de acometimento do SNC, para que fosse possível avaliar a melhor resposta e dar seguimento ao protocolo. E por causa do tipo histológico agressivo, sendo desenvolvido após uma descarga hormonal intensa, pensou-se em um prognóstico reservado com a necessidade de uma terapêutica mais agressiva. CONTRIBUIÇÕES Laís Gomes Ferreira, Isabela Nagime Barros Gomes, Bruno de Almeida Castro Soares e Camila Fleckner Navarro Rodrigues Caldas contribuíram na concepção e/ou no planejamento do estudo; na obtenção, análise e interpretação dos dados; na redação e revisão crítica. Luciana Ximenes Bonani Alvim Brito e Rogério Martins de Castro contribuíram na revisão crítica. Todos os autores aprovaram a versão final a ser publicada. Ao se discutir o caso, percebe-se a importância de um maior embasamento científico quanto à propedêutica do LPM ao se manifestar como linfoma de Burkitt pela agressividade e raridade da neoplasia. DECLARAÇÃO DE CONFLITO DE INTERESSES Os LPM são neoplasias raras que apresentam crescimento rápido, progressivo e prognóstico reservado. O diagnóstico de certeza consiste em biópsia dirigida e análise imuno-histoquímica. Em razão da raridade desses tipos tumorais, ainda não há uma padronização Nada a declarar. Revista Brasileira de Cancerologia 2022; 68(2): e-151961 Linfoma de Burkitt Primário de Mama: Relato de Caso Linfoma de Burkitt Primário de Mama: Relato de Caso Revista Brasileira de Cancerologia 2022; 68(2): e-151961 FONTES DE FINANCIAMENTO Não há. Revista Brasileira de Cancerologia 2022; 68(2): e-151961 REFERÊNCIAS 11. Jennings WC, Baker RS, Murray SS, et al. Primary breast lymphoma: the role of mastectomy and the importance of lymph node status. Ann Surg. 2007;245(5):784-9. doi: https://doi.org/10.1097/01.sla.0000254418.90192.59 11. Jennings WC, Baker RS, Murray SS, et al. Primary breast lymphoma: the role of mastectomy and the importance of lymph node status. Ann Surg. 2007;245(5):784-9. doi: https://doi.org/10.1097/01.sla.0000254418.90192.59 1. Scheliga AAS, Reinert T, Santos ALS, et al. Linfoma primário da mama: apresentação clínica e características histopatológicas e moleculares. Rev Bras Oncol Clín [Internet]. 2012 [acesso 2020 abr 15];8(28):79-87. Disponível em: https://www.sboc.org.br/sboc-site/ revista-sboc/pdfs/28/artigo4.pdf 2. Joks M, Myśliwiec K, Lewandowski K. Primary breast lymphoma - a review of the literature and report of three cases. Arch Med Sci. 2011;7(1):27-33. doi: https://doi. org/10.5114/aoms.2011.20600 3. Surov A, Holzhausen HJ, Wienke A, et al. Primary and secondary breast lymphoma: prevalence, clinical signs and radiological features. Br J Radiol. 2012;85(1014):e195- 205. doi: https://doi.org/10.1259/bjr/78413721 4. Avenia N, Sanguinetti A, Cirocchi R, et al. Primary breast lymphomas: a multicentric experience. World J Surg Oncol. 2010;8:53. doi: https://doi.org/10.1186/1477- 7819-8-53 5. Gonçalves JTF, Giordani RR, Lima PL, et al. Linfoma primário de mama: relato de caso. Rev Bras Mastologia [Internet]. 2011 [acesso 2020 maio 22];21(4):70-2. Disponível em: https://www.mastology.org/wp-content/ uploads/2015/06/MAS_v21n4_178-180.pdf 6. Vallejo Díaz JF, Rodríguez R, Orduz R, et al. Linfoma de Burkitt primario de mama. Presentación de un caso. Rev Colomb Radiol [Internet]. 2014 [acesso 2020 abr 19];25(3):4036-9. Disponible en: http:// contenido.acronline.org/Publicaciones/RCR/RCR25- 3/11_Linfoma.pdf 7. Jeanneret-Sozzi W, Taghian A, Epelbaum R, et al. Primary breast lymphoma: patient profile, outcome and prognostic factors. A multicentre rare cancer network study. BMC Cancer. 2008;8:86. doi: https://doi. org/10.1186/1471-2407-8-86 8. Horowitz NA, Benyamini N, Wohlfart K, et al. Reproductive organ involvement in non-Hodgkin lymphoma during pregnancy: a systematic review. Lancet Oncol. 2013;14(7):e275-e82. doi: https://doi. org/10.1016/S1470-2045(12)70589-2 9. Yi JI, Chae BJ, Bae JS, et al. Bilateral primary breast lymphoma. Chin Med J [Internet]. 2010 [cited 2020 Jun 10];123(11):1482-4. Available from: https://journals. lww.com/cmj/Fulltext/2010/06010/Bilateral_primary_ breast_lymphoma.28.aspx 10. Wiserman C, Liao KT. Primary lymphoma of the breast. 1972;29(6):1705-12. doi: https://doi. org/10.1002/1097-0142(197206)29:6<1705::AID- CNCR2820290640>3.0.CO;2-I Editora-científica: Anke Bergmann. Orcid iD: https://orcid.org/0000-0002-1972-8777 Revista Brasileira de Cancerologia 2022; 68(2): e-151961
https://openalex.org/W4299844030	https://dergipark.org.tr/tr/download/article-file/304784	Latin	null	Efficiency analysis of Turkey’s Road Transportation System Using Data Envelopment Method	DergiPark (Istanbul University)	2,015	cc-by	7,185	EFFICIENCY ANALYSIS OF TURKEY’S ROAD TRANSPORTATION SYSTEM USING DATA ENVELOPMENT METHOD Fatih Çipil Emniyet Genel Müdürlüğü, Konya Keywords: Transportation, Efficiency, Road, Performance, DEA Gazi Mühendislik Bilimleri Dergisi ABSTRACT This study investigates whether Turkey utilizes its road transportation indicators efficiently or not within the framework of the European Union (EU) accession process. In addition, it aims to demonstrate Turkey’s current position compared to other EU countries by performing relative efficiency analysis on road transportation indicators. Data Envelopment Analysis, which is among efficiency measuring methods and used in this research, is a non‐parametric method that is based on linear programming principles. In the analysis, three different models were formed in order to determine the dimensions of different road transportation parameters with the aim of minimizing the number of accidents and the number of casualties in accidents. As a candidate for the EU membership, Turkey’s road transportation performance was demonstrated in this study using Data Envelopment Analysis in comparison to members (27 countries whose data were fully accessed) of the EU, which requires its members of fulfilling certain criteria and targets. Gazi Mühendislik Bilimleri Dergisi F. Çipil 1/1 (2015) 143‐172 144 Gazi Journal of Engineering Sciences ÖZET Bu çalışmada Avrupa Birliği'ne katılım sürecinde Türkiye'nin karayolu ulaşım göstergelerini etkin olarak kullanıp kullanmadığı araştırılmıştır. Buna ilaveten karayolu ulaşım göstergelerinin bağıl performansıyla Avrupa Birliği ülkeleri arasındaki konumu gösterilmiştir. Çalışmada parametrik olmayan lineer programlama özellikleri olan Veri Zarflama Analizi kullanılmıştır. Analizde, üç farklı model oluşturulmuştur: farklı yol taşımacılığı parametrelerinin boyutlarını belirlemek, kazaların sayısını en aza indirmek ve kazalarda yaralıların sayısını minimize etmek. AB için bir aday olarak Türkiye'nin karayolu taşımacılığı performansı 25 Avrupa Ülkesine göre karşılaştırılmıştır. Anahtar Kelimeler: Ulaştırma, Etkinlik, Karayolu, Performans, Veri Zarflama Gazi Journal of Engineering Sciences F. Çipil 1/1 (2015) 143‐172 145 1. INTRODUCTION Traffic is among the prioritized issues for not only Turkey but also other countries. According to the European Transport Safety Council (ETSC), traffic accidents are the main cause of death in all EU countries for those aged 45 and below. It is indicated in various reports published by World Bank that Turkey loses around 2% of its Gross National Product (GNP) every year in traffic accidents, which suggests that the annual socioeconomic cost of traffic accidents in Turkey is nearly 10 billion dollars [1,2]. Number of vehicles per capita, which is among the indicators of economic welfare and development, has reached its peak density especially in cities in recent years. This situation has unsurprisingly increased traffic problems in and out of towns. Given the fact that the volume of road transportation in Turkey is high, Turkey is among the countries with the highest number of traffic accidents. Statistics indicate that over 4000 people are killed and over 200,000 people are injured in traffic accidents every year in Turkey on average. In order to attain a stable performance in preventing accidents and establishing road safety; the characteristics of vehicles, drivers and roads, which are the three main components of the transportation system, need to be addressed as a whole. More than one factor have an impact on the frequency and violence of traffic accidents such as driver behaviors, characteristics of vehicles and roads, environmental factors and traffic characteristics. The causes of traffic accidents in Turkey are distributed as follows: 96,82% drivers, 2,38% pedestrians, 0,16% passengers, 0,32% vehicles, and 0,32% roads. These statistics suggest that the human factor is responsible for traffic accidents by 99,36% [3]. Traffic safety is among the issues prioritized by Turkey and other countries in the world. In the EU summit held in Helsinki on 10‐11 December 1999, Turkey was admitted to candidacy having equal rights with other candidate countries. Thus began Turkey’s accession process to the EU. With this development, Turkey would be required to make extensive adjustments in numerous areas including the transportation industry for a long period. The transportation sector is among the areas on which the creation and implementation of a common Gazi Mühendislik Bilimleri Dergisi Gazi Mühendislik Bilimleri Dergisi F. Çipil 1/1 (2015) 143‐172 146 policy was put as an obligation in the Rome Treaty. 1. INTRODUCTION Since the Rome Treaty, which is the foundation of the EU, the primary policy that has been pursued by members is the free circulation of individuals and products. These countries make significant efforts to alleviate the obstacles in their borders. The main components of the common transportation policy addressed within this framework were defined as reducing employment, protecting the environment, being open to competition and providing the best service to the customer with the largest perspective. The main purpose of transportation is to provide the service necessitated by the economic and social targets of national development; with the most convenient qualities demanded by the user; in a way to satisfy national security requirements; in a safe and environmentally‐ friendly manner; for the cheapest price; using contemporary technologies; and in harmony with international regulations and EU policies; continuously and without any interruption [4]. It is aimed in this study to determine Turkey’s position among EU member countries and to define its pros and cons in terms of road transportation indicators. This way, it is believed that this study will contribute to the efforts towards harmonization. Therefore, a relative efficiency analysis was conducted using the method of Data Envelopment Analysis (DEA) for 27 EU member countries and Turkey by considering their transportation and traffic structures. Thus, Turkey’s position among EU member countries with respect to traffic indicators was determined. Based on the concrete findings, the policies that Turkey should develop in order to increase harmonization with the transportation and traffic structures of EU member countries were discussed. Gazi Journal of Engineering Sciences 1.1. TRAFFIC IN TURKEY As in all other countries, traffic accidents in Turkey are among the top problems. According to a research by the World Bank, the socioeconomic cost of traffic accidents in Turkey constitutes 2,2% of the entire GNP [5]. Traffic indicators by years based on the data provided by Turkish Statistics Institute (TUIK) are given in Figure 1 and Table 1. In recent years, numbers of drivers and vehicles as well as the number of accidents are on the rise. However, deaths caused by accidents slightly declined; which might have F. Çipil 1/1 (2015) 143‐172 147 stemmed from the fact that most new vehicles are automobiles whose equipments have been designed with an increased focus on safety (Table 2). Table 1.Traffic Indicators Year Number of Motor Vehicles Increase (%) Population Increase (%) Number of Vehicles per 1000 people Number of Driver’s Licences Increase (%) Number of Accidents Increase (%) 2005 11145826 8,88 72065000 1,28 155 16604724 2,81 621183 15,59 2006 12227393 9,70 72987400 1,28 168 17962895 8,17 728756 17,31 2007 13022945 6,51 70586256 ‐3,29 184 18877354 5,09 825583 13,29 2008 13765395 5,70 71517100 1,32 192 19924442 5,55 942105 14,11 2009 14316700 3,85 72561312 1,46 197 20460739 2,69 1050011 11,44 G i Müh di lik Bili l i D i i Table 1.Traffic Indicators Year Number of Motor Vehicles Increase (%) Population Increase (%) Number of Vehicles per 1000 people Number of Driver’s Licences Increase (%) Number of Accidents Increase (%) 2005 11145826 8,88 72065000 1,28 155 16604724 2,81 621183 15,59 2006 12227393 9,70 72987400 1,28 168 17962895 8,17 728756 17,31 2007 13022945 6,51 70586256 ‐3,29 184 18877354 5,09 825583 13,29 2008 13765395 5,70 71517100 1,32 192 19924442 5,55 942105 14,11 2009 14316700 3,85 72561312 1,46 197 20460739 2,69 1050011 11,44 Figure 1.Traffic indicators in Turkey Table 1.Traffic Indicators Year Number of Motor Vehicles Increase (%) Population Increase (%) Number of Vehicles per 1000 people Number of Driver’s Licences Increase (%) Number of Accidents Increase (%) 2005 11145826 8,88 72065000 1,28 155 16604724 2,81 621183 15,59 2006 12227393 9,70 72987400 1,28 168 17962895 8,17 728756 17,31 2007 13022945 6,51 70586256 ‐3,29 184 18877354 5,09 825583 13,29 2008 13765395 5,70 71517100 1,32 192 19924442 5,55 942105 14,11 2009 14316700 3,85 72561312 1,46 197 20460739 2,69 1050011 11,44 Figure 1.Traffic indicators in Turkey Figure 1.Traffic indicators in Turkey Gazi Mühendislik Bilimleri Dergisi F. Gazi Journal of Engineering Sciences 1.1. TRAFFIC IN TURKEY Çipil 1/1 (2015) 143‐172 148 Table 2.Distribution of Vehicles on Roads Motorbike Automobile Minibus Bus Light‐duty truck Truck Tractor Special‐Purpose Vehicle Total 2008 2181383 6796629 383548 199934 2066007 744217 1358577 35100 13765395 2009 2303261 7093964 384053 201033 2204951 727302 1368032 34104 14316700 Increase (%) 5,29 4,19 0,13 0,54 6,30 ‐2,27 0,69 ‐0,69 3,85 Table 2.Distribution of Vehicles on Roads Table 3 shows the distributions of both passengers and cargo on roads in Turkey. Figure 2 demonstrates the distribution of passengers and cargo with respect to the system of transportation in Turkey. As Figure 2 suggests, both passenger and cargo transportation in Turkey are performed on roads, and thus, researches should primarily focus on the road transportation industry. Table 3.Road cargo and passenger statistics Year Vehicle x Km (Millions) Ton x Km (Millions) Passenger x Km (Millions) Motorway State Highway Provincial Road Motorway State Highway Provincial Road Motorway State Highway Provincial Road 2005 9466 45818 5845 28504 128343 9964 31606 134681 15865 2006 11528 47055 5994 32926 134361 9265 37994 133608 15991 2007 12727 50459 6423 34452 136967 9911 43873 147694 17548 2008 13131 50255 8385 36925 135607 9403 44394 144378 17326 2009 13908 51932 6592 40515 127211 6729 47481 147253 17730 Table 3.Road cargo and passenger statistics Year Vehicle x Km (Millions) Ton x Km (Millions) Passenger x Km (Millions) Motorway State Highway Provincial Road Motorway State Highway Provincial Road Motorway State Highway Provincial Road 2005 9466 45818 5845 28504 128343 9964 31606 134681 15865 2006 11528 47055 5994 32926 134361 9265 37994 133608 15991 2007 12727 50459 6423 34452 136967 9911 43873 147694 17548 2008 13131 50255 8385 36925 135607 9403 44394 144378 17326 2009 13908 51932 6592 40515 127211 6729 47481 147253 17730 Gazi Journal of Engineering Sciences Gazi Journal of Engineering Sciences F. Çipil 1/1 (2015) 143‐172 149 Figure 2.Distribution of transportation sectors Figure 2.Distribution of transportation sectors The transportation sector constitutes 7% of the EU’s Gross Product, 7% of employment, 40% of member countries’ investments and 30% of energy consumption. Across the EU, in the last twenty years, an annual demand of 2,3% for goods and of 3,1% for passengers emerged on average. Steps towards the liberalization of the EU’s economy such as the elimination of boundaries between countries and the liberalization of maritime transportation have rendered inevitable the creation of a Common Transportation Policy. 1.1. TRAFFIC IN TURKEY These steps are of importance in terms of maintaining growth and overcoming problems such as deadlock and market saturation. 2.1. MATERİAL In this study, DEA, which is a method used in many studies to evaluate performance, was employed in order to determine Turkey’s performance in traffic indicators in comparison with EU member countries. Using the DEA models of CRS (CCR) and VRS (BCC), a section (cross‐section) analysis was performed and efficiency scores were ranked. In the study; the main road traffic indicators were used as variables whereas Turkey and 27 EU member Gazi Mühendislik Bilimleri Dergisi F. Çipil 1/1 (2015) 143‐172 150 countries were regarded as decision making units (DMU) [6]. To determine Turkey’s efficiency, three different efficiency models were formulated taking into consideration nine input and various output variables. These models were analyzed according to their input and output values, as presented in Table 4. Table 4. Input‐output variables used in the analysis Main Road Traffic Indicators MODEL 1 (Input/ Output) MODEL 2 (Input/ Output) MODEL 3 (Input/ Output) Length of Motorways I I I Length of Road Network‐Main or National Roads I I I Length of Road Network‐Secondary or Regional Roads I I I Motorization I I I Passenger Cars‐Stock of Registered Vehicles I I I Buses and Coaches‐Stock of Registered Vehicles I I I Goods Vehicles‐Stock of Registered Vehicles I I I Road share of inland freight transport I I I Passenger Cars‐ New Vehicle Registrations I I I Road Fatalities O Road Fatalities Country Rankings‐Fatalities per Million Inhabitants Road Fatalities Country Rankings‐Fatalities per 10 Billion pkm O Road Fatalities Country Rankings‐Fatalities per Million Passenger Cars O Number of Accidents Involving Personal Injury O Injuries‐Numbers O Table 4. Input‐output variables used in the analysis F. Gazi Mühendislik Bilimleri Dergisi 2.1. MATERİAL Çipil 1/1 (2015) 143‐172 151 The aim here is; The aim here is; Model 1: Determining the efficiency that takes the number of accidents as the output and the main road indicators as the input Model 2: Determining the efficiency that takes the number of casualties on road as the output and the main road indicators as the input Model 3: Determining the efficiency that takes the number of deaths per vehicle and per kilometer as the output and the main road indicators as the input DEA analyses are performed in two directions: DEA analyses are performed in two directions:  Input‐oriented efficiency measures the rate of inputs (main road indicators) that should be used less by decision making units (countries) that are not 100% efficient in order to attain 100% efficiency; when a constant output level is given.  Output‐oriented efficiency measures the rate of outputs that should be produced more by decision making units that are not 100% efficient in order to attain 100% efficiency; when a constant input level is given.  In the input‐oriented approach, if it is not possible to decrease the amount of any input belonging to a DMU without increasing the amount of another input and/or decreasing the amount of an output, this DMU is regarded aspareto‐efficient. In the output‐oriented approach, if it is not possible to decrease the amount of any output belonging to a DMU without decreasing the amount of another output and/or increasing the amount of an input, this DMU is regarded aspareto‐efficient. Figure 3 illustrates the efficiency values calculated oriented towards input and output. Gazi Mühendislik Bilimleri Dergisi Gazi Mühendislik Bilimleri Dergisi 152 F. Çipil 1/1 (2015) 143‐172 Figure 3. Input‐ and output‐oriented efficiency Figure 3. Input‐ and output‐oriented efficiency Here, in measuring the input‐oriented efficiency, both CRS (constant returns to scale) and VRS (variable returns to scale) models were employed. Constant Returns to Scale (CRS) Constant Returns to Scale (CRS)  Here, any radial increase in the input vector creates a radial increase in the output vector at the same rate.  Radial increase: means the increase of all input components at the same rate. Variable/Decreasing/Increasing Returns to Scale (VRS, DRS, IRS)  Variable Returns to Scale  Variable Returns to Scale  It means that increasing, decreasing and constant returns are possible. Gazi Journal of Engineering Sciences 2.1. MATERİAL  Decreasing Returns to Scale  Decreasing Returns to Scale  Decreasing Returns to Scale  Any radial increase in the input vector creates a radial increase in the output vector at a lower rate.  Increasing Returns to Scale  Increasing Returns to Scale  Increasing Returns to Scale Any radial increase in the input vector creates a radial increase in the output vector at a higher rate (Figure4). 153 F. Çipil 1/1 (2015) 143‐172 Figure4. Variable to scale model Figure4. Variable to scale model Scale efficiency measures the difference between the CRS and VRS efficiency scores. The scale efficiency of a DMU is calculated by dividing VRS to CRS efficiency score. A scale efficiency score smaller than 1 point to scale inefficiency; while a DMU is regarded as scale‐efficient if it is equal to 1 (100%) and at the same time CRS and VRS efficiency scores are equal to 1. Thus, it is understood that the above DMUs B and D are technically efficient whereas they are not scale‐efficient. Only the DMU C is scale‐efficient [7]. Constraint on the weights of decision units equates to 1 linear programming model, boundary event "the best observation", and the relative efficiency of the formation of multiple linear combinations allows defining a less strict. For, increasing and decreasing returns to scale are also included in the scale, that is, within the efficiency limits. In general, a comparison of efficiency in the case of constant returns to scale displays a picture with a lower performance; because a DMU needs to have both technical and scale efficiency in order to reach to the efficiency value of "1". In the case of variable returns to scale, on the other hand, if a unit that is not scale efficient has technical efficiency, it can be located over the frontier as the “best observation” [8]. Therefore, it can be stated that the technical efficiency measure for the same DMU is lower in the case of constant returns to scale than in the case of variable returns to scale. Gazi Mühendislik Bilimleri Dergisi Gazi Mühendislik Bilimleri Dergisi 154 F. Çipil 1/1 (2015) 143‐172 Gazi Journal of Engineering Sciences 3.1. ACCIDENT PERFORMANCE (MODEL 1) ANALYSIS RESULTS It measures the number of accidents in an outcome‐oriented and the efficiency in an input‐oriented way; that is, it is the method that focuses on the degree that those inputs that minimize output should be decreased. Moreover, both CRS and VRS efficiency scores were calculated for each model. That is, it is determined in the CRS model whether increasing the amount of input influences the output at the same rate or not. In the VRS model, inputs and outputs are considered as variables. DMUs’ (countries’) both CRS and VRS efficiencies according to the Model 1 analysis results conducted for determining accident performance are presented in Figure 5. Figure 5. Model 1 analysis results Figure 5. Model 1 analysis results Figure 5. Model 1 analysis results By performing input‐oriented DEA for Model 1; the DMU that ensures minimum number of accidents with its existing inputs was determined. If the efficiency score found through input‐oriented model is 1 or 100% and if the inputs or outputs of this DMU do not have mixed inefficiency, this country is deemed efficient. Countries with efficiency scores of higher or lower than 100 are not efficient. Yet another meaning of the efficiency score of an inefficient F. Çipil 1/1 (2015) 143‐172 155 country is the following: this unit can become efficient if it decreases its inputs as much as its efficiency score. According to the Model 1 CRS analysis results, 13 countries including Belgium, Germany, Italy and Turkey have an efficiency score of 100%. According to the Model 1 VRS analysis results, on the other hand, 19 countries are efficient such as Romania, Denmark and France. These countries were ranked using super efficiency analysis. This ranking is presented in Table 5. The table ranks the countries that produce the highest amount of outputs using the lowest amount of inputs. In the ranking of countries’ efficiencies based on CRS analysis, Belgium ranked first whereas Turkey ranked fourth. Romania, on the other hand, ranked first based on VRS analysis. Countries that are not found efficient in neither of the analyses need to decrease their inputs as much as the weights of the input values of their reference countries presented in Table 6 (for CRS) and Table 7 (for VRS). Table 5. Gazi Mühendislik Bilimleri Dergisi Gazi Mühendislik Bilimleri Dergisi 3.1. ACCIDENT PERFORMANCE (MODEL 1) ANALYSIS RESULTS Super‐efficiency ranking DMU CRS DMU VRS 1 BE 374,88% RO 800% 2 LV 249,45% MT 757,79% 3 DE 246,04% BE 474,81% 4 TR 206,49% DK 298,31% 5 RO 196,04% NL 252,15% 6 PT 174,81% EL 202,77% 7 IT 143,49% PT 181,56% 8 HU 141,35% CY 178,23% 9 UK 131,50% ES 146,88% 10 SI 124,50% AT 144,19% 11 AT 113,94% BG 142,83% 12 PL 112,87% LU 139,29% 13 NL 103,92% IE 134,50% 14 FR 125,17% 15 SE 124,98% 16 SK 118,89% 17 LT 117,54% 18 LV 112,71% 19 CZ 109,15% Gazi Mühendislik Bilimleri Dergisi 156 F. Çipil 1/1 (2015) 143‐172 Table 6. Weights of inefficient countries based on CRS analysis results compared to efficient countries and the number of efficient countries’ being references DMU Number for efficient countries to be references (dark) Name and weight of reference country for inefficient countries (light) 1 BE 0 2 BG LV (0,53) HU (0,13) TR (0,01) 3 CZ DE (0,01) IT (0,05) LV (0,34) RO (0,22) 4 DK DE(0,00) PT (0,05) SI (0,13) TR (0,00) 5 DE 3 6 EE LV (0,11) AT (0,01) RO (0,02) 7 IE IT (0,02) PL (0,00) PT (0,04) RO (0,05) TR (0,00) 8 EL 20 (0,02) PT (0,20) RO (0,23) TR (0,00) 9 ES IT (0,34) PT (0,39) TR (0,01) 10 FR IT(0,34) 11 IT 6 12 CY LV (0,04) HU (0,03) PT (0,03) TR (0,00) 13 LV 7 14 LT LV (0,43) HU (0,05) TR (0,00) 15 LU AT (0,02) TR (0,00) 16 HU 4 17 MT LV (0,02) HU (0,00) PT (0,01) TR (0,00) 18 NL 0 19 AT 5 20 PL 2 21 PT 9 22 RO 7 23 SI 1 24 SK IT (0,01) LV(0,07) AT (0,02) PT (0,04) RO (0,08) 25 FI IT(0,01) AT (0,02) PT (0,07) RO (0,05) TR(0,00) 26 SE DE (0,00) AT (0,37) PT (0,06) RO (0,05) TR(0,00) 27 UK 0 28 TR 11 157 F. Çipil 1/1 (2015) 143‐172 Table 7. Gazi Mühendislik Bilimleri Dergisi 3.1. ACCIDENT PERFORMANCE (MODEL 1) ANALYSIS RESULTS Weights of inefficient countries based on VRS analysis results compared to efficient countries and the number of efficient countries’ being references DMU Number for efficient countries to be references (dark) Name and weight of reference country for inefficient countries (light) 1 BE 3 2 BG 0 3 CZ EE (0,28) IT (0,06) LV (0,52) RO(0,13) UK (0,01) 4 DK LV (0,53) SK (0,47) 5 DE 4 6 EE 5 7 IE BE (0,06) EE (0,20) MT (0,21) PL (0,01) SK (0,53) 8 EL DE (0,01) LV (0,19) HU (0,16) RO (0,04) SK (0,58) TR (0,01) 9 ES DE (0,03) IT (0,34) SK (0,62) 10 FR DE (0,02) IT (0,29) LV (0,29) SK (0,40) 11 IT 3 12 CY BE (0,01) EE (0,11) MT(0,83) PL (0,00) PT (0,01) SK(0,04) TR(0,00) 13 LV 5 14 LT 0 15 LU 0 16 HU 1 17 MT 2 18 NL 0 19 AT 0 20 PL 2 21 PT 2 22 RO 2 23 SI 0 24 SK 8 25 FI BE (0,12) EE (0,83) PT (0,04) SK (0,01) 26 SE DE (0,04) EE(0,24) LV (0,45) SK(0,27) 27 UK 1 28 TR 2 Gazi Mühendislik Bilimleri Dergisi 158 F. Çipil 1/1 (2015) 143‐172 Table 6 and Table 7 also illustrate the times efficient countries became references for inefficient ones (shaded in grey). For example, based on CRS analysis results (for Model 1), Turkey became a reference as an efficient country for 11 inefficient countries (Table 6). These countries, including Bulgaria, Denmark and Spain, need to decrease their inputs as much as the multiplication of Turkey’s input values by their weights given in brackets. Based on VRS analysis results, similarly, Turkey became a reference for Cyprus and Greece. In other words, inefficient countries should take the capacity of efficient countries to convert their inputs into outputs as a reference. They should decrease their inputs as much as the values given in blankets nearby the references in both tables. Gazi Mühendislik Bilimleri Dergisi 3.2. RESULTS OF EFFICIENCY ANALYSIS FOR CASUALTIES IN ACCIDENTS (MODEL 2) According to the performance analysis results of Model 2, which takes the number of casualties in accidents as output and road transportation indicators as input, both CRS and VRS efficiencies of DMUs are presented in Figure 6. Figure 6. Model 2 analysis results Figure 6. Model 2 analysis results Gazi Journal of Engineering Sciences Gazi Journal of Engineering Sciences F. Çipil 1/1 (2015) 143‐172 159 By performing input‐oriented DEA for Model 2; the DMU that ensures minimum number of casualties with its existing inputs was determined. If the efficiency score found through input‐oriented model is 1 or 100% and if the inputs or outputs of this DMU do not have mixed inefficiency, this country is deemed efficient. Countries with efficiency scores of higher or lower than 100 are not efficient. Yet another meaning of the efficiency score of an inefficient country is the following: this unit can become efficient if it decreases its inputs as much as its efficiency scores. According to the Model 2 CRS analysis results, 16 countries including Belgium, Germany, France, Italy and Turkey have an efficiency score of 100%. According to the Model 2 VRS analysis results, on the other hand, 20 countries are efficient such as Romania, Poland, Hungary and France. These countries were ranked using super efficiency analysis. This ranking is presented in Table 8. The table ranks the countries that produce the highest amount of outputs using the lowest amount of inputs. In the ranking of countries’ efficiencies based on CRS analysis, Belgium ranked first whereas Turkey ranked second. Based on VRS analysis, on the other hand, Poland ranked first whereas Turkey ranked fifth. Countries that are not found efficient in neither of the analyses need to decrease their inputs as much as the weights of the input values of their reference countries presented in Table 9 (for CRS) and Table 10 (for VRS). Gazi Mühendislik Bilimleri Dergisi F. Çipil 1/1 (2015) 143‐172 160 Table 8. Gazi Journal of Engineering Sciences 3.2. RESULTS OF EFFICIENCY ANALYSIS FOR CASUALTIES IN ACCIDENTS (MODEL 2) Super‐efficiency ranking for Model 2 DMU CRS DMU VRS 1 BE 471,04% PL 800,00% 2 TR 399,71% DE 800% 3 PL 251,88% MT 757,79% 4 RO 233,55% BE 602,05% 5 DE 226,23% TR 451,72% 6 BG 185,26% RO 241,35% 7 HU 173,55% BG 185,77% 8 FR 167,97% EE 182,39% 9 IT 165,10% LV 174,47% 10 PT 142,96% HU 173,97% 11 SI 136,09% FR 173,54% 12 LT 126,10% IT 167,89% 13 NL 123,12% SI 157,19% 14 UK 122,21% LU 154,33% 15 AT 112,22% LT 152,96% 16 MT 101,68% PT 144,38% 17 NL 132,31% 18 SK 126,74% 19 UK 126,15% 20 AT 120,24% Table 8. Super‐efficiency ranking for Model 2 Table 9 and Table 10 also illustrate the times efficient countries became references for inefficient ones (shaded in grey). For example, based on CRS analysis results (for Model 2), Turkey became a reference as an efficient country for 5 inefficient countries (Table 9). These countries, including Czech Republic and Cyprus, need to decrease their inputs as much as the multiplication of Turkey’s input values by their weights given in brackets. F. Çipil 1/1 (2015) 143‐172 161 Table 9. Weights of inefficient countries based on CRS analysis results compared to efficient countries and the number of efficient countries’ being references DMU Number for efficient countries to be references (dark) Name and weight of reference country for inefficient countries (light) 1 BE 2 2 BG 2 3 CZ IT (0,06) HU (0,11) RO (0,20) TR (0,00) 4 DK BG (0,14) PL (0,02) RO (0,02) SI (0,20) 5 DE 1 6 EE HU (0,04) RO (0,04) TR (0,00) 7 IE BE (0,03) IT (0,00) AT (0,03) PT (0,02) RO (0,06) 8 EL PL (0,03) PT (0,31) RO (0,37) 9 ES FR (0,08) IT (0,35) PL (0,16) RO (0,05) SI (0,24) 10 FR 1 11 IT 5 12 CY HU (0,02) PL (0,00) PT (0,02) RO (0,01) TR (0,00) 13 LV BG (0,28) RO (0,00) 14 LT 0 15 LU BE(0,01) RO (0,01) SI (0,01) 16 HU 6 17 MT 0 18 NL 0 19 AT 3 20 PL 5 21 PT 5 22 RO 11 23 SI 5 24 SK IT(0,01) HU(0,06) AT(0,02) RO(0,10) SI (0,11) TR(0,00) 25 FI IT(0,01) HU(0,01) PL (0,02) PT(0,03) RO(0,04) SI (0,16) 26 SE DE(0,00) HU(0,06) AT (0,39) PT(0,06) TR (0,00) 27 UK 0 28 TR 5 Gazi Mühendislik Bilimleri Dergisi 162 F. Çipil 1/1 (2015) 143‐172 Table 10. 3.2. RESULTS OF EFFICIENCY ANALYSIS FOR CASUALTIES IN ACCIDENTS (MODEL 2) Weights of inefficient countries based on VRS analysis results compared to efficient countries and the number of efficient countries’ being references DMU Number for efficient countries to be references (dark) Name and weight of reference country for inefficient countries (light) 1 BE 4 2 BG 4 3 CZ BG (0,06) EE(0,25) IT (0,06) LV (0,08) MT (0,19) PL (0,01) RO(0,14) SK (0,20) 4 DK LV (0,47) SI (0,04) SK (0,49) 5 DE 1 6 EE 4 7 IE BE(0,02) MT (0,28) SK (0,70) 8 EL BE (0,00) MT (0,19) PL (0,04) PT (0,26) RO (0,36) RO (0,14) 9 ES BG (0,25) 10 (0,09) IT (0,35) PL (0,06) RO(0,11) SI (0,14) 10 FR 1 11 IT 2 12 CY BE (0,01) BG (0,01) EE (0,10) MT (0,83) PT (0,01) SK (0,04) 13 LV 3 14 LT 1 15 LU 0 16 HU 0 17 MT 5 18 NL 0 19 AT 0 20 PL 3 21 PT 2 22 RO 3 23 SI 3 24 SK 5 25 FI BE (0,14) BG(0,07) EE (0,74) LT (0,02) MT (0,03) 26 SE DE (0,06) EE (0,38) LV (0,34) SK (0,22) 27 UK 0 28 TR 0 Based on VRS analysis results, similarly, Turkey did not become a reference for other countries although it was an efficient country. In other words, inefficient countries should take the capacity of efficient countries to convert Gazi Journal of Engineering Sciences Gazi Journal of Engineering Sciences F. Çipil 1/1 (2015) 143‐172 163 their inputs into outputs as a reference. They should decrease their inputs as much as the values given in blankets nearby the references in both tables. their inputs into outputs as a reference. They should decrease their inputs as much as the values given in blankets nearby the references in both tables. By performing input‐oriented CRS and VRS analyses with Model 2; it was aimed to minimize the number of deaths and the injured. If the efficiency score found through input‐oriented model is 1 or 100% and if the inputs or outputs of this DMU do not have mixed inefficiency, it is concluded that the main road traffic indicators of this country cause less casualties in accidents than those of other countries, and this country is deemed efficient. Countries with efficiency scores of higher or lower than 100 are not efficient. Gazi Mühendislik Bilimleri Dergisi Gazi Mühendislik Bilimleri Dergisi 3.2. RESULTS OF EFFICIENCY ANALYSIS FOR CASUALTIES IN ACCIDENTS (MODEL 2) Yet another meaning of the efficiency score of an inefficient country is the following: this unit can become efficient if it decreases its inputs, that is its main road traffic indicators, as much as its efficiency score. For this reason, the input‐oriented inefficiency score is called contraction coefficient. For example, Czech Republic is not efficient for Model 2 according to CRS analysis with the efficiency score of 84,41%. In order to become efficient, this country needs to reduce its inputs (e.g. number of passengers/vehicles or rate of motorization) by 15.59%. Table 11. Model 3 super‐efficiency analysis results 1 DMU CRS DMU VRS 2 MT 720,66% RO 800,00% 3 RO 306,80% MT 757,79% 4 LU 204,30% BE 307,39% 5 LV 196,36% LU 226,79% 6 TR 186,19% TR 205,69% 7 BE 180,90% EE 202,22% 8 EE 162,11% LV 198,48% 9 BG 152,36% CY 184,25% 10 CY 120,54% BG 182,30% 11 SI 118,72% SK 125,03% 12 SK 116,83% SI 119,35% 13 HU 105,09% NL 111,77% 14 PT 100,93% PT 110,16% 15 LT 109,15% 16 HU 105,18% Table 11. Model 3 super‐efficiency analysis results Gazi Mühendislik Bilimleri Dergisi 164 F. Çipil 1/1 (2015) 143‐172 Gazi Journal of Engineering Sciences Gazi Mühendislik Bilimleri Dergisi 3.3. RESULTS OF EFFICIENCY ANALYSIS FOR LOSSES PER ROAD TRANSPORTATION INDICATOR (MODEL 3) According to the performance analysis results of Model 3, which takes the number of deaths on roads per one million vehicles for passenger transportation and the number of deaths on roads per one billion passengers‐ km as outputs and road transportation indicators as input, both CRS and VRS efficiencies of DMUs are presented in Figure 7. Figure 7. Model 3 analysis results Figure 7. Model 3 analysis results Figure 7. Model 3 analysis results By performing input‐oriented DEA for Model 3; the DMU that ensures minimum number of deaths on roads with its existing inputs was determined. If the efficiency score found through input‐oriented model is 1 or 100% and if the inputs or outputs of this DMU do not have mixed inefficiency, this country is deemed efficient. Countries with efficiency scores of higher or lower than 100 are not efficient. Yet another meaning of the efficiency score of an inefficient country is the following: this unit can become efficient if it decreases its inputs as much as its efficiency score. According to the Model 3 CRS analysis results, 14 countries including Belgium, Hungary, Lithuania, Bulgaria and Turkey have F. Çipil 1/1 (2015) 143‐172 165 an efficiency score of 100%. According to the Model 3 VRS analysis results, on the other hand, 16 countries are efficient such as Romania, Belgium and Turkey. These countries were ranked using super efficiency analysis. This ranking is presented in Table 11. The table ranks the countries that produce the highest amount of outputs using the lowest amount of inputs. In the ranking of countries’ efficiencies based on CRS and VRS analyses, Turkey ranked sixth. Countries that are not found efficient in neither of the analyses need to decrease their inputs as much as the weights of the input values of their reference countries presented in Table 12 (for CRS) and Table 13 (for VRS). Gazi Mühendislik Bilimleri Dergisi 166 F. Çipil 1/1 (2015) 143‐172 Table 12. Gazi Journal of Engineering Sciences Gazi Mühendislik Bilimleri Dergisi 3.3. RESULTS OF EFFICIENCY ANALYSIS FOR LOSSES PER ROAD TRANSPORTATION INDICATOR (MODEL 3) Weights of inefficient countries based on CRS analysis results compared to efficient countries and the number of efficient countries’ being references DMU Number for efficient countries to be references (dark) Name and weight of reference country for inefficient countries (light) 1 BE 2 2 BG 10 3 CZ BG (0,03) LV (0,26) RO (0,10) SK (0,26) 4 DK BG (0,09) LV (0,34) RO (0,02) 5 DE RO (0,15) 6 EE 0 7 IE BG (0,02) CY (0,15) SI (0,05) SK (0,35) 8 EL BG (0,30) LV (0,11) RO (0,09) SK (0,34) 9 ES BG (0,11) LV (0,08) RO (0,11) 10 FR BG (0,05) LV (0,18) RO(0,10) 11 IT LV (0,04) RO(0,25) 12 CY 1 13 LV 12 14 LT BG (0,20) LV (0,51) LU(0,00) MT(0,03) 15 LU 2 16 HU 0 17 MT 2 18 NL BE(0,03) BG (0,15) LV (0,02) LU (0,10) SI (0,01) 19 AT LV (0,51) MT(0,11) 20 PL BG (0,48) LV (0,23) RO (0,09) 21 PT 0 22 RO 10 23 SI 3 24 SK 4 25 FI BE (0,05) BG (0,08) LV(0,14) SI (0,14) 26 SE LV(0,19) RO(0,01) SK (0,10) 27 UK RO (0,12) 28 TR 0 Gazi Journal of Engineering Sciences F. Çipil 1/1 (2015) 143‐172 167 Table 13. Weights of inefficient countries based on VRS analysis results compared to efficient countries and the number of efficient countries’ being references DMU Number for efficient countries to be references (dark) Name and weight of reference country for inefficient countries (light) 1 BE 2 2 BG 0 3 CZ LV(0,41) RO(0,16) SK(0,43) 4 DK LV (0,53) SK (0,47) 5 DE LV (0,78) RO (0,22) 6 EE 2 7 IE BE (0,02) MT (0,28) SK (0,70) 8 EL LV (0,23) RO (0,11) SK (0,65) TR(0,01) 9 ES LV (0,36) RO (0,36) SK (0,28) 10 FR LV (0,57) RO (0,31) SK (0,12) 11 IT LV (0,67) RO (0,33) 12 CY 0 13 LV 11 14 LT 0 15 LU 0 16 HU 0 17 MT 1 18 NL 0 19 AT EE (0,40) LV (0,60) SK (0,00) 20 PL LV (0,53) RO (0,06) SK (0,30) TR (0,10) 21 PT 1 22 RO 9 23 SI 0 24 SK 10 25 FI BE (0,12) EE (0,83) PT (0,04) SK(0,01) 26 SE LV (0,65) RO (0,03) SK (0,32) 27 UK LV (0,47) RO (0,53) 28 TR 2 Gazi Mühendislik Bilimleri Dergisi 168 F. 3.3. RESULTS OF EFFICIENCY ANALYSIS FOR LOSSES PER ROAD TRANSPORTATION INDICATOR (MODEL 3) Çipil 1/1 (2015) 143‐172 Table 12 and Table 13 also illustrate the times efficient countries became references for inefficient ones (shaded in grey). For example, based on CRS analysis results (for Model 3), Turkey could not become a reference for other countries although it was efficient (Table 12). According to VRS analysis results, on the other hand, Turkey became a reference as an efficient country for Poland and Greece (Table 13). Gazi Journal of Engineering Sciences Gazi Mühendislik Bilimleri Dergisi 3.4. GENERAL EVALUATION OF DATA ENVELOPMENT ANALYSIS RESULTS Through three different input‐oriented models (Model 1‐3) and two different DEA models (CRS and VRS), it was concluded that Turkey is more efficient than EU countries in terms of converting its road transportation indicators into useful outputs (Table 14). Turkey had an efficiency score of 100% in all the three models we worked on. Along with Turkey, Romania and Slovenia are other efficient countries. That is, their road transportation indicators caused less number of accidents, less number of casualties and less number of accidents with casualties within residential areas. In addition, they managed to keep the share of transportation within GNP in its maximum with respect to main indicators; which suggests that they utilized traffic indicators efficiently. Gazi Journal of Engineering Sciences F. Çipil 1/1 (2015) 143‐172 169 Table 14. General evaluation MODEL 1 MODEL 2 MODEL 3 DMU CRS VRS CRS VRS CRS VRS BE 100,00% 100,00% 100,00% 100,00% 100,00% 100,00% BG 82,66% 100,00% 100,00% 100,00% 100,00% 100,00% CZ 87,43% 92,00% 84,41% 92,82% 50,28% 76,53% DK 23,83% 91,69% 46,12% 92,19% 44,98% 91,69% DE 100,00% 100,00% 100,00% 100,00% 15,85% 69,86% EE 46,19% 100,00% 57,02% 100,00% 100,00% 100,00% IE 54,36% 88,94% 43,29% 86,55% 50,72% 86,55% EL 54,38% 68,91% 85,53% 86,37% 57,05% 67,37% ES 77,37% 85,14% 95,08% 96,23% 19,68% 62,40% FR 66,32% 83,70% 100,00% 100,00% 21,64% 65,25% IT 100,00% 100,00% 100,00% 100,00% 21,22% 55,46% CY 54,60% 93,92% 47,15% 93,90% 100,00% 100,00% LV 100,00% 100,00% 77,58% 100,00% 100,00% 100,00% LT 63,07% 100,00% 100,00% 100,00% 87,36% 100,00% LU 29,43% 100,00% 47,65% 100,00% 100,00% 100,00% HU 100,00% 100,00% 100,00% 100,00% 100,00% 100,00% MT 73,37% 100,00% 100,00% 100,00% 100,00% 100,00% NL 100,00% 100,00% 100,00% 100,00% 40,23% 100,00% AT 100,00% 100,00% 100,00% 100,00% 52,96% 77,52% PL 100,00% 100,00% 100,00% 100,00% 62,47% 75,18% PT 100,00% 100,00% 100,00% 100,00% 100,00% 100,00% RO 100,00% 100,00% 100,00% 100,00% 100,00% 100,00% SI 100,00% 100,00% 100,00% 100,00% 100,00% 100,00% SK 59,77% 100,00% 75,75% 100,00% 100,00% 100,00% FI 39,00% 80,06% 38,67% 80,60% 35,27% 80,06% SE 49,43% 82,22% 54,83% 83,87% 23,41% 78,54% UK 100,00% 100,00% 100,00% 100,00% 9,39% 62,56% TR 100,00% 100,00% 100,00% 100,00% 100,00% 100,00% Table 14. General evaluation Gazi Mühendislik Bilimleri Dergisi 170 F. Çipil 1/1 (2015) 143‐172 Gazi Journal of Engineering Sciences 4. CONCLUSIONS Attaining a high level of performance is a key factor of success for every organization. To this end; it is today an important managerial instrument to define the criteria necessary for development, improve the existing performance and understand why some units in the organization are operating inefficiently. In the study, using Data Envelopment Analysis that is a technique for measuring efficiency and total factor productivity, performance was demonstrated subjectively. The accession period to the EU, which has a significant place especially in the recent years in Turkey’s social and economic development, has accelerated harmonization efforts. “Transportation” is among the chapters opened as part of this process of harmonization and works are ongoing under this chapter. This study will contribute to the adaptation of road transportation indicators into the EU criteria. DEA is employed in order to determine the performance of many enterprises and to define the policies to be employed to improve this performance. In this study, it was aimed to determine the performances of road transportation indicators comparatively in terms of their positive and negative consequences, and thus Turkey’s position among EU countries was determined. It is aimed, through the subjective results obtained, to provide information about the limitations of main indicators that will maximize the performance of transportation indicators and about the strategies and targets to be followed. For countries’ efficiencies, three different models were developed. Therefore, main road transportation elements such as countries’ numbers of accidents, numbers of deaths and injuries caused by accidents on roads, and numbers of these incidents in residential areas were analyzed subjectively. The input‐oriented model investigates to what extent the inputs belonging to the DMU whose efficiency is explored can be reduced in order to measure a certain level of output. Therefore, through four different input‐oriented models employed in this study, it was investigated how much the inputs (main road traffic indicators) should be reduced by keeping output levels (e.g. number of accidents) constant. Also, countries that were found to be efficient were ranked F. Çipil 1/1 (2015) 143‐172 171 among themselves. For this ranking, the concept of scale‐efficiency was employed. among themselves. For this ranking, the concept of scale‐efficiency was employed. Outcomes of this study will provide those who work on and make policies about this issue with information regarding Turkey’s current situation in comparison to EU member countries in concrete (quantitative) values. Gazi Mühendislik Bilimleri Dergisi 4. CONCLUSIONS In addition, it was concluded in this study that the intense utilization of road transportation in Turkey does not create a disadvantage vis‐a‐vis other countries. When the road transportation data, which is an important component of the chapter of transportation for Turkey, are compared with EU countries with respect to multiple parameters; it is observed that Turkey’s young and high population, existing road transportation infrastructure and main road indicators such as road‐km and passenger‐km are balanced. This analysis produced important data for developing road transportation policies in Turkey’s accession period to the EU. Useful conclusions were drawn for the use of policy‐makers within the field of transportation in Turkey. Gazi Mühendislik Bilimleri Dergisi 172 F. Çipil 1/1 (2015) 143‐172 Gazi Journal of Engineering Sciences REFERENCES 1. Ministry of Transportation, “Transportation Strategy Report”,ITÜ, Ministry of Transportation, Ankara, 23‐30 (2004). 1. Ministry of Transportation, “Transportation Strategy Report”,ITÜ, Ministry of Transportation, Ankara, 23‐30 (2004). 2. Çubuk, K. and Cansız, Ö.F.“Energy Status between Transportation Systems in Turkey”, Energy Efficiency Conference, Ministry of Energy and Natural Resources, Ankara, 47‐49 (2005) 2. Çubuk, K. and Cansız, Ö.F.“Energy Status between Transportation Systems in Turkey”, Energy Efficiency Conference, Ministry of Energy and Natural Resources, Ankara, 47‐49 (2005) 3. J.A Beasley, Data Envelopment Analysis, http://mscmga.ms.ic.ac.uk/jep/jep.html (03.04.2004) 4. Aydoğdu, A., (2006), “Transportation and energy policies that Turkey should develop in order to reduce the number of traffic accidents in the EU accession period”, Master’s Thesis, GaziUniversity, Institute of Science 5. Bedrettin, M., (2004) “Traffic Law and Fundamental Traffic Knowledge”, General Directorate of Security 5. Bedrettin, M., (2004) “Traffic Law and Fundamental Traffic Knowledge”, General Directorate of Security 6. “EU transport in figures”, Statistical Pocket Book 2011, European Commission 6. “EU transport in figures”, Statistical Pocket Book 2011, European Commission 7. Chen, Y., Liang, L., Yang, F., Zhu, J., “Evaluation Of Information Technology Investment: A Data Envelopment Analysis Approach”, Computers& Operations Research, 33 :1368– 1379 (2006). 7. Chen, Y., Liang, L., Yang, F., Zhu, J., “Evaluation Of Information Technology Investment: A Data Envelopment Analysis Approach”, Computers& Operations Research, 33 :1368– 1379 (2006). 8. uggiero, J., “Measurement Error, Education Production And Data Envelopment Analysis”, Economics of Education Review, 42:653‐662 (2005) . 8. uggiero, J., “Measurement Error, Education Production And Data Envelopment Analysis”, Economics of Education Review, 42:653‐662 (2005) . Gazi Journal of Engineering Sciences
https://openalex.org/W2806808987	https://europepmc.org/articles/pmc6025628?pdf=render	English	null	Influence of Absorbable Calcium Sulfate-Based Bone Substitute Materials on Human Haemostasis—In Vitro Biological Behavior of Antibiotic Loaded Implants	Materials	2,018	cc-by	4,299	Received: 20 April 2018; Accepted: 30 May 2018; Published: 1 June 2018 Abstract: Calcium sulfate (CS) formulations are frequently implanted as antibiotically impregnated bone substitutes in orthopedic and trauma surgery to prevent or treat bone infections. Calcium ions have been discussed as candidates to accelerate blood coagulation. The goal of this study is to evaluate substance-speciﬁc inﬂuences of CS formulations on blood coagulation. Speciﬁc ELISAs were conducted to determine markers of activated blood coagulation after incubation of human blood with CS beads. Additionally, wettability with freshly drawn human blood was measured. Three different types of CS bone substitute beads were compared (CS dihydrate with tripalmitin, containing Gentamicin (Heraﬁll®-G: Group A) or Vancomycin (CaSO4-V: Group B); and a CS hemihydrate with Tobramycin (Osteoset®: Group C)). Examinations were performed by ELISA assays for F1+2, FXIIa and C3a. Our results prove that none of the CS preparations accelerated single speciﬁc assays for activated coagulation markers. This allows the conclusion that neither Heraﬁll®-G (CaSO4-G) nor CaSO4-V alter haemostasis negatively. Blood samples incubated with Osteoset® display an elevated F1+2-activity. The addition of tripalmitin in Heraﬁll®-G shifts the original into a signiﬁcantly hydrophobic formulation. This was additionally proven by contact angle examination of the three substances with freshly drawn human blood, showing that acceleration of plasmatic coagulation is hindered by lipids and induced by surface effects caused by presence of rapidly soluble calcium ions in the Osteoset® preparation. Keywords: calcium sulfate formulations; calcium carbonate; tripalmitin; coagulation; in vitro; Heraﬁll®-G; Osteoset®, gentamicin; vancomycin; tobramycin; FXIIa; C3a; F1+2 Received: 20 April 2018; Accepted: 30 May 2018; Published: 1 June 2018 materials materials Influence of Absorbable Calcium Sulfate-Based Bone Substitute Materials on Human Haemostasis—In Vitro Biological Behavior of Antibiotic Loaded Implants Dominik Pförringer 1,,†, Norbert Harrasser 2,†, Marc Beirer 1, Moritz Crönlein 1, Dominik Pförringer 1,,†, Norbert Harrasser 2,†, Marc Beirer 1, Moritz Crönlein 1, Axel Stemberger 2, Martijn van Griensven 1 ID , Martin Lucke 3, Rainer Burgkart 2,‡ and Andreas Obermeier 2,‡ ID 1 Klinikum rechts der Isar der Technischen Universität München, Klinik und Poliklinik für Unfallchirurgie, Ismaninger Str. 22, 81675 München, Germany; Marc.Beirer@mri.tum.de (M.B.); Moritz.Croenlein@mri.tum.de (M.C.); Martijn.vanGriensven@tum.de (M.v.G.) 2 1 Klinikum rechts der Isar der Technischen Universität München, Klinik und Poliklinik für Unfallchirurgie, Ismaninger Str. 22, 81675 München, Germany; Marc.Beirer@mri.tum.de (M.B.); Moritz.Croenlein@mri.tum.de (M.C.); Martijn.vanGriensven@tum.de (M.v.G.) 2 Kli ik h d I d T h i h U i i M h Kli ik f O h di d S h di 2 Klinikum rechts der Isar der Technischen Universität München, Klinik für Orthopädie und Sportorthopädie, Ismaninger Str. 22, 81675 München, Germany; norbert.harrasser@mri.tum.de (N.H.); axel.stemberger@lrz.tum.de (A.S.); burgkart@tum.de (R.B.); aobermeier@tum.de (A.O.) g ( ) g ( ) ( ) 3 Chirurgisches Klinikum München Süd, Am Isarkanal 30, 81379 München, Germany; martin.lucke@artemed.de * Correspondence: Dominik.pfoerringer@mri.tum.de or dominik@pfoerringer.de; Tel.: +49-89-4140-5 * Correspondence: Dominik.pfoerringer@mri.tum.de or dominik@pfoerringer.de; Tel.: +49-89-4140-5539 † These authors contributed equally to this work. † These authors contributed equally to this work. † These authors contributed equally to this work. ‡ These authors also contributed equally to this work. 1. Introduction Infected bone defects are frequently addressed through implantation of various bone grafts, often based on calcium sulfate preparations containing antibiotics. The clinical side effects of such Materials 2018, 11, 935; doi:10.3390/ma11060935 www.mdpi.com/journal/materials www.mdpi.com/journal/materials 2 of 8 Materials 2018, 11, 935 formulations are highly relevant to usability, as well as to potential risks and opportunities in defect healing situations. In this study, two formulations which are commercially available in Germany, as well as a third, prospective formulation containing vancomycin to address MRSA-infections, were compared, regarding their speciﬁc effects on blood coagulation after implantation [1]. The blood coagulation cascade requires calcium ions, and efﬁciently acts on the activation of platelets [2]. Three antibiotically loaded [3] calcium-based formulations were compared for this experiment: Calcium Sulfate (CS) dihydrate with tripalmitin, containing gentamicin (Heraﬁll®-G: Group A) and vancomycin (CaSO4-V: Group B); CS hemihydrate containing tobramycin (Osteoset®: Group C). CS is an inexpensive material known for its high biocompatibility [4], and functions as a carrier material [5,6] by incorporation of therapeutic substances. Calcium preparations are known to inﬂuence blood coagulation, rendering it meaningful to compare the substances’ effects on human blood coagulation. Markers of activated coagulation and of the complement system (also a serin protease system) were determined using speciﬁc ELISA assays. Furthermore, a comparison of the wettability of the substances using contact angle measurements allows conclusions to be drawn regarding superﬁcial material-blood interaction. 2. Materials and Methods Workﬂow of preparing blood samples for ELISA assays and testing markers of activated coagulation and complement system. the prothrombin activation quantitatively. To determine the degree of the activation of the extrinsic haemostasis pathway via Factor FXII activation into FXIIa quantitatively, an “amidolytic substrate assay” was used to measure the Factor XIIa-like activity (UNITEST, Haemochrom Diagnostica, Essen, Germany). Inside prepared human plasma, the FXIIa-like activity is measured by αXIIa bound to α2-macroglobulin using a chromogenic substrate. Photometrical measures determine the release of p-nitroanaline (pNA); with that, the amount of FXIIa-like activity can be calculated. Using the commercially available and reliable complement C3a-desArg-ELISA (PROGEN biotechnology, Heidelberg, Germany), the content of C3a-desArg, as a stable version of the short-lived C3a, is quantified in plasma. This ELISA is based on the H13 antibody, which blocks the short-living C3a, allowing us to determine its quantity in plasma [8]. This gives us an insight into the initial complement activation of novel formulated bone substitute materials. Contact angle measurements with freshly drawn human blood were conducted to evaluate the material specific wettability in comparison to that of untreated glass. For this purpose, bone substitute beads were crushed to fine powder using a mortar, and dissolved in 3 mL of 70% ethanol and 1 mL H2O. To achieve solutions of comparable mass contents or Herafill® G 4 beads were used; for Osteoset®, 10 beads were used Subsequently, the calcium sulfate dispersions were homogenized ELISA assay (Enzygnost® F1+2 Assay, Dade Behring, Marburg, Germany) was used to measure the prothrombin activation quantitatively. To determine the degree of the activation of the extrinsic haemostasis pathway via Factor FXII activation into FXIIa quantitatively, an “amidolytic substrate assay” was used to measure the Factor XIIa-like activity (UNITEST, Haemochrom Diagnostica, Essen, Germany). Inside prepared human plasma, the FXIIa-like activity is measured by αXIIa bound to α2-macroglobulin using a chromogenic substrate. Photometrical measures determine the release of p-nitroanaline (pNA); with that, the amount of FXIIa-like activity can be calculated. Using the commercially available and reliable complement C3a-desArg-ELISA (PROGEN biotechnology, Heidelberg, Germany), the content of C3a-desArg, as a stable version of the short-lived C3a, is quantified in plasma. This ELISA is based on the H13 antibody, which blocks the short-living C3a, allowing us to determine its quantity in plasma [8]. This gives us an insight into the initial complement activation of novel formulated bone substitute materials. for Osteoset , 10 beads were used. 2. Materials and Methods For this controlled clinical trial, resorbable bone substitute materials consisting of CS dihydrate, gentamicin and tripalmitin (Herafill®-G, Heraeus, Wehrheim, Germany), CS dihydrate, vancomycin, and tripalmitin, as well as commercially available CS hemihydrate with tobramycin (Osteoset®, Wright Medical Group Inc., Memphis, Tennessee, USA), were compared. The beads are composed as follows: Herafill®-G has a 6.0 mm diameter, at 250 mg weight per unit, consisting of calcium sulfate dihydrate (71.6%), calcium carbonate (17.9%), tripalmitin (8.8%), and gentamicin sulfate (1.7%). The second group has a 3.0 mm diameter, at 35 mg per unit, consisting of calcium sulfate dihydrate (72.0%), calcium carbonate (18.0%), tripalmitin (8.9%), and vancomycin hydrochloride (1.1%). Osteoset® has a 4.8 mm diameter, with 107.5 mg weight per unit, consisting of a hemihydrate modification of calcium sulfate (96.0%) and tobramycin sulfate (4.0%). The composition of bead implants is given in contents per weight. For the ELISA assays, all three substances were compared. For the contact angle measurement a reduced comparison between Heraﬁll®-G and Osteoset® as well as a glass control group was conducted. To understand the influence of calcium carriers on blood coagulation, samples were tested using ELISA assays for quantitative detection of relevant coagulation factors in 16 samples of freshly drawn human blood after contact with the bone substitute materials. The blood was drawn using a regular polyethylene syringe without addition of any anticoagulation substances. Prior to blood drawing, two sets of seven 15 mL falcon tubes were equipped with one bead and one additional pure blood control sample. Subsequently, they were filled with 4 mL fresh, untreated human blood, and incubated for 8 min. Incubation time was chosen in accordance with previous literature by Obermeier et al. 2012 [7]. p y Samples were then prepared for testing the markers of activated coagulation and complement activation according to the workﬂow shown in Figure 1. 3 of 8 Materials 2018, 11, 935 Figure 1. Workflow of preparing blood samples for ELISA assays and testing markers of activated coagulation and complement system. ELISA assay (Enzygnost® F1+2 Assay Dade Behring Marburg Germany) was used to measure Figure 1. Workﬂow of preparing blood samples for ELISA assays and testing markers of activated coagulation and complement system. Figure 1. Workflow of preparing blood samples for ELISA assays and testing markers of activated coagulation and complement system. ELISA (E t® F A D d B h i M b G ) d t Figure 1. 2. Materials and Methods Subsequently, the calcium sulfate dispersions were homogenized with a ptfe pestle. Three hundred microliters of the resulting suspension were pipetted on glass slides and dried to achieve homogenous surface layers of the examined bone substitute materials. Ten microliters of freshly drawn blood were pipetted onto relevant test surfaces with a Herafill®-G or Osteoset® layer; as a comparison control surface, untreated glass was used. The contact angles were determined on eight independent samples per type of material by means of magnified photographs; the software ImageJ v1.47 (ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA), in combination with the DropSnake plugin, facilitated contact angle measurements. Contact angle measurements with freshly drawn human blood were conducted to evaluate the material speciﬁc wettability in comparison to that of untreated glass. For this purpose, bone substitute beads were crushed to ﬁne powder using a mortar, and dissolved in 3 mL of 70% ethanol and 1 mL H2O. To achieve solutions of comparable mass contents or Heraﬁll® G 4 beads were used; for Osteoset®, 10 beads were used. Subsequently, the calcium sulfate dispersions were homogenized with a ptfe pestle. Three hundred microliters of the resulting suspension were pipetted on glass slides and dried to achieve homogenous surface layers of the examined bone substitute materials. Ten microliters of freshly drawn blood were pipetted onto relevant test surfaces with a Heraﬁll®-G or Osteoset® layer; as a comparison control surface, untreated glass was used. The contact angles were determined on eight independent samples per type of material by means of magniﬁed photographs; the software ImageJ v1.47 (ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA), in combination with the DropSnake plugin, facilitated contact angle measurements. 4 of 8 Materials 2018, 11, 935 3. Results Fragment F1+2 is a peptide, being split from inactive prothrombin during coagulation, forming the active thrombin. The amount of thrombin is proportional to the amount of F1+2, allowing us to quantify the coagulation process. Figure 2 displays the F1+2–concentration, as well as the FXIIa and C3a-desArg, after 8 min of incubation of human blood with the bone substitute formulations, as well as the control groups consisting of untreated blood samples. The reference range for F1+2 concentration was between 69 and 229 pmol/L. Reference values are below 150 ng/L plasma EDTA; values are regarded as elevated if above 200 ng/L plasma. Figure 2. Coagulation markers following incubation of blood with tested beads (n = 16). Levels of signiﬁcance used were : p < 0.01. Figure 2. Coagulation markers following incubation of blood with tested beads (n = 16). Levels of signiﬁcance used were : p < 0.01. The Osteoset® group showed the highest value of F1+2 activation; at 274.3 pmol/L, it was highly signiﬁcant (p < 0.01; ), compared to pure blood activation and outside the assay’s reference range. Both tested calcium sulphate formulations containing tripalmitin (Heraﬁll®-G and CaS04-V) remained at 178.0 and 211.1 pmol/L, i.e., within the range of reference. Activation of factor XII in the endogenous coagulation cascade leads, via the shedding of fragment F1+2, to a development of prothrombin into active thrombin, catalyzing the formation of ﬁbrin. The reference range is between 14 and 27 u/L; all tested substances remained at a range of between 10.5 and 12.2 u/L, i.e., below the reference range, and thus, without any inﬂuence on this coagulation factor. The complement C3a-desArg ELISA quantiﬁes the content of C3a-desArg as a stable form of short lived C3a in plasma. The results showed values between 132.8 and 158.0 ng/mL, i.e., all remaining below the upper reference level of 200 ng/mL. Contact angle measurements yielded statistically signiﬁcant results, underlining the different degrees of wettability. Heraﬁll®-G displays the lowest degree of wettability (high hydrophobicity), represented by the highest contact angle, followed by Osteoset® (medium hydrophobicity), and the glass as negative control (highly hydrophilic). The speciﬁc results are displayed in Table 1 and Figure 3. 5 of 8 Materials 2018, 11, 935 Table 1. Blood contact angle measurement (n = 8) mean results in degrees (± standard deviations) and p-values of student’s t-test referred to glass (Levels of signiﬁcance used were : p < 0.001). Intern 4. Discussion reconstruction for more than a century [1]. While offering high biocompatibility, its influence on coagulation by dissociation has been discussed, but not examined thoroughly to date [9]. A surface-induced coagulation is triggered by factor XIIa, itself having further influence on the complement system, as well as fibrinolysis. The presented results prove that the tested dihydrate preparations do not accelerate the global hemostasis nor the tested specific markers of coagulation Osteoset® samples yielded elevated F1+2 Internal use of calcium sulfate (CS), also known as plaster of Paris, has been employed for bone reconstruction for more than a century [1]. While offering high biocompatibility, its inﬂuence on coagulation by dissociation has been discussed, but not examined thoroughly to date [9]. A surface-induced coagulation is triggered by factor XIIa, itself having further inﬂuence on the complement system, as well as ﬁbrinolysis. hemostasis, nor the tested specific markers of coagulation. Osteoset samples yielded elevated F1+2 concentrations, indicating elevated thrombin formation. Simultaneously, no elevated FXIIa activity is measurable. The observed acceleration of plasmatic coagulation is triggered by the elevated concentration of calcium-ions in the Osteoset® preparation. Addition of hydrophobic tripalmitate renders the originally hydrophilic calcium sulfate formulation more hydrophobic. Thus, no relevant quantities of calcium ions are dissolved into the blood, and coagulation processes are not relevantly influenced. The observed contact angle measurement results clearly underline how the tripalmitate addition increases the hydrophobicity, and thus, reduces wettability. Moreover, this could be a hint for one root cause of delayed resorption of beads, as shown by Pförringer et al. [10]. Wettability and dissolution are closely related, and thus, in the observed setup play an important role in the scaffold dissolving process, since contact angles are good indicators for dissolution transition of solid dispersions [11]. In this context, lipid content plays a significant role, The presented results prove that the tested dihydrate preparations do not accelerate the global hemostasis, nor the tested speciﬁc markers of coagulation. Osteoset® samples yielded elevated F1+2 concentrations, indicating elevated thrombin formation. Simultaneously, no elevated FXIIa activity is measurable. The observed acceleration of plasmatic coagulation is triggered by the elevated concentration of calcium-ions in the Osteoset® preparation. Addition of hydrophobic tripalmitate renders the originally hydrophilic calcium sulfate formulation more hydrophobic. Thus, no relevant quantities of calcium ions are dissolved into the blood, and coagulation processes are not relevantly inﬂuenced. 3. Results p g ( g p ) Interfaces Contact Angle (±SD) Signiﬁcance Glass 41.1 (±9.6)◦ Heraﬁll®-G 119.6 (±10.4)◦ p < 0.001; Osteoset® 86.6 (±8.4)◦ p < 0.001; * Materials 2018, 11, x FOR PEER REVIEW 5 of 7 Figure 3. Contact angle measurement in graphic comparison. Magnifications showing average contact angles of fresh human blood on test surfaces (n = 8). White scale bar indicating 1 mm. 4 Discussion Figure 3. Contact angle measurement in graphic comparison. Magniﬁcations showing average contact angles of fresh human blood on test surfaces (n = 8). White scale bar indicating 1 mm. Figure 3. Contact angle measurement in graphic comparison. Magnifications showing average contact angles of fresh human blood on test surfaces (n = 8). White scale bar indicating 1 mm. 4 Di i Figure 3. Contact angle measurement in graphic comparison. Magniﬁcations showing average contact angles of fresh human blood on test surfaces (n = 8). White scale bar indicating 1 mm. Intern 4. Discussion The observed contact angle measurement results clearly underline how the tripalmitate addition increases the hydrophobicity, and thus, reduces wettability. Moreover, this could be a hint for one root cause of delayed resorption of beads, as shown by Pförringer et al. [10]. p [ ] , p p y g , as it does in vital body functions, to purposely influence interaction between liquids and solids [12], as shown in our experiment. Interaction between bone substitute material and the surrounding bone tissue has been the focus of recent research, and deserves further attention [13]. The aqueous interaction has been researched, and can partially be connected to the dissolution examination of the compared material [14] Wettability and dissolution are closely related, and thus, in the observed setup play an important role in the scaffold dissolving process, since contact angles are good indicators for dissolution transition of solid dispersions [11]. In this context, lipid content plays a signiﬁcant role, as it does in vital body functions, to purposely inﬂuence interaction between liquids and solids [12], as shown in our Materials 2018, 11, 935 6 of 8 experiment. Interaction between bone substitute material and the surrounding bone tissue has been the focus of recent research, and deserves further attention [13]. The aqueous interaction has been researched, and can partially be connected to the dissolution examination of the compared material [14]. The presence of blood, as well as hematoma, and their effects on the complex process of bone healing, have been discussed in literature [15]. The effects of the presence and addition of platelet rich plasma (PRP) with released growth factors have been tested and discussed, showing the importance of the presence of a scaffold [16] for bone regeneration. Bone’s healing properties after trauma have been investigated [17], yielding a multitude of inﬂuencing and decisive factors in the healing process. Shiu et al. even propose that the bone healing response becomes dysregulated if the blood response, and subsequent formation and properties of a hematoma, are altered [18]. Effects of calcium compositions on osteoinduction and—conduction have been researched and described [10], yet the co-inﬂuence of bleeding times remains to be tested. 5. Conclusions Modulation of coagulation may inﬂuence bone healing. The underlying research compares the interaction of freshly drawn human blood with two calcium dihydrates and one hemihydrate formulation with differing calcium compositions and antimicrobial contents. The two dihydrates showed no inﬂuence, while the hemihydrate signiﬁcantly prolonged coagulation. This can partially be accounted for by the reduced solubility of calcium through the addition of tripalmitate to the formulation [10]. Further research may focus on the inﬂuence of coagulation on the resulting hematoma and subsequent bone healing properties. Author Contributions: Conceptualization, A.S., A.O. and D.P.; Methodology, R.B., N.H. and M.v.G.; Software, A.O.; Validation, A.O., A.S., R.B. and D.P.; Formal Analysis, A.O. and M.L.; Investigation, M.C., M.B. and D.P.; Resources, M.L.; Data Curation, M.v.G., A.O. and D.P.; Writing-Original Draft Preparation, A.O., N.H. and D.P.; Writing-Review & Editing, R.B., A.S., M.v.G., N.H. and M.C.; Visualization, A.O. and D.P.; Supervision, M.B., A.S., R.B., M.L., M.v.G.; Project Administration, A.O., R.B., A.S., M.v.G. and D.P. Author Contributions: Conceptualization, A.S., A.O. and D.P.; Methodology, R.B., N.H. and M.v.G.; Software, A.O.; Validation, A.O., A.S., R.B. and D.P.; Formal Analysis, A.O. and M.L.; Investigation, M.C., M.B. and D.P.; Resources, M.L.; Data Curation, M.v.G., A.O. and D.P.; Writing-Original Draft Preparation, A.O., N.H. and D.P.; Writing-Review & Editing, R.B., A.S., M.v.G., N.H. and M.C.; Visualization, A.O. and D.P.; Supervision, M.B., A.S., R.B., M.L., M.v.G.; Project Administration, A.O., R.B., A.S., M.v.G. and D.P. Funding: This work was supported by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program (www.ub.tum.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments: We would like to thank the working group of implant associated infection research at Klinikum rechts der Isar for their continued support of our test and research and especially Meredith Kiokekli for her extensive laboratory work. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Pforringer, D.; Obermeier, A.; Kiokekli, M.; Buchner, H.; Vogt, S.; Stemberger, A.; Burgkart, R.; Lucke, M. Antimicrobial formulations of absorbable bone substitute materials as drug carriers based on calcium sulfate. Antimicrob. Agents Chemother. 2016, 60, 3897–3905. [CrossRef] [PubMed] 1. Pforringer, D.; Obermeier, A.; Kiokekli, M.; Buchner, H.; Vogt, S.; Stemberger, A.; Burgkart, R.; Lucke, M. Antimicrobial formulations of absorbable bone substitute materials as drug carriers based on calcium sulfate. Antimicrob. Agents Chemother. 2016, 60, 3897–3905. [CrossRef] [PubMed] 2. Hashiguchi, T. Basic concept for evaluating coagulation and ﬁbrinolysis data. Rinsho Ketsueki 2017, 58, 2096–2103. [PubMed] 2. Hashiguchi, T. Basic concept for evaluating coagulation and ﬁbrinolysis data. Rinsho Ketsueki 2017, 58, 2096–2103. [PubMed] 3. Lalidou, F.; Kolios, G.; Drosos, G.I. Bone infections and bone graft substitutes for local antibiotic therapy. Surg. Technol. Int. 2014, 24, 353–362. [PubMed] 3. Lalidou, F.; Kolios, G.; Drosos, G.I. Bone infections and bone graft substitutes for local antibiotic therapy. Surg. Technol. Int. 2014, 24, 353–362. [PubMed] g 4. Peltier, L.F. The use of plaster of Paris to ﬁll large defects in bone. Am. J. Surg. 1959, 97, 311–315. [CrossRef] 5 Helgeson M D ; Potter B K ; Tucker C J ; Frisch H M ; Shawen S B Antibiotic impregnated calcium sulfate . Peltier, L.F. The use of plaster of Paris to ﬁll large defects in bone. Am. J. Surg. 1959, 97, 311–315. [Cross 4. Peltier, L.F. The use of plaster of Paris to ﬁll large defects in bone. Am. J. Surg. 1959, 97, 311–315. [CrossRef] 5. Helgeson, M.D.; Potter, B.K.; Tucker, C.J.; Frisch, H.M.; Shawen, S.B. Antibiotic-impregnated calcium sulfate use in combat-related open fractures. Orthopedics 2009, 32, 323. [CrossRef] [PubMed] 4. Peltier, L.F. The use of plaster of Paris to ﬁll large defects in bone. Am. J. Surg. 1959, 97, 311–315. [CrossRef] 5. Helgeson, M.D.; Potter, B.K.; Tucker, C.J.; Frisch, H.M.; Shawen, S.B. Antibiotic-impregnated calcium sulfate use in combat-related open fractures. Orthopedics 2009, 32, 323. [CrossRef] [PubMed] 6. Beuerlein, M.J.; McKee, M.D. Calcium sulfates: What is the evidence? Orthop. Trauma 2010, 24 (Suppl. S1), S46–S51. [CrossRef] [PubMed] 7. Obermeier, A.; Matl, F.D.; Schwabe, J.; Zimmermann, A.; Kuhn, K.D.; Lakemeier, S.; von Eisenhart-Rothe, R.; Stemberger, A.; Burgkart, R. Novel fatty acid gentamicin salts as slow-release drug carrier systems for anti-infective protection of vascular biomaterials. J. Mater. Sci. Mater. Med. 2012, 23, 1675–1683. [CrossRef] [PubMed] 7. 8. Burger, R.; Bader, A.; Kirschﬁnk, M.; Rother, U.; Schrod, L.; Worner, I.; Zilow, G. Functional analysis and quantiﬁcation of the complement c3 derived anaphylatoxin c3a with a monoclonal antibody. Clin. Exp. Immunol. 1987, 68, 703–711. [PubMed] 9. Scarano, A.; Sinjari, B.; Murmura, G.; Mijiritsky, E.; Iaculli, F.; Mortellaro, C.; Tete, S. Hemostasis control in dental extractions in patients receiving oral anticoagulant therapy: An approach with calcium sulfate. J. Craniofac. Surg. 2014, 25, 843–846. [CrossRef] [PubMed] References Obermeier, A.; Matl, F.D.; Schwabe, J.; Zimmermann, A.; Kuhn, K.D.; Lakemeier, S.; von Eisenhart-Rothe, R.; Stemberger, A.; Burgkart, R. Novel fatty acid gentamicin salts as slow-release drug carrier systems for anti-infective protection of vascular biomaterials. J. Mater. Sci. Mater. Med. 2012, 23, 1675–1683. [CrossRef] [PubMed] Materials 2018, 11, 935 7 of 8 8 of 8 Materials 2018, 11, 935 10. Pforringer, D.; Harrasser, N.; Muhlhofer, H.; Kiokekli, M.; Stemberger, A.; van Griensven, M.; Lucke, M.; Burgkart, R.; Obermeier, A. Osteoinduction and -conduction through absorbable bone substitute materials based on calcium sulfate: In vivo biological behavior in a rabbit model. J. Mater. Sci. Mater Med. 2018, 29, 17. [CrossRef] [PubMed] 11. Aparicio, C.; Maazouz, Y.; Yang, D. Measuring wettability of biosurfaces at the microscale. Nanotechnol. Regen. Med. 2012, 811, 163–177. [CrossRef] 12. Lu, Y.; Tang, N.; Lian, R.; Qi, J.; Wu, W. Understanding the relationship between wettability and dissolution of solid dispersion. Int. J. Pharm. 2014, 465, 25–31. [CrossRef] [PubMed] 13. Bhamla, M.S.; Nash, W.L.; Elliott, S.; Fuller, G.G. Inﬂuence of lipid coatings on surface wettability characteristics of silicone hydrogels. Langmuir 2015, 31, 3820–3828. [CrossRef] [PubMed] 14. Sheikh, Z.; Najeeb, S.; Khurshid, Z.; Verma, V.; Rashid, H.; Glogauer, M. Biodegradable materials for bone repair and tissue engineering applications. Materials 2015, 8, 5744–5794. [CrossRef] [PubMed] 15. Remedios, A. Bone and bone healing. Vet. Clin. N. Am. Small Anim. Pract. 1999, 29, 1029–1044. [C 15. Remedios, A. Bone and bone healing. Vet. Clin. N. Am. Small Anim. Pract. 1999, 29, 1029–1044. [CrossRef] 16. Malhotra, A.; Pelletier, M.H.; Yu, Y.; Walsh, W.R. Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes. Arch. Orthop. Trauma Surg. 2013, 133, 153–165. [CrossRef] [PubMed] Dittmer, K.E.; Firth, E.C. Mechanisms of bone response to injury. J. Vet. Diagn. Investig. 2017, 29, 385–395 [CrossRef] [PubMed] 18. Shiu, H.T.; Leung, P.C.; Ko, C.H. The roles of cellular and molecular components of a hematoma at early stage of bone healing. J. Tissue Eng. Regen. Med. 2018, 12, e1911–e1925. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. 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Open Access Intraoperative hypothermia in patients undergoing Total knee arthroplasty: a cross-sectional study from a developing country Ronika Devi Ukrani1, Aiman Arif1, Anum Sadruddin2, Obada Hasan2 and Shahryar Noordin2 Ronika Devi Ukrani1, Aiman Arif1, Anum Sadruddin2, Obada Hasan2 and Shahryar Noord Abstract Background: Intraoperative hypothermia is associated with various risk factors, morbidity, and mortality in patients undergoing total knee arthroplasty (TKA), increasing the emotional and financial burden on patients. This study aimed to identify risk factors of intraoperative hypothermia in patients undergoing TKA. Materials and methods: All adult patients (⩾18 years) who underwent TKA from January 2016 to December 2017 at a tertiary-care hospital in Pakistan were included in this retrospective, cross-sectional study. Temperature < 36 °C was defined as hypothermia. Results: The study included 286 patients (77.6% female) with a mean age of 61.4 ± 10.4 years. The overall proportion of intraoperative hypothermia was 26.6%. Of the total patients, 66.1% underwent bilateral TKA whereas 33.9% underwent unilateral TKA. 73.8% of the patients were ASA Level 2. Only 13.3% of patients had postoperative hypothermia. Conclusion: Intraoperative hypothermia was significantly associated with age, bilateral procedure, ASA level and postoperative hypothermia in patients undergoing TKA. The surgeon and the operative team should be aware of the risk factors and the adverse outcomes associated with intraoperative hypothermia, especially in resource constrained settings to plan preventive strategies. Trial registration: This study was retrospectively registered on ClinicalTrials.gov on 3rd October 2020. The registration ID is NCT04575246. Keywords: Developing country, Hypothermia, Intraoperative hypothermia, SSI, Total knee arthro Intraoperative hypothermia is a common complication of anaesthesia administration that affects normal thermal autoregulation in the body [4, 5]. Moreover, further heat loss due to the body surface area exposed to the cold operating room environment [6, 7] means that normal metabolic heat production is inadequate to maintain the core body temperature of the patient leading to numer- ous avoidable complications by altering normal physiologic processes in the body. The result of drastic heat loss is systemic vasoconstriction and reduction in Introduction Hypothermia, defined as core body temperature < 36 °C [1, 2], is a phenomenon in which the rate of heat loss is greater than the rate of heat production. It results from increased heat redistribution from core to periphery, coupled with decreased metabolic heat production [3]. * Correspondence: aimanarif15@gmail.com 1Medical College, Aga Khan University Hospital, Karachi 74800, Pakistan Full list of author information is available at the end of the article * Correspondence: aimanarif15@gmail.com 1Medical College, Aga Khan University Hospital, Karachi 74800, Pakistan Full list of author information is available at the end of the article (2021) 22:504 (2021) 22:504 Ukrani et al. BMC Musculoskeletal Disorders https://doi.org/10.1186/s12891-021-04390-7 Results The study comprised of a total of 286 patients with 77.6% of the patients being female. The mean age of the patients was 61.4 ± 10.4 years. 64.3% of the patients were hypertensive and 27.6% were diabetic. The indication for performing TKA was predominantly osteoarthritis. Of the 286 patients, 189 underwent bilateral TKA, while 97 patients underwent unilateral TKA (right: 46; left: 51). The majority of the patients were classified as ASA Level 2 (73.8%) followed by ASA Level 3 (20.3%) and ASA Level 1 (5.9%). The mean duration of surgery was 201.2 ± 66.2 min. The mean duration of surgery in pa- tients who underwent unilateral TKA was 131.4 ± 36.1 min as opposed to 237.0 ± 46.6 min in patients who had bilateral TKA (p = 0.032). Preoperatively, 62 (21.7%) pa- tients had hypothermia. However, 76 (26.6%) patients developed intraoperative hypothermia while 38 (13.3%) patients were noted to be hypothermic postoperatively. Statistical analysis IBM SPSS (Statistical Package for Social Sciences) Ver- sion 23.0 was used for all statistical analysis. Continuous data is presented as mean ± standard deviation with comparison using independent sample t-test. Nominal and ordinal data is presented as N (%) with percentages compared using Chi-Square test. A p-value of < 0.05 was considered significant for all analyses. Patients and methods A standardized proforma was used to collect data from an online patient data system about patients’ preopera- tive, intraoperative, and postoperative details. Preopera- tive data included age, gender, and comorbid conditions. Intraoperative data was collected from the surgical and anaesthesia records and included surgical site, American Society of Anesthesiologists (ASA) Level, duration of surgery, preoperative body temperature, intraoperative body temperature and duration of surgery. Postoperative data included postoperative body temperature and the presence of any surgical site infection within the 1 year follow-up period. The occurrence of perioperative hypothermia in total joint arthroplasty has been assessed in several studies and has yielded a range of results ranging from 11 to 72% [4, 10–12] to even up to 90% [7]. A study reported that 34% of patients undergoing total knee arthroplasty (TKA) have hypothermia at the time of incision and 33% of the patients stayed hypothermic throughout surgery [7]. Moreover, in the absence of active warming tech- niques, a 1–2 °C fall in temperature has been observed in surgical procedures [3]. Standard operating conditions All TKA procedures at AKUH are conducted under a standard protocol. The operating room temperature is set at 20 °C. Moreover, all patients are given a bear hug- ger with K-thermia underneath set at 37 °C. The increase in the incidence of untoward complica- tions as a result of hypothermia are a cause of concern for patients and surgeons. TKA is a procedure frequently done in elderly patients with end-stage osteoarthritis at our University Hospital. Although previous literature on TKA and hypothermia exists from developed countries, there is a scarcity of data from developing countries for complications hypothermia. The differences in settings, resources and risk factors in developing countries merit further research to enhance the clinical course of these patients. The purpose of this study is to determine the frequency of intraoperative hypothermia and factors as- sociated with intraoperative hypothermia in patients undergoing TKA at a tertiary care hospital in Karachi, Pakistan. This can allow surgical teams in developing countries in the future to determine the risk factors that can be controlled to improve postoperative clinical course with regards to intraoperative hypothermia. Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 Page 2 of 6 Ukrani et al. BMC Musculoskeletal Disorders prior to the initiation of this study. The ERC study protocol number is 2019–1274-3270. The study was ap- proved with an exemption of informed consent from pa- tients due to lack of contact with them in a retrospective clinical study. Data was collected from patient charts and files without any interaction with the patient. peripheral circulation which leads to several different events such as tissue hypoxia [8], failure of immune cells to reach target cells and altered neutrophil function which are a major cause of post-operative infection [6, 8, 9]. Other complications include platelet dysfunction leading to bleeding tendencies and increased require- ment for blood transfusions [3, 8, 9], and cardiac events such as tachycardia due to increased catecholamine levels causing a detrimental impact on morbidity and mortality of patients [6, 8, 9]. Post-operative shivering can also lead to unnecessary discomfort and prolonged hospitalizations for the patients [6, 8, 9]. Materials and methods Study location and sample We conducted a retrospective cross-sectional study of patients who underwent total knee arthroplasty at the Aga Khan University Hospital (AKUH) between the period January 2016 to December 2017. Nonprobability consecutive sampling was used for all patients fulfilling the inclusion criteria which was adults ⩾18 years under- going TKA at our University Hospital. The study was conducted in accordance with the eth- ical standards described in the 1964 Declaration of Helsinki and its later amendments. Aga Khan University Ethics Review Committee (ERC) approval was obtained Page 3 of 6 Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 Page 3 of 6 Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 The mean age of patients in the intraoperative hypothermia group was 63.7 ± 10.1 years which was sig- nificantly higher than the mean age of patients in the normothermia group (60.5 ± 10.5 years; p = 0.023). In the intraoperative hypothermia group, 53.9% of patients underwent bilateral TKA (p = 0.018) which was noted to be statistically significant. There were more patients classified as ASA Level 2 in the intraoperative group than in the normothermia group (84.2% vs. 70.0%; p = 0.049). A significantly lower proportion of intraoperative hypothermia patients had hypothermia postoperatively (23.7% with p = 0.002). Only 2 patients developed surgi- cal site infection postoperatively. is also a risk factor for hypothermia due to smaller re- serve of body fat [7, 18]. The findings of our study show that among the patients in the intraoperative hypothermia group, majority of the patients were female (72.4%) and only 27.6% were male. This is also evidenced by the high percentage of 79% females in the study at AKU Hospital by Usmani et al. [19] The patients in our study had multiple co-morbidities, with hypertension (64.3%) being the most common. The prevalence of hypertension in our study is similar to the prevalence of hypertension previously reported in patients undergoing TKA [20]. Majority of the patients in our study had bilateral TKA (66.1%) and a significantly greater number of pa- tients in intraoperative hypothermia group had bilateral TKA (53.9%; p-value = 0.018). Although our findings show that bilateral TKA increased the risk of intraopera- tive hypothermia compared to a unilateral procedure, there is lack of data showing the correlation of bilateral procedures and intraoperative hypothermia in patients for total knee arthroplasty. Discussion The incidence of intraoperative hypothermia in patients undergoing TKA is increasing and is expected to rise to 600% by 2030 [13]. We conducted a retrospective single centre study to determine risk factors of intraoperative hypothermia in patients undergoing TKA. The results of our study highlight that age, bilateral procedure, ASA level and postoperative hypothermia were significantly associated with intraoperative hypothermia. The incidence of intraoperative hypothermia reported in our study was 26.6%. The findings of our study are similar to the results of a study conducted by Frisch et al. who reported 32.6% patients with intraoperative hypothermia during TKA [13]. According to the results of our study, the most com- mon ASA Level was Level 2. In a study conducted by Vural et al. inadvertent hypothermia, in patients from several different fields including orthopedic surgery, was most commonly associated with ASA Level 2 which is consistent with our findings [22]. This study also re- ported that ASA level had an impact on intraoperative body temperature and was a risk factor of development of hypothermia in univariate analysis [22]. However, in multivariate analysis, ASA Level had no effect on hypothermia [22]. Moreover, studies conducted by Belayneh et al. [23] and Monzón et al. [24] also reported that ASA Level was a risk factor for development of hypothermia. The results of our study reported that ASA Level was significantly associated with intraopera- tive hypothermia (p = 0.049). However, more studies are still required to establish an association between ASA classification and development of intraoperative hypothermia in a developing country. The mean age of patients in our study was 61.4 ± 10.4 years, with intraoperative hypothermia group having a significantly higher age than normothermia group (p < 0.023). A previously reported study also had mean age of patients 62 ± 13 years which is similar to the finding of our study [14]. Another study conducted in United Kingdom had higher age in hypothermia group com- pared to normothermia group [2]. Yinan Li et al. also conducted a retrospective study including 1467 patients who underwent video assisted thoracoscopic surgery and reported an increased risk of intraoperative hypothermia with advanced age [15]. Age increases the risk of osteoarthritis and as a result, patients get TKA for man- agement. This association between intraoperative hypothermia and older patients could also be due to de- crease in thermoregulatory vasoconstriction threshold in patients under general anaesthesia with age [15]. Materials and methods Study location and sample Theoretically, in bilateral procedure, the surgical site is exposed to lower tempera- tures for a total longer duration during sequential sur- gery, hence there is an increased risk of hypothermia. A study conducted by Qadir et al. reported a higher incidence of sequential bilateral TKA compared to staged TKA in Pakistan [21]. However, more research is required to assess the effect of bilateral TKA on hypothermia. The results of the study are shown in Table 1. Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 Discussion The percentage of female patients who have had TKA reported in literature vary between approximately 56– 74%. Although, the reason for higher prevalence of TKA among females is unclear, the greater percentage of fe- males in our cohort (77.6%) could be attributed to women being more prone to injuries [12, 13, 16, 17]. Fe- male gender is also considered the strongest risk factor for knee osteoarthritis [17].. In addition, female gender According to Vural et al., intraoperative hypothermia is a risk factor for postoperative hypothermia [22]. Although our results showed that postoperative hypothermia is significantly associated with intraoperative hypothermia (p = 0.002), the incidence of postoperative hypothermia in intraoperative hypothermia group was lower than in Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 Page 4 of 6 normothermia group. The difference in the results can be due to differences in postoperative care. Only 2 (0 7%) patients developed SSI post operatively between intraoperative hypothermia with wound infec- tion, as suggested by other studies [13]. Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available since the Ethical Review Committee guidelines do not allow institutional data to be dispersed. However, the data is available on reasonable request to the corresponding author. 12. Mohib Y, Zahid M, Ashraf I, Noordin S. Does hypothermia really contributes to infection in hip and knee arthroplasty? A tertiary care experience. Int J Surg Open. 2017;8:15–7. https://doi.org/10.1016/j.ijso.2017.06.001. 12. Mohib Y, Zahid M, Ashraf I, Noordin S. Does hypothermia really contributes to infection in hip and knee arthroplasty? A tertiary care experience. Int J Surg Open. 2017;8:15–7. https://doi.org/10.1016/j.ijso.2017.06.001. 13. Frisch NB, Pepper AM, Rooney E, Silverton C. Intraoperative hypothermia in Total hip and knee arthroplasty. Orthopedics. 2017;40(1):56–63. https://doi. org/10.3928/01477447-20161017-04. 13. Frisch NB, Pepper AM, Rooney E, Silverton C. Intraoperative hypothermia in Total hip and knee arthroplasty. Orthopedics. 2017;40(1):56–63. https://doi. org/10.3928/01477447-20161017-04. Funding Funding No funding was obtained during this research. Acknowledgements 8. Giuliano KK, Hendricks J. Inadvertent perioperative hypothermia: current nursing knowledge. AORN J. 2017;105(5):453–63. https://doi.org/10.1016/j.a orn.2017.03.003. Received: 2 December 2020 Accepted: 19 May 2021 Received: 2 December 2020 Accepted: 19 May 2021 Received: 2 December 2020 Accepted: 19 May 2021 References 1. Kleimeyer JP, Harris AHS, Sanford J, Maloney WJ, Kadry B, Bishop JA. Incidence and risk factors for postoperative hypothermia after Orthopaedic surgery. J Am Acad Orthop Surg. 2018;26(24):e497–503. https://doi.org/10. 5435/JAAOS-D-16-00742. 1. Kleimeyer JP, Harris AHS, Sanford J, Maloney WJ, Kadry B, Bishop JA. Incidence and risk factors for postoperative hypothermia after Orthopaedic surgery. J Am Acad Orthop Surg. 2018;26(24):e497–503. https://doi.org/10. 5435/JAAOS-D-16-00742. 2. Williams M, El-Houdiri Y. Inadvertent hypothermia in hip and knee total joint arthroplasty. J Orthop. 2018;15(1):151–8. https://doi.org/10.1016/j.jor.2 018.01.035. 2. Williams M, El-Houdiri Y. Inadvertent hypothermia in hip and knee total joint arthroplasty. J Orthop. 2018;15(1):151–8. https://doi.org/10.1016/j.jor.2 018.01.035. 3. Dan M, Martos SM, Beller E, Jones P, Randle R, Liu D. Blood loss in primary total knee arthroplasty--body temperature is not a significant risk factor--a prospective, consecutive, observational cohort study. J Orthop Surg Res. 2015;10(1):97. https://doi.org/10.1186/s13018-015-0241-5. 3. Dan M, Martos SM, Beller E, Jones P, Randle R, Liu D. Blood loss in primary total knee arthroplasty--body temperature is not a significant risk factor--a prospective, consecutive, observational cohort study. J Orthop Surg Res. 2015;10(1):97. https://doi.org/10.1186/s13018-015-0241-5. Conclusion Intraoperative hypothermia is significantly associated with age, bilateral procedures, ASA level and postopera- tive hypothermia in patients undergoing TKA. The sur- geon and the operative team should be aware of the risk factors and the adverse outcomes associated with intra- operative hypothermia, especially in a resource con- strained developing country like Pakistan. 4. Aksu C, Kuş A, Gürkan Y, Solak M, Toker K. Survey on postoperative hypothermia incidence In operating theatres of Kocaeli University. Turk J Anaesthesiol Reanim. 2014;42(2):66–70. https://doi.org/10.5152/TJAR.2 014.15010. 4. Aksu C, Kuş A, Gürkan Y, Solak M, Toker K. Survey on postoperative hypothermia incidence In operating theatres of Kocaeli University. Turk J Anaesthesiol Reanim. 2014;42(2):66–70. https://doi.org/10.5152/TJAR.2 014.15010. 5. Yi J, Xiang Z, Deng X, Fan T, Fu R, Geng W, et al. Incidence of inadvertent intraoperative hypothermia and its risk factors in patients undergoing general anesthesia in Beijing: a prospective regional survey. PLoS One. 2015; 10(9):e0136136. https://doi.org/10.1371/journal.pone.0136136. 5. Yi J, Xiang Z, Deng X, Fan T, Fu R, Geng W, et al. Incidence of inadvertent intraoperative hypothermia and its risk factors in patients undergoing general anesthesia in Beijing: a prospective regional survey. PLoS One. 2015; 10(9):e0136136. https://doi.org/10.1371/journal.pone.0136136. 6. Madrid E, Urrútia G, Roqué i Figuls M, Pardo-Hernandez H, Campos JM, Paniagua P, et al. Active body surface warming systems for preventing complications caused by inadvertent perioperative hypothermia in adults. Cochrane Database Syst Rev. 2016;4:Cd009016. 6. Madrid E, Urrútia G, Roqué i Figuls M, Pardo-Hernandez H, Campos JM, Paniagua P, et al. Active body surface warming systems for preventing complications caused by inadvertent perioperative hypothermia in adults. Cochrane Database Syst Rev. 2016;4:Cd009016. Authors’ contributions RDU and AA contributed to the study concept, design, data analysis and manuscript writing. AS was involved in the study concept, design and data analysis. OH contributed to data collection and analysis. SN contributed to the study concept, design and final approval of the paper. All authors approved the submitted version and have agreed to be personally accountable for their contributions. The authors read and approved the final manuscript. 9. Leslie K, Sessler DI. Perioperative hypothermia in the high-risk surgical patient. Best Pract Res Clin Anaesthesiol. 2003;17(4):485–98. https://doi.org/1 0.1016/S1521-6896(03)00049-1. 10. Leijtens B, Koëter M, Kremers K, Koëter S. High incidence of postoperative hypothermia in total knee and total hip arthroplasty: a prospective observational study. J Arthroplast. 2013;28(6):895–8. https://doi.org/10.1016/ j.arth.2012.10.006. 11. Long KC, Tanner EJ, Frey M, Leitao MM Jr, Levine DA, Gardner GJ, et al. Intraoperative hypothermia during primary surgical cytoreduction for advanced ovarian cancer: risk factors and associations with postoperative morbidity. Gynecol Oncol. 2013;131(3):525–30. https://doi.org/10.1016/j. ygyno.2013.08.034. 11. Long KC, Tanner EJ, Frey M, Leitao MM Jr, Levine DA, Gardner GJ, et al. Intraoperative hypothermia during primary surgical cytoreduction for advanced ovarian cancer: risk factors and associations with postoperative morbidity. Gynecol Oncol. 2013;131(3):525–30. https://doi.org/10.1016/j. ygyno.2013.08.034. Declarations 14. Ashraf I, Mohib Y, Hasan O, Malik A, Ahmad K, Noordin S. Surgical site infection surveillance following total knee arthroplasty: Tertiary care hospital experience. Ann Med Surg (Lond). 2018;31:14–6. Discussion Further studies are required in order to better under Table 1 Characteristics of TKA Patients Variables Overall (N = 286) Intraoperative Hypothermia p- value Yes (N = 76) No (N = 210) Age 61.4 ± 10.4 63.7 ± 10.1 60.5 ± 10.5 0.023 Gender 0.200 Male 64 (22.4) 21 (27.6) 43 (20.5) Female 222 (77.6) 55 (72.4) 167 (79.5) HTN 0.418 Yes 184 (64.3) 46 (60.5) 138 (65.7) No 102 (35.7) 30 (39.5) 72 (34.3) DM 0.551 Yes 79 (27.6) 19 (25.0) 60 (28.6) No 207 (72.4) 57 (75.0) 150 (71.4) Rheumatoid Arthritis Yes 2 (0.7) 0 (0) 2 (1.0) > 0.999 No 284 (99.3) 76 (100) 208 (99.0) Osteoarthritis 0.252 Yes 9 (3.1) 4 (5.3) 5 (2.4) No 277 (96.9) 72 (94.7) 205 (97.6) Site 0.018 Right 46 (16.1) 19 (25.0) 27 (12.9) Left 51 (17.8) 16 (21.1) 35 (16.7) Bilateral 189 (66.1) 41 (53.9) 148 (70.5) ASA Level 0.049 Level 1 17 (5.9) 2 (2.6) 15 (7.1) Level 2 211 (73.8) 64 (84.2) 147 (70.0) Level 3 58 (20.3) 10 (13.2) 48 (22.9) Preoperative Hypothermia 0.620 Yes 62 (21.7) 18 (23.7) 44 (21.1) No 224 (78.3) 58 (76.3) 166 (79.0) Postoperative Hypothermia 0.002 Yes 38 (13.3) 18 (23.7) 20 (9.5) No 248 (86.7) 58 (76.3) 190 (90.5) SSI 0.462 Yes 2 (0.7) 1 (1.3) 1 (0.5) No 284 (99.3) 75 (98.7) 209 (99.5) Duration of Surgery (mins) 201.2 ± 66.2 Unilateral 131.4 ± 36.1 126.4 ± 38.6 135.2 ± 33.8 0.2466 Bilateral 237.0 ± 46.6 228.3 ± 49.2 240.4 ± 45.3 0.1246 between intraoperative hypothermia with wound infec- tion, as suggested by other studies [13]. normothermia group. The difference in the results can be due to differences in postoperative care. Only 2 (0.7%) patients developed SSI post-operatively with only 1 (1.3%) out of total 76 hypothermic patients having suffered infection which was non-significant. Thereby, our study could not find a positive association Further studies are required in order to better under- stand the risk factors associated with hypothermia and its impact on different post-operative outcomes. In addition, further studies can also help devise Page 5 of 6 Page 5 of 6 Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 Ukrani et al. Abbreviations TKA: Total Knee Arthroplasty; AKUH: Aga Khan University Hospital; ASA Level: American Society of Anesthesiologists (ASA) Level; SSI: Surgical Site Infection; BMI: Body Mass Index; HTN: Hypertension; DM: Diabetes Mellitus 7. Simpson JB, Thomas VS, Ismaily SK, Muradov PI, Noble PC, Incavo SJ. Hypothermia in Total joint arthroplasty: a wake-up call. J Arthroplast. 2018; 33(4):1012–8. https://doi.org/10.1016/j.arth.2017.10.057. 7. Simpson JB, Thomas VS, Ismaily SK, Muradov PI, Noble PC, Incavo SJ. Hypothermia in Total joint arthroplasty: a wake-up call. J Arthroplast. 2018; 33(4):1012–8. https://doi.org/10.1016/j.arth.2017.10.057. Author details 1 Author details 1Medical College, Aga Khan University Hospital, Karachi 74800, Pakistan. 2Department of Orthopaedic Surgery, Aga Khan University Hospital, Karachi 74800, Pakistan. There were several limitations in this study. The sample size was small which could be one of the reasons the post-operative hypothermia results were inconsistent with the literature. Body surface area and BMI was not included in the study. Measurement of temperature was not standardized and data on duration of hypothermia and rewarming strategies was also not recorded. ASA Level 4 category patients are not candidates for quality of life surgery and hence were not included which potentially skews the understanding of the association between ASA and hypothermia as the entire spectrum is not included. Consent for publication Not applicable. Discussion BMC Musculoskeletal Disorders standardized protocols such as effective warming tech- niques as preventative measures to reduce hypothermia and to reduce the risk of complications that may arise due to it. Consent for publication Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Qadir I, Shah B, Waqas M, Ahmad U, Javed S, Aziz A. Component alignment in simultaneous bilateral versus unilateral Total knee arthroplasty. Knee Surg Relat Res. 2019;31(1):31–6. https://doi.org/10.5792/ksrr.18.049. 22. Vural F, Çelik B, Deveci Z, Yasak K. Investigation of inadvertent hypothermia incidence and risk factors. Turk J Surg. 2018;34(4):300–5. https://doi.org/10. 5152/turkjsurg.2018.3992. 23. Belayneh T, Gebeyehu A, Abdissa Z. Post-operative hypothermia in surgical patients at University of Gondar Hospital, Ethiopia. J Anesth Clin Res. 2014; 5(11):1–4. 24. Castillo Monzón CG, Candia Arana CA, Marroquín Valz HA, Aguilar Rodríguez F, Benavides Mejía JJ, Alvarez Gómez JA. Temperature management during the perioperative period and frequency of inadvertent hypothermia in a general hospital. Colombian J Anesthesiol. 2013;41(2):97–103. Ethics approval and consent to participate The study was approved by the Institutional Ethical Review Committee at Aga Khan University Hospital, Karachi with an exemption of informed consent from patients as it is a retrospective study which did not involve any contact with the patient. The reference number for the study was 2019– 1274-3270. The study was approved by the Institutional Ethical Review Committee at Aga Khan University Hospital, Karachi with an exemption of informed consent from patients as it is a retrospective study which did not involve any contact with the patient. The reference number for the study was 2019– 1274-3270. 15. Li Y, Liang H, Feng Y. Prevalence and multivariable factors associated with inadvertent intraoperative hypothermia in video-assisted thoracoscopic surgery: a single-center retrospective study. BMC Anesthesiol. 2020;20(1):25. https://doi.org/10.1186/s12871-020-0953-x. Page 6 of 6 Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 Ukrani et al. BMC Musculoskeletal Disorders (2021) 22:504 16. Martin JR, Beahrs TR, Stuhlman CR, Trousdale RT. Complex primary Total knee arthroplasty: Long-term outcomes. J Bone Joint Surg Am. 2016;98(17): 1459–70. https://doi.org/10.2106/JBJS.15.01173. 17. Koh IJ, Kim TK, Chang CB, Cho HJ, In Y. Trends in Use of Total Knee Arthroplasty in Korea From 2001 to 2010. Clin Orthopaed Related Res. 2013; 471(5):1441–50. 18. Scholten R, Leijtens B, Kremers K, Snoeck M, Koëter S. The incidence of mild hypothermia after total knee or hip arthroplasty: a study of 2600 patients. J Orthop. 2018;15(2):408–11. https://doi.org/10.1016/j.jor.2018.03.014. 19. Usmani MA, Zahid M, Ahmad T, Umer M, Hu R, Hashmi PM, et al. Surgical care improvement program and surgical site infections: initiatives to improve outcome in patients with joint replacements in a tertiary care center in Pakistan. IJS Glob Health. 2019;2(2):e05. 19. Usmani MA, Zahid M, Ahmad T, Umer M, Hu R, Hashmi PM, et al. Surgical care improvement program and surgical site infections: initiatives to improve outcome in patients with joint replacements in a tertiary care center in Pakistan. IJS Glob Health. 2019;2(2):e05. 20. Raddaoui K, Khedhri W, Zoghlami K, Radhouani M, Trigui E, Kaabachi O. Perioperative morbidity in total knee arthroplasty. Pan Afr Med J. 2019;33:233. 20. Raddaoui K, Khedhri W, Zoghlami K, Radhouani M, Trigui E, Kaabachi O. Perioperative morbidity in total knee arthroplasty. Pan Afr Med J. 2019;33:233. 21. Qadir I, Shah B, Waqas M, Ahmad U, Javed S, Aziz A. Component alignment in simultaneous bilateral versus unilateral Total knee arthroplasty. Knee Surg Relat Res. 2019;31(1):31–6. https://doi.org/10.5792/ksrr.18.049. 21. 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4301169259	https://eprints.whiterose.ac.uk/167248/1/s13063_016_1732_3.pdf	English	null	Effectiveness of an implementation optimisation intervention aimed at increasing parent engagement in HENRY, a childhood obesity prevention programme - the Optimising Family Engagement in HENRY (OFTEN) trial: study protocol for a randomised controlled trial	Carolina Digital Repository (University of North Carolina at Chapel Hill)	2,017	cc-by	11,123	This is a repository copy of Effectiveness of an implementation optimisation intervention aimed at increasing parent engagement in HENRY, a childhood obesity prevention programme - the Optimising Family Engagement in HENRY (OFTEN) trial: study protocol for a randomised controlled trial. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/167248/ Version: Published Version Article: Article: Bryant, Maria orcid.org/0000-0001-7690-4098, Burton, Wendy orcid.org/0000-0001-7885- 5971, Cundill, Bonnie et al. (10 more authors) (2017) Effectiveness of an implementation optimisation intervention aimed at increasing parent engagement in HENRY, a childhood obesity prevention programme - the Optimising Family Engagement in HENRY (OFTEN) trial: study protocol for a randomised controlled trial. Trials. 40. ISSN 1745-6215 https://doi.org/10.1186/s13063-016-1732-3 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. 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Farrin1, Jane Nixon1, June Stevens2,3, Kim Roberts4, Robbie Foy5, Harry Rutter6, Suzanne Hartley1, Sandy Tubeuf7, Michelle Collinson1 and Julia Brown1 Abstract Open Access © The Author(s). 2016 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. Effectiveness of an implementation optimisation intervention aimed at increasing parent engagement in HENRY, a childhood obesity prevention programme - the Optimising Family Engagement in HENRY (OFTEN) trial: study protocol for a randomised controlled trial Maria Bryant1* , Wendy Burton1, Bonnie Cundill1, Amanda J. Farrin1, Jane Nixon1, June Stevens2,3, Kim Roberts4, Robbie Foy5, Harry Rutter6, Suzanne Hartley1, Sandy Tubeuf7, Michelle Collinson1 and Julia Brown1 * Correspondence: m.j.bryant@leeds.ac.uk 1Leeds Institute of Clinical Trials Research, University of Leeds, Leeds LS29JT, UK Full list of author information is available at the end of the article Background all parenting interventions delivered in community set- tings to optimise their impact and so that the degree to which they are effective can be assessed with confidence. In contrast to conventional pathways, in which imple- mentation research is conducted following trials for clin- ical effectiveness [23], it is argued that there is a need for comprehensive early-phase (evidentiary) evaluation and enhancement of complex interventions such as those designed to prevent childhood obesity [24], prior to the conduct of large randomised controlled trials (RCTs). This novel approach ensures that factors which limit trial outcomes, such as low adherence, are mini- mised prior to dedicating the resources required to con- duct a large trial which may identify no evidence of effectiveness, perhaps as a result of poor compliance. Although the most recent data suggest a levelling off of the prevalence of childhood obesity in the United Kingdom, levels remain high and there are clear inequal- ities, with rates continuing to rise in more deprived and ethnic minority groups [1]. Childhood obesity impacts physiological and psychological health, which tracks into adulthood [2, 3], increasing the risk of morbidity and mortality [4, 5]. It imposes significant costs on the U.K. economy, with an expected sevenfold increase in related NHS costs by 2020 and a forecasted £2 billion annual spend by 2030 [6]. Tackling obesity is a national public health priority in terms of both treatment and preven- tion. Establishing healthy behaviours in early childhood is critical for optimum growth and development [7]. Fur- ther, poor eating patterns developed early can persist and are associated with chronic diseases in adulthood (e.g., cardiovascular disease, type 2 diabetes mellitus [8]). Once established, obesity is difficult to reverse [9], strengthening the case for primary prevention [10, 11]. To reverse trends in obesity, there is a clear need to en- gage parents in shaping healthy weight-related behav- iours in their children. The present trial involves the evaluation of an imple- mentation enhancement ‘optimisation intervention’ of an existing preschool obesity prevention group programme, HENRY (Health Exercise Nutrition for the Really Young), to promote parent enrolment and attend- ance prior to establishing its clinical effectiveness and cost-effectiveness. HENRY is an 8-week group-delivered programme provided to parents of preschool children. (Continued from previous page) (Continued from previous page) Discussion: This innovative trial will provide evidence on the implementation of a theory-informed optimisation intervention to promote parent engagement in HENRY, a community-based childhood obesity prevention programme. The findings will be generalisable to other interventions delivered to parents in other community-based environments. This research meets the expressed needs of commissioners, children’s centres and parents to optimise the potential impact that HENRY has on obesity prevention. A subsequent cluster randomised controlled pilot trial is planned to determine the practicality of undertaking a definitive trial to robustly evaluate the effectiveness and cost-effectiveness of the optimised intervention on childhood obesity prevention. Trial registration: ClinicalTrials.gov identifier: NCT02675699. Registered on 4 February 2016. Keywords: Engagement, Trial, Implementation, Childhood obesity, Parent, Recruitment Abstract Background: Family-based interventions to prevent childhood obesity depend upon parents’ taking action to improve diet and other lifestyle behaviours in their families. Programmes that attract and retain high numbers of parents provide an enhanced opportunity to improve public health and are also likely to be more cost-effective than those that do not. We have developed a theory-informed optimisation intervention to promote parent engagement within an existing childhood obesity prevention group programme, HENRY (Health Exercise Nutrition for the Really Young). Here, we describe a proposal to evaluate the effectiveness of this optimisation intervention in regard to the engagement of parents and cost-effectiveness. Methods/design: The Optimising Family Engagement in HENRY (OFTEN) trial is a cluster randomised controlled trial being conducted across 24 local authorities (approximately 144 children’s centres) which currently deliver HENRY programmes. The primary outcome will be parental enrolment and attendance at the HENRY programme, assessed using routinely collected process data. Cost-effectiveness will be presented in terms of primary outcomes using acceptability curves and through eliciting the willingness to pay for the optimisation from HENRY commissioners. Secondary outcomes include the longitudinal impact of the optimisation, parent-reported infant intake of fruits and vegetables (as a proxy to compliance) and other parent-reported family habits and lifestyle. (Continued on next page) Page 2 of 13 Bryant et al. Trials (2017) 18:40 Background It was developed in 2006 with joint funds from the Depart- ment of Health and the Department for Education (then called the Department of Children, Schools and Families) and is currently commissioned and delivered across the United Kingdom by 32 local authorities providing more than 150 programmes each year. It is delivered in com- munity settings, often in children’s centres by children’s centre staff [25]. HENRY uses a responsive approach to provide practical guidance and improve parenting skills aimed at enhancing family lifestyle and children’s centre environments. Preliminary data indicate that HENRY may be effective at preventing childhood obesity and im- proving family health [25], although there is not yet evi- dence from an RCT. Design, implementation and evaluation of interven- tions to prevent or treat obesity may be expensive and time-consuming, and researchers often report a lack of effectiveness [12–15]. Within the literature on childhood obesity, low parent enrolment and attendance at group- delivered programmes, along with a lack of reported be- haviour change, are commonly described to have had a substantial impact on group dynamics, effectiveness and cost-effectiveness [16–18]. There is a need for pragmatic, ‘real-world’ evaluations of interventions to understand the generalisability of the interventions across everyday practice [19, 20]. Further, in order for interventions to be accurately implemented and interpreted, the underpinning behaviour change techniques need to be clearly defined [21]. Implementa- tion optimisation strategies can then be adopted to tar- get a specific intervention component [22]. It is imperative that parent engagement be improved across Despite some indications of success of HENRY from audit [25] and qualitative data [26–28], process evalu- ation indicates implementation targets are often not met. Lack of involvement by some parents may be a Bryant et al. Trials (2017) 18:40 Page 3 of 13 Page 3 of 13 developed to optimise parent engagement in obesity pre- vention programmes, and which have been evaluated using an RCT design. To meet this need, we developed a parent engagement optimisation intervention following Medical Research Council (MRC) guidance for develop- ment of complex interventions [38]. The Consolidated Framework for Implementation Research was used to understand contextual factors associated with the imple- mentation of HENRY and their impact on parent en- gagement [43]. Background The Behaviour Change Wheel [44] provided guidance for the development of the interven- tion, which features use of the ‘COM-B’ model (capabil- ity, opportunity and motivation) to dissect behaviours into their individual and interacting components and consider which of these should be targeted in an inter- vention to bring about the desired behaviour change. A rapid ethnography was conducted in five children’s cen- tres to understand the environmental, social, psycho- social, political and economic factors associated with parent engagement in HENRY. This study gathered data from approximately 190 h of observations, 22 interviews with children’s centre managers and staff, HENRY com- missioners, and HENRY coordinators and facilitators. Six focus groups were also conducted with parents who had attended the programme. We convened an intervention development team consisting of experts in intervention development, obesity, applied health and behaviour change, a local authority representative, a HENRY parent champion, a parent who has attended the programme, and a representative from the HENRY team. The inter- vention team used evidence from the ethnography, from the literature, and from their own experiences and ex- pertise to develop a multi-component optimisation intervention which attempts to promote parent engage- ment and positive behaviour change in families by sup- porting local authorities, children’s centres, HENRY staff, and parents using candidate implementation and deliv- ery strategies (Career Development Fellowship CDF- 2014-07-052). Here we report the protocol of our cluster randomised controlled trial (cRCT) (using routine process data) to evaluate the optimisation intervention for its ability to engage parents in HENRY prior to con- ducting a further evaluation of its clinical effectiveness. threat to the viability of this programme and other, simi- lar community-based interventions. The effectiveness of interventions in real-world conditions may not match the efficacy found in research studies [29]. This may be particularly relevant to population-based prevention pro- grammes, in which parents may be less likely to be moti- vated to attend because their children show no clinical symptoms [30, 31]. Thus, it is imperative to create tai- lored methods to maximise parent participation and en- hance successful implementation in childhood obesity prevention programmes. g Failure to attend interventions is complicated by issues of health inequalities; socio-demographic attributes; and, in some cases, challenges related to safeguarding [30, 32, 33]. Literature on non-attendance in other areas (clinical appointments) indicates that attendance is lower in cer- tain populations and may be an indicator of vulnerability [34]. Background This is pertinent to obesity because prevalence rates continue to rise in children in lower socio- economic or ethnic minority groups. In addition to the anticipated public health benefits, the economic benefits of implementation optimisation in programmes such as HENRY are substantial, with one parenting intervention calculating an extra £800 cost per child in programmes that run with 8 parents compared with those running at the intended capacity of 12 parents [35]. Unlike the development of other robust behaviour change interventions, initiatives to improve parental at- tendance at clinical appointments have been confined largely to simple approaches such as text reminders [34, 36, 37]. To promote behaviour change, enhance the transparency of interventions and guide their generalis- ability, it is recognised that a systematic approach is used during intervention development, which is under- pinned by theories of behaviour change and guided by an intervention-planning framework such as the Behav- iour Change Wheel [24, 38]. Lessons can be learnt from child mental health research, in which many interven- tions have been developed and tested using robust theory-based approaches which carefully consider parent engagement [33]. Research in this field has explored per- ceived barriers to participation with suggested strategies to engage parents, including promotion of self-efficacy and treatment motivation. Such strategies perceive par- ents as the central agents of change and aim to modify the pre-treatment experience [33, 39]. RCTs evaluating parent engagement in mental health services [40] and drug misuse [41] indicate that parental engagement can be increased with a dedicated optimisation component, although a further trial focused on cultural relevance for child behaviour problems to optimise engagement [42] did not find any increase. Methods/design Design This study is a two-arm, multi-centre cRCT across 24 local authorities (local governments) (supporting ap- proximately 144 children’s centres) in the United Kingdom to determine the effectiveness and cost- effectiveness of an optimisation intervention to promote parent engagement in the HENRY programme com- pared with standard HENRY practice (Fig. 1). Although the children’s centres deliver the HENRY programme, To our knowledge, there are no theory-based, multi- component optimisation interventions that have been Bryant et al. Trials (2017) 18:40 Page 4 of 13 Fig. 1 Trial design flow diagram. HENRY Health Exercise Nutrition for the Really Young, ITT Intention to treat Fig. 1 Trial design flow diagram. HENRY Health Exercise Nutrition for the Really Young, ITT Intention to treat Fig. 1 Trial design flow diagram. HENRY Health Exercise Nutrition for the Really Young, ITT Intention to treat 1. The effect of the optimisation intervention on achieving combined parent enrolment, attrition and compliance targets 1. The effect of the optimisation intervention on achieving combined parent enrolment, attrition and compliance targets owing to the multi-component and interactive nature of the optimisation intervention, local authorities will be the units of randomisation (i.e., clusters) to prevent con- tamination between the randomised groups. Outcomes will be assessed through routinely collected data avail- able for each HENRY programme at the parent and chil- dren’s centre levels. Information on commissioners’ willingness to pay (within local authorities) related to the optimisation intervention will be elicited. A process evaluation will be conducted alongside the cRCT in ac- cordance with the MRC guidance on evaluating process in complex interventions [19]. 2. The effect of the optimisation intervention on parent compliance with HENRY programme content 3. The effect of the optimisation intervention on parent-reported family habits and lifestyle y y 4. The potential longitudinal impact (sustainability) of the optimisation intervention on enrolment and attrition in centres that provide data from more than one programme Setting The study will be conducted in local authorities with children’s centres where staff are currently trained to de- liver the HENRY programme. There are currently 32 local authorities (approximately 144 centres) in the United Kingdom running the programme, all of which provide process data to the central HENRY office for monitoring/quality assurance (QA) purposes. These data will be anonymised and transferred for analysis. Objectives Primary objectives of the trial are to determine the following: 1. The effect of the optimisation intervention applied to HENRY compared with standard HENRY in regard to increasing parent enrolment in HENRY programmes or reducing parent attrition within HENRY programmes Inclusion criteria Local authorities comprising the following: Secondary objectives of the study are to determine: Recruitment and consent Fig. 2 Recruitment/opt-out process. HENRY Health Exercise Nutrition for the Really Young, LA Local authorities Local authorities and their centres across the United Kingdom will be identified from an existing database of HENRY delivery sites (see Fig. 2). Those meeting the eli- gibility criteria will be contacted by invitation letter (pos- tal and electronic) issued jointly by the HENRY central office and the University of Leeds. HENRY is currently commissioned across the whole of the United Kingdom, with most sites situated within England. conducted in public health. The Leeds Institute of Clin- ical Trials Research (LICTR) has experience in using an opt-out approach and has found that it (1) is more likely than opt-in methods to provide a more represen- tative or ‘typical’ participating sites; (2) appears more effective and efficient in promoting research participa- tion by sites; and (3) is acceptable, provided that suffi- cient sensitivity and safeguards are applied [46]. In this instance, individual-/family-level consent is not re- quired, because all patient-level data will be unlinked and anonymous and is routinely collected by centres and submitted to the central HENRY office. Centres will not be asked to conduct any additional data collec- tion from parents for the purposes of this trial. Our parent advisory panel (patient and public involvement group) and trial steering committee (TSC) have both approved this process. Explicit consent will not be sought. Instead, opt-out consent will be sought at the cluster level (local author- ities) and from the centres within each local authority. It will be possible for centres to decline participation in the randomisation, even if they are based within a con- senting local authority. However, if a local authority de- clines to take part in the trial, none of its centres will be eligible to take part. Exclusion criteria related to fur- ther commissioning of HENRY (local authorities) and plans to run HENRY programmes (centres) will be self- reported by commissioners and centres, respectively. This opt-out method has been chosen because this is a low-risk trial in which anonymised data will be ex- tracted remotely. It has been advocated to promote a simple and efficient trial design, and literature suggests that it is acceptable to participants [45]. Exclusion criteria Exclusion criteria Local authorities: Exclusion criteria Local authorities: Local authorities which plan to decommission the HENRY intervention during the period of the trial or which are not planning to run any HENRY programmes during the trial period Children’s centres: Centres which were recruited in the development phase of the optimisation intervention (participating in the rapid ethnography) p g p y Centres which are not planning to run any HENRY programmes during the trial period There will be no exclusions based on the demograph- ics of children’s centres, but location will be monitored to ensure inclusion of those with diverse social and environmental characteristics. Randomisation will also stratify by area-level deprivation to ensure balance be- tween trial arms. Inclusion criteria Inclusion criteria Local authorities comprising the following: 2. The willingness to pay of commissioners of the HENRY programme per additional level of parent engagement related to the optimisation intervention (Costs will be presented as an incremental cost per increase of parent engagement, so that commissioners are able to identify the point at which additional costs are acceptably offset by the benefits of engaging effectively with parents.) Local authorities comprising the following: Local authorities that commission HENRY and consent for their centres to be involved in the research Commissioning HENRY programmes delivered by trained staff who have been certified by HENRY Children’s centres comprising the following: Children’s centres comprising the following: Secondary objectives of the study are to determine: Bryant et al. Trials (2017) 18:40 Page 5 of 13 Centres providing routinely collected data for the most recent HENRY programme that they ran at the point of randomisation Fig. 2 Recruitment/opt-out process. HENRY Health Exercise Nutrition for the Really Young, LA Local authorities Recruitment and consent It also repli- cates, as far as possible, the ‘real-life’ conditions under which such quality improvement initiatives are usually Local authorities and children’s centres will be given 30 days from the date on which the initial information letter is sent to decline participation (confirmed by re- corded delivery). Centres that opt out after randomisa- tion will be treated as ‘withdrawn’. Bryant et al. Trials (2017) 18:40 Page 6 of 13 Page 6 of 13 Unit of randomisation for the duration of the trial, the HENRY QA team have agreed to reallocated areas that they are responsible for, so that only one person will have contact with areas allocated to the optimisation intervention. Other QA staff will be aware that they have standard QA responsibility (e.g., an- swering questions, providing encouragement, checking on progress) for areas not randomised to the optimisation, but will not know the details or content of the optimisa- tion intervention. To further avoid contamination, the de- tails of the optimisation intervention will not be published until after the final analysis. Unplanned masking (unblind- ing) will be monitored and reported to the TSC. Local authorities will be randomised in a 1:1 allocation ratio (HENRY + optimisation intervention, HENRY as standard) by a statistician at LICTR, using an algorithm for covariate-constrained randomisation [47] to achieve a balanced allocation between the trial arms on (1) local authority baseline level of parental engagement with HENRY (proportion of centres recruiting a minimum of eight parents per programme, proportion of centres retaining at least 75% of parents for a minimum of five of eight sessions), (2) proportion of centres running at least one HENRY programme in 2015, (3) size of local authorities (number of children’s centres participating), and (4) area deprivation (proportion of centres in the least/most deprived quintiles as ranked by the 2015 Index of Multiple Deprivation at the Lower Layer Super Output Area) [48]. Randomisation will be performed for all local authorities at a single time point after baseline data are transferred, prior to the implementation of the intervention. All possible enumerations of allocation to the trial arms will be generated, and an imbalance statis- tic will be calculated for each allocation. A set of optimal allocations which minimise the imbalance will be pro- vided, and, in accordance with the principles of Inter- national Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidance on randomness, the final alloca- tion will be randomly selected from among this optimal set [49]. Results of the randomisation procedure will be sent by the LICTR statistician to the HENRY central of- fice, which will be responsible for informing local au- thorities of their treatment allocation. Intervention HENRY HENRY is an 8-week programme delivered in children’s centres with the aim of providing parents with skills, knowledge and confidence to support healthy lifestyles among their preschool children and their families, in- cluding parenting skills, emotional well-being and activ- ity. The programme was set up in 2006 with the aim of reversing rising trends in school entry age obesity. HENRY is currently delivered within 32 local authorities across England and Wales by trained health and com- munity practitioners who undergo two stages of training [26]. Stage 1, centre-level training, is a 2-day workshop designed to equip centre staff with knowledge and skills to promote and provide healthy nutrition within early years settings and support parents to provide healthy family lifestyles and nutrition for their families. The the- oretical underpinning combines proven models of be- haviour change, including the Family Partnership Model, motivational interviewing and solution-focused support. Stage 2, practitioner-level training to deliver the HENRY programme to families, is provided to approximately four practitioners per centre after completing Stage 1 to deliver the 8-week HENRY programme. The aim of this stage is to build parents’ skills, knowledge and confidence to change old habits, provide healthier nutrition for their young children, and encourage healthier lifestyles [25, 27]. Programme content includes sessions on lifestyle and eat- ing habits (e.g., family meals), balancing healthy meals and snacks, child-appropriate portion sizes, parenting, physical activity and emotional well-being. Blinding Owing to the nature of the intervention, it will not be possible to blind allocation within intervention sites or of those involved in the optimisation intervention train- ing. Families attending the HENRY programme have been made aware that HENRY uses data anonymously for research, but they have not been told explicitly about the Optimising Family Engagement in HENRY (OFTEN) trial or whether their local authority has been assigned to the optimisation. HENRY is coordinated by a central team that is responsible for coordinating training and for providing support and QA to areas that commission HENRY. Because the HENRY central office is integral to the delivery of the optimisation intervention, it will not be possible to blind the HENRY central office (as it will communicate the randomisation allocation to all those responsible for its implementation); however, staff who are responsible for collating data and transferring it to LICTR will remain blinded to allocation. The chief ex- ecutive officer of HENRY will be blinded to treatment allocation and will not attend TSC meetings. In addition, Current HENRY QA practice involves the review of process data by dedicated individuals in the HENRY cen- tral office with provision of written and oral feedback. This QA will continue in both trial arms and will be monitored. Staff delivering current QA activities will be blinded to treatment allocation. HENRY + parent engagement optimisation intervention We summarise only the key features of the optimisation intervention in the trial protocol to minimise Bryant et al. Trials (2017) 18:40 Bryant et al. Trials (2017) 18:40 Page 7 of 13 – Parenting self-efficacy (assessed via four parenting confidence items with 5-point Likert scales, modified from the validated Parenting Self-Agency Measure [50]). This measure assesses the parents’ estimate of their ability to influence their child and environment to lead to positive development, which is a key goal of the HENRY approach. In order for the questionnaire to be feasible to administer as part of the service, HENRY chose to reduce the number of items to a single scale of parenting confidence. Internal consistency of these items in a previous HENRY evaluation was high, and internal consistency in this sample was high (Cronbach’s alpha = 0.82 at baseline, 0.74 at completion) [51]. contamination (i.e., uptake of initiatives of the interven- tion by local authorities randomised to the control arm) and maintain blinding. The optimisation intervention consists of interacting components delivered to local authorities, children’s centres and HENRY facilitators. It includes additional support to stakeholders, modifi- cation of HENRY marketing to clarify perceptions of the programme, and methods to modify the pre- programme experience for parents. Strategies have been developed to increase parent motivation to enrol in HENRY and promote parent self-efficacy to con- tinue to attend. Implementation of the optimisation intervention will begin immediately following random- isation. All components of the optimisation interven- tion will be implemented within 6 months following randomisation. Eating behaviours (based on the Golan Family Eating and Activity Habits Questionnaire [52]). This validated measure is sensitive to change and demonstrates high levels of reliability and validity. HENRY facilitators routinely administer six items related to family behaviours, included parent report of family sitting together for meals, watching television during mealtimes, consuming take-out meals, consumption of home-cooked foods, stopping eating when full and choosing healthy meals. Individual evaluation of these items will be undertaken because reliability for the scale was poor when previously tested using HENRY families (Cronbach’s alpha = 0.52 at baseline, 0.56 at completion). Secondary outcomes Secondary outcomes, measured 12 months post- randomisation (unless stated otherwise), include some further assessment of parent engagement to explore the degree to which the intervention was effective. Data from additional questionnaires which are routinely ad- ministered by HENRY facilitators at the beginning and end of each 8-week course will also been analysed to ex- plore the potential impact of the optimisation on behav- ioural outcomes, outlined below: – Child screen time (via a parent reported using the bespoke HENRY questionnaire). This assesses use of televisions, DVDs, computers, smartphones, and so forth with response categories ranging from none to <1 h, 1–2 h, 2–3 h, or >3 h. – Intake of key indicator foods per day (assessed via 14 items from a modified validated Food Frequency Questionnaire [53] completed in relation to the parent and child). Parents are asked to estimate how often (never, once per month, once per fortnight, 1–7 days per week) they consumed each of the 14 items or groups of – Child screen time (via a parent reported using the bespoke HENRY questionnaire). This assesses use of televisions, DVDs, computers, smartphones, and so forth with response categories ranging from none to <1 h, 1–2 h, 2–3 h, or >3 h. – Intake of key indicator foods per day (assessed via 14 items from a modified validated Food Frequency Questionnaire [53] completed in relation to the parent and child). Parents are asked to estimate how often (never, once per month, once per fortnight, 1–7 days per week) they consumed each of the 14 items or groups of – Child screen time (via a parent reported using the bespoke HENRY questionnaire). This assesses use of televisions, DVDs, computers, smartphones, and so forth with response categories ranging from none to <1 h, 1–2 h, 2–3 h, or >3 h. – Child screen time (via a parent reported using the bespoke HENRY questionnaire). This assesses use of televisions, DVDs, computers, smartphones, and so forth with response categories ranging from none to <1 h, 1–2 h, 2–3 h, or >3 h. Proportion of centres achieving all targets for enrolment, attrition and parent compliance – Intake of key indicator foods per day (assessed via 14 items from a modified validated Food Frequency Questionnaire [53] completed in relation to the parent and child). Primary outcome The effectiveness of the optimisation intervention will be determined by comparing parent engagement in HENRY + optimisation intervention vs HENRY alone. The co-primary outcomes are (1) the proportion of cen- tres enrolling at least eight parents per programme and (2) the proportion of centres with at least 75% of parents attending five of eight sessions per programme. Both will be evaluated 12 months post-randomisation. The opti- misation intervention will be deemed effective if either the enrolment or attrition goals are met. p Family activity (via a brief HENRY questionnaire asking parents to report the frequency that parents and children engage in exercise). This questionnaire has been developed bespoke to use within the HENRY evaluations and includes parental report of their own activities that result in breathlessness, with response categories ranging from none to <1 h, 2 h, 3 h or >3 h. Children’s activity is assessed using parental report as energetic play, with response categories ranging from none to 5–15 minutes, 20–30 minutes, 30 minutes to 1 h, or >1 h. Family activity (via a brief HENRY questionnaire asking parents to report the frequency that parents and children engage in exercise). This questionnaire has been developed bespoke to use within the HENRY evaluations and includes parental report of their own activities that result in breathlessness, with response categories ranging from none to <1 h, 2 h, 3 h or >3 h. Children’s activity is assessed using parental report as energetic play, with response categories ranging from none to 5–15 minutes, 20–30 minutes, 30 minutes to 1 h, or >1 h. HENRY as standard Local authorities randomised to the control arm will continue to deliver HENRY programmes as per standard practice. Secondary outcomes Parents are asked to estimate how often (never, once per month, once per fortnight, 1–7 days per week) they consumed each of the 14 items or groups of Parent adherence to HENRY programme content, defined as the proportion of parents reporting an increase of 0.5 in the daily frequency of consumption of fruits and vegetables by children per programme g y p p g Impact of HENRY on parenting and family health, assessed by parent-report, including the following: Bryant et al. Trials (2017) 18:40 Page 8 of 13 Page 8 of 13 foods (e.g., fresh fruit, sweets, chocolate, water), with space to report the number of times consumed. It has previously demonstrated sensitivity to change with parents attending HENRY sessions [25]. foods (e.g., fresh fruit, sweets, chocolate, water), with space to report the number of times consumed. It has previously demonstrated sensitivity to change with parents attending HENRY sessions [25]. optimisation intervention is cost-effective for a range of maximum monetary values that a decision-maker might be willing to pay for a particular unit change in outcome. Cost-effectiveness analysis leaves it to decision-makers to form their own view of the relative importance of these results [54]; therefore, in a second part of the work, we will conduct a meeting with commissioners to determine their willingness to pay per additional unit of effectiveness (parent engagement) before concluding whether the optimisation intervention can be considered cost-effective. We will use contingent valuation tech- niques and design an experiment to estimate the point at which commissioners consider that the costs of the programme are acceptably offset by the benefits of en- gaging effectively with parents. Longitudinal impact of HENRY + optimisation intervention vs HENRY alone (parent engagement [enrolment and attrition] within centres during the 12-month follow-up in local authorities which provide data for more than one programme per centre at follow-up) (This evaluation will explore whether the optimisation intervention needs time to ‘bed-in’ and/or whether any early effects are maintained over time.) Longitudinal impact of HENRY + optimisation intervention vs HENRY alone (parent engagement [enrolment and attrition] within centres during the 12-month follow-up in local authorities which provide data for more than one programme per centre at follow-up) (This evaluation will explore whether the optimisation intervention needs time to ‘bed-in’ and/or whether any early effects are maintained over time.) Sample size W d p We assumed that 25% of the 32 local authorities cur- rently running HENRY will not be eligible or will opt out of the study, leaving 24 local authorities (12 per arm). Power calculations for this fixed sample size (with all eligible centres in the United Kingdom invited to take part) were therefore conducted to examine anticipated power for various intervention effects, at the 5% signifi- cance level, in each of the primary outcomes and for the composite endpoint (enrol at least eight parents per programme and retain ≥75% of parents attending five of eight sessions) (see Table 1 for scenarios). On the basis of data from previous HENRY programmes (all terms in 2014), we assumed an average of 6 children’s centres per local authority providing a total of 144 children’s centres (72 per arm), an intra-cluster correlation coefficient (ICC) between 0.05 and 0.1, a coefficient of variation in cluster size of 0.54, and the following estimates of the outcomes in the control sites (HENRY alone): 55% of centres will enrol at least eight parents per programme; 50% of centres will retain ≥75% of parents attending five of eight sessions; and 25% of centres will enrol at least eight parents per programme and retain ≥75% of parents attending five of eight sessions. Thus, with the antici- pated number of centres (24 local authorities, 144 chil- dren’s centres), we will have at least 80% power to detect meaningful improvements in differences of between 25% and 30% in either of the primary endpoints or the com- posite endpoint at the 5% significance level, assuming the ICC is no greater than 0.05. Process evaluation A process evaluation will be conducted by an LICTR re- searcher in accordance with MRC guidance on evaluat- ing process in complex interventions [19]. In brief, the aims of this evaluation will be to (1) examine the uptake, delivery and acceptability of the optimisation interven- tion across local authorities/children’s centres (reach); (2) explore the effects of individual intervention compo- nents at increasing parent engagement, along with po- tential unintended consequences; and (3) consider the utility of theories underpinning the intervention and ap- proach used to develop the intervention design. Process measures will include quantitative measurement of centre uptake and fidelity of delivered trial components; qualitative and quantitative analysis of stakeholder perceptions of intervention components considering individual, organisational and contextual factors; quanti- tative measurement of enrolment methods used to re- cruit HENRY programme participants; and qualitative and/or quantitative measurement of predicted behaviour change outcomes among stakeholders targeted in the implementation optimisation. Quantitative data required for process evaluation will be gathered and reported on paper case report forms by a researcher within the LICTR, who will also collect qualitative data from a ran- dom selection of sites in person, including interviews and observations. Economic evaluation Economic evaluation will model the benefits of optimisa- tion intervention over the costs of implementation and will seek commissioners’ willingness to pay for this. It will include the costs required to deliver the intervention (gathered and transferred from HENRY) and the rou- tinely collected outcome data. Data collection and transfer Data collection and transfer In this trial, we will use routinely collected data for all primary and secondary outcomes, except for the process evaluation data, which will be gathered by an independ- ent LICTR researcher. HENRY has designed a centra- lised process for evaluating programmes, developed for QA purposes and reporting to commissioners of the The cost-effectiveness analysis in the present trial will include an array of trial endpoints (including parent en- rolment and attrition rates) and costs. Cost-effectiveness acceptability curves will describe the probability that the Page 9 of 13 Page 9 of 13 Bryant et al. Trials (2017) 18:40 Table 1 Power calculations for enrolment and attrition endpoints for various estimates of the intervention effect and a fixed sample size of 144 children’s centres in 24 local authorities Outcome in the control Percentage point increase in intervention Power for ICC = 0.1 Power for ICC = 0.05 Enrolment (≥8 parents per programme) 55% 5% 6% 7% 10% 15% 18% 15% 29% 35% 20% 49% 58% 25% 70% 79% 30% 87% 93% Attrition (≥75% of parents attending five of eight sessions) 50% 5% 6% 7% 10% 15% 17% 15% 28% 34% 20% 47% 55% 25% 67% 76% 30% 84% 91% Enrolment and attrition (at least eight parents per programme and 75% of parents attending five of eight sessions) 25% 5% 7% 8% 10% 17% 19% 15% 31% 37% 20% 49% 58% 25% 67% 76% 30% 82% 89% CC l l ff Table 1 Power calculations for enrolment and attrition endpoints for various estimates of the intervention effect and a fixed sample size of 144 children’s centres in 24 local authorities Table 1 Power calculations for enrolment and attrition endpoints for various estimates of the intervention effect and a fixed sample size of 144 children’s centres in 24 local authorities information on participant satisfaction, parent-reported compliance, parenting and family lifestyle (Table 2), which are gathered in questionnaires on the first and last days of each HENRY programme. This information is submitted to the central HENRY office (without any personal iden- tifiers for the families), which check the data for complete- ness and collate the information in a.csv file. For the trial period, centre-level baseline data from the most recent programme delivered prior to randomisa- tion (winter term 2015) will be used. Follow-up data will be gathered 12 months after randomisation. Data collection and transfer It will in- clude routine data collected from the autumn and spring terms (2016–17), allowing 6 months for intervention de- livery at all sites followed by the delivery of two terms of HENRY programmes. In accordance with the planned intention-to-treat (ITT) analysis, data will be collected from centres that are not compliant with the interven- tion. For those withdrawing from the trial, only data col- lected up to the point of withdrawal will be used. 50% 5% 6% 7% 10% 15% 17% 15% 28% 34% 20% 47% 55% 25% 67% 76% 30% 84% 91% Enrolment and attrition (at least eight parents per programme and 75% of parents attending five of eight sessions) All data transferred from HENRY to the LICTR will be in the form of unlinked, anonymised datasets and will be transferred via a secure, encrypted system. Only data for centres participating in the trial will be transferred. Data transfer agreements (including details on the sender, re- cipient, content of transfer/general purpose of transfer and any data-processing limitations) will be set up be- tween HENRY and the LICTR prior to the transfer of any data. Queries pertaining to outliers or missing data will be sent to HENRY in accordance with LICTR stand- ard operating procedures. Any new data that are identi- fied following queries will also be transferred via the secure, encrypted system. Missing data, except individual data items collected via parent questionnaires, will be chased until it is received, confirmed as not available, or the trial is at analysis. For missing item-level data within questionnaires (item non-response), no attempts will be Enrolment and attrition (at least eight parents per programme and 75% of parents attending five of eight sessions) 25% 5% 7% 8% 10% 17% 19% 15% 31% 37% 20% 49% 58% 25% 67% 76% 30% 82% 89% ICC Intra-cluster correlation coefficient ICC Intra-cluster correlation coefficient service. For the purpose of this trial, baseline data at the centre level include routine data on enrolment and at- tendance at programmes that have been run before randomisation. Analysis Statistical analysis of the quantitative elements of the trial is the responsibility of the LICTR statisticians. The analysis plan outlined in this section will be reviewed, and a detailed statistical analysis plan will be written and approved, before any formal analyses are undertaken. Statistical analyses will be carried out by the ITT principle, and statistical significance will be assessed at the two-sided 5% significance level. The ITT population is defined as analysis according to the randomisation and regardless of compliance with the protocol or with- drawal from the trial. Appropriate scoring manuals will be followed, and missing items within individual outcome measures will be treated according to instructions for that particular measure. For all other outcomes (without instructions for dealing with missing data), half rule will be used, substitution of the mean of the answered questions for that specific subscale for the missing responses as long as at least half the questions are answered [57]. If more than 50% of the items are missing, the outcome will be assigned as missing. Data collection and transfer These data also include anonymous Table 2 Data collection summary Data Screening Randomisation Baseline On-going Follow-up Local authority level Cluster eligibility X Stratification factors X Programme enrolment X X Programme attrition X X Implementation data (e.g., fidelity) X Parent level (anonymised routine) Infant diet (fruit and vegetable intake) X X Family habits and lifestyle X X Implementation/process data X Intervention level Costs for intervention delivery for economic evaluation X X X X X X indicates when data are gathered X indicates when data are gathered Bryant et al. Trials (2017) 18:40 Page 10 of 13 Page 10 of 13 made to retrieve missing data; only missing question- naires will be chased. of the data structure, and the reliability of two-level (par- ents nested within centres or local authorities) and three-level (parents nested within centres within local authorities) random-effects models will also be investi- gated. The proportion of centres achieving combined en- rolment, attrition and parent adherence will be analysed using the same methods described for the primary out- comes. The longitudinal impact of the intervention on parental engagement will be analysed among those local authorities providing data for more than one programme per centre at follow-up, using logistic regression with random effects adjusting for the time point of the programme. All data provided will be stored, handled and processed in accordance with the principles of the 1998 Data Protec- tion Act, the operation of the agreement and the study publication policy. The rights for this data belong to the study sponsor, and no processing, including further data transfer in whole or in part to a third party, is permitted other than as stated in the data transfer agreements. Data monitoring T i l i i The primary outcomes associated with enrolment or attri- tion will be compared between the intervention and con- trol arms through a cluster-level analysis, using a weighted t test of cluster-level proportions and a two-stage process to adjust for covariates [55]. Intervention effects and corresponding 95% confidence intervals will be presented. As part of a sensitivity analysis, the reliability of random effects logistic regression will also be investigated. Trial supervision includes a core project team, a study management group, and a TSC. The core project team will comprise the chief investigator and a research assist- ant, with oversight from all other monitoring groups. The study management group will comprise the chief in- vestigator, clinical trial research unit team (research as- sistant [WB], statisticians [BC and MC], data management, trial management [SH] and health econo- mist [ST]). The TSC will comprise an independent chair (a professor of chronic disease and public health) plus independent expertise in statistics, qualitative and mixed-methods research, behaviour change and a parent (a volunteer from our parent advisory group). Because the trial is using routine data, a separate data monitoring and ethics committee was not convened. Rather, the in- dependent TSC will adopt a safety monitoring role, and will establish a subcommittee to review safety issues should this become necessary. Serious adverse events are not anticipated, and, as such, a separate protocol for unmasking has not be produced. This will, however, be reviewed by the TSC as a regular item on the meeting agenda. In the primary analyses, missing data will be assumed to be missing completely at random; that is, analyses will be performed using complete cases (including those where sufficient item-level data exists). The extent of unit non-response (data missing from a whole course) will be assessed, and the reason for missingness and the missing data mechanism will be investigated. A sensitivity analysis accounting for all participants in the ITT population assuming data missing at random, using multiple imputation, will be performed, and poten- tial predictors of missingness will be investigated. The number of imputations will be determined using Bodner rule of thumb, where number of imputations is similar to the percentage of cases that are complete [56]. Secondary analysis The TSC operates in line with the LICTR terms of ref- erence as amended and agreed by TSC members at their first meeting. Data provided to the LICTR will be moni- tored for quality and completeness by the LICTR, using established verification, validation and checking pro- cesses. Clinical governance issues pertaining to all as- pects of routine management will be brought to the Adherence to HENRY programme content and the im- pact of the HENRY optimisation on parenting and fam- ily health will be analysed using a cluster-level analysis of either proportions or means, depending on the out- come, as described for the primary analysis. As part of sensitivity analyses, the degree of clustering at each level Page 11 of 13 Page 11 of 13 Bryant et al. Trials (2017) 18:40 attention of the TSC and, where applicable, individual local authorities. to estimate the willingness of commissioners to pay for any additional costs borne by additional engagement activities. Ultimately, the true value of the optimised intervention will be ascertained following these discussions with the commissioners. Abbreviations d l Abbreviations COM-B model: Capability, opportunity and motivation; cRCT: Cluster randomised controlled trial; HENRY: Health Exercise Nutrition for the Really Young; ICC: Intra-cluster correlation coefficient; ITT: Intention to treat; LA: Local authorities; LICTR: Leeds Institute of Clinical Trials Research; MRC: Medical Research Council; NIHR: National Institute for Health Research; OFTEN: Optimising Family Engagement in HENRY; QA: Quality assurance; RCT: Randomised controlled trial; TSC: Trial steering committee Trial organisation and administration The trial is sponsored by the University of Leeds and co- ordinated by the LICTR (University of Leeds). The man- agement group consists of the chief investigator and the study management group. Protocol amendments will be handled in accordance with LICTR standard operating procedures. Amendments required to the protocol and ethically approved documents will be identified by the trial researcher and the chief investigator. Amendments will be drafted and reviewed by members of the study management group and sponsor as appropriate. Sub- stantial amendments to ethically approved documents will be submitted to the School of Medicine Research Committee for ethical opinion prior to implementation. A list of amendments will be included in the final report. Implementation research is usually focused on the im- plementation of interventions of known effectiveness into routine practice and policy [23]. However, we in- tend to evaluate an approach to optimising the imple- mentation of HENRY prior to a further evaluation of its clinical effectiveness. This approach is relatively novel and follows literature suggesting that complex interven- tion components should ideally be optimised and tested in evidentiary research [24]. Such an approach ensures that the future clinical effectiveness data relate to the impact that interventions can have once components have been enhanced. Similarly to other interventions (particularly prevention programmes), we recognised a need to increase parent engagement to enrol and attend HENRY programmes in order to improve the chances of a more successful, cost-effective definitive trial of clinical effectiveness. However, other components within com- plex interventions, such as content or delivery aspects, could warrant optimisation prior to effectiveness testing. A future pilot RCT is planned to test the feasibility of conducting a definitive trial of the effectiveness of HENRY to prevent childhood obesity (also within the National Institute for Health Research [NIHR] under Career Development Fellowship CDF-2014-07-052). This will occur regardless of the outcome of the present optimisation trial because there remains a need to estab- lish an evidence base of an intervention that is already widely commissioned. Trial status Recruitment and randomisation of local authorities is complete. Implementation is on-going and family enrol- ment (providing data on enrolment and attendance to the HENRY programme) was started in September 2016 and will continue until April 2017. Dissemination plan A full dissemination plan will be written in accordance with those agreed by the funders. This includes dissem- ination to all stakeholders, including parents, and will be supported by our parent advisory group. The dissemin- ation plan and publication plan will be developed by the study management group and agreed by the TSC. The policy will detail appropriate methods for determining writing groups, lead authors standard acknowledgement text to be included and necessary funding disclosure. The publication plan will detail potential paper titles (main findings and additional papers), authors (highlighting lead author) and time lines. Health Research Authority guidance for informing participants of the trial results will be applied [58]. Discussion Engaging parents to enrol and attend programmes to prevent obesity in their children’s early years is a chal- lenge. This resounds with literature exploring the diffi- culties of recruiting parents to attend population-based programmes in the absence of health problems in their children [9, 30]. Early childhood provides an opportunity to intervene to establish healthy behaviours that are crit- ical for optimum growth and development [7], but the failure to attract parents to programmes such as HENRY (particularly in populations theorised to have the great- est benefit) is a threat to the success and viability of such programmes. Our work has developed a theory-based intervention specifically targeting parent engagement with HENRY, with clear, transferable components for community-based interventions delivered to parents of young children. The early-phase evaluation of this opti- misation intervention is described here, including methods Authors’ information 12. LeBlanc ES, O’Connor E, Whitlock EP, Patnode CD, Kapka T. Effectiveness of primary care–relevant treatments for obesity in adults: a systematic evidence review for the U.S. Preventive Services Task Force. Ann Intern Med. 2011;155(7):434–47. doi:10.7326/0003-4819-155-7-201110040-00006. Ethics approval and consent to participate 17. Clarke AT, Marshall SA, Mautone JA, Soffer SL, Jones HA, Costigan TE, et al. Parent attendance and homework adherence predict response to a family- school intervention for children with ADHD. J Clin Child Adolesc Psychol. 2015;44(1):58–67. doi:10.1080/15374416.2013.794697. This study was approved by the School of Medicine Research Ethics Committee at the University of Leeds (MREC15-017). This study was approved by the School of Medicine Research Ethics Committee at the University of Leeds (MREC15-017). Availability of data and materials 8. Freedman DS, Dietz WH, Srinivasan SR, Berenson GS. The relation of overweight to cardiovascular risk factors among children and adolescents: the Bogalusa Heart Study. Pediatrics. 1999;103(6 Pt 1):1175–82. Not applicable. 9. Oude Luttikhuis H, Baur L, Jansen H, Shrewsbury VA, O’Malley C, Stolk RP, et al. Interventions for treating obesity in children. Cochrane Database Syst Rev. 2009;(1):CD001872. doi:10.1002/14651858.CD001872.pub2 Consent for publication Not applicable. 16. Williams NA, Coday M, Somes G, Tylavsky FA, Richey PA, Hare M. Risk factors for poor attendance in a family-based pediatric obesity intervention program for young children. J Dev Behav Pediatr. 2010;31(9):705–12. doi:10. 1097/DBP.0b013e3181f17b1c. Received: 27 April 2016 Accepted: 27 November 2016 Received: 27 April 2016 Accepted: 27 November 2016 Acknowledgements We acknowledge our parent advisory team for their support in developing the intervention and ongoing advice in study design and recruitment (Amal Najlat, Chloe Anderson, Kelly Milner, Claire Donkin, Sarah Young, Terri Francis and Rachael Baptista). We thank members of the trials steering committee (TSC), including Professor Peymane Adab (TSC Chair, University of Birmingham), Professor Alicia O’Cathain (mixed methods expert, University of Sheffield), Professor Kelvin Jordan (statistical expertise, Keele University), Dr Page 12 of 13 Page 12 of 13 Page 12 of 13 Bryant et al. Trials (2017) 18:40 Bryant et al. Trials (2017) 18:40 Bryant et al. Trials (2017) 18:40 Thomas Willis (behaviour change expertise, University of Leeds) and Amal Najlat (parent representative). We are also grateful to the children’s centres that permitted observations which support the intervention development (anonymous). We acknowledge the optimisation intervention development team for their significant contributions (Professor Pinki Sahota, Dr Maureen Twiddy, Jackie Moores and Chloe Anderson). We thank the HENRY team for their support in developing and delivering the intervention, providing data and liaising with centres (Rebecca Nourse, Sian Livsey and Kim Roberts). 3. Juonala M, Magnussen CG, Berenson GS, Venn A, Burns TL, Sabin MA, et al. Childhood adiposity, adult adiposity, and cardiovascular risk factors. N Engl J Med. 2011;365(20):1876–85. doi:10.1056/NEJMoa1010112. 4. Singh G, Kogan M, van Dyck P. A multilevel analysis of state and regional disparities in childhood and adolescent obesity in the United States. J Community Health. 2008;33:90–102. 5. Whitaker RC, Wright JA, Pepe MS, Seidel KD, Dietz WH. Predicting obesity in young adulthood from childhood and parental obesity. N Engl J Med. 1997; 337(13):869–73. doi:10.1056/NEJM199709253371301. Funding 6. Wang YC, McPherson K, Marsh T, Gortmaker SL, Brown M. Health and economic burden of the projected obesity trends in the USA and the UK. Lancet. 2011;378(9793):815–25. doi:10.1016/s0140-6736(11)60814-3. g The trial is funded by the NIHR Trainees Coordinating Programme awarded to the chief investigator (MB) (CDF-2014-07-052). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. 7. School Food Trust. Advisory Panel on Food and Nutrition in Early Years. Laying the table: recommendations for national food and nutrition guidance for early years settings in England. Sheffield: School Food Trust; 2010. Authors’ contributions MB and JS conceived of the study. MB secured funding with support from JN, AJF, JS, JB, RF and HR. WB, KR, JB and MB participated in the design of the intervention and the development of the recruitment strategy. All authors contributed to the design of the trial. BC, AJF, JB, MB, MC and SH advised on the statistical design and sample size calculation and also developed the recruitment strategy. ST led the economic evaluation design. All authors contributed to the writing of the manuscript and prepared a draft manuscript. All authors read and approved the final manuscript. 10. Waters E, de Silva-Sanigorski A, Hall BJ, Brown T, Campbell KJ, Gao Y, et al. Interventions for preventing obesity in children. Cochrane Database Syst Rev. 2011;(12):CD001871. doi:10.1002/14651858.CD001871.pub3 11. Morandi A, Meyre D, Lobbens S, Kleinman K, Kaakinen M, Rifas-Shiman SL, et al. Estimation of newborn risk for child or adolescent obesity: lessons from longitudinal birth cohorts. PLoS One. 2012;7(11):e49919. doi:10.1371/ journal.pone.0049919. Competing interests 13. Loveman E, Frampton GK, Shepherd J, Picot J, Cooper K, Bryant J, et al. The clinical effectiveness and cost-effectiveness of long-term weight management schemes for adults: a systematic review. Health Technol Assess. 2011;15(2). doi:10.3310/hta15020 KR is the chief executive of HENRY, which may receive increased publicity and consequent commissioning as a result of this research. KR is blinded to treatment allocation and is excluded from steering committee meetings. Involvement of KR was essential for the development of the optimisation intervention under investigation (ensuring feasible approaches were recommended and agreeing on related incurred costs). HR is supported by the NIHR Collaboration for Leadership in Applied Health Research and Care (CLAHRC) North Thames at Barts Health NHS Trust. The other authors declare that they have no competing interests. 14. Summerbell CD, Ashton V, Campbell KJ, Edmunds L, Kelly S, Waters E. Interventions for treating obesity in children. Cochrane Database Syst Rev. 2003;3:CD001872. doi:10.1002/14651858.CD001872. 15. Summerbell CD, Waters E, Edmunds LD, Kelly S, Brown T, Campbell KJ. Interventions for preventing obesity in children. Cochrane Database Syst Rev. 2005;(3):CD001871. doi: 10.1002/14651858.CD001871 Author details 1 1Leeds Institute of Clinical Trials Research, University of Leeds, Leeds LS29JT, UK. 2Department of Nutrition, Gillings School of Public Health, University of North Carolina, Chapel Hill, NC 27599, USA. 3Department of Epidemiology, Gillings School of Public Health, University of North Carolina, Chapel Hill, NC 27599, USA. 4HENRY Head Office, 8 Elm Place, Old Witney Road, Eynsham OX29 4BD, UK. 5Academic Unit of Primary Care, Institute of Health Sciences, University of Leeds, Leeds LS2 9JT, UK. 6London School of Hygiene and Tropical Medicine, 15-17 Tavistock Place, London WC1H 9SH, UK. 7Academic Unit of Health Economics, Leeds Institute of Health Sciences, University of Leeds, Leeds LS2 9JT, UK. 18. Hillier F, Pedley C, Summerbell C. Evidence base for primary prevention of obesity in children and adolescents. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz. 2011;54(3):259–64. doi:10.1007/ s00103-010-1227-4. 19. Moore GF, Audrey S, Barker M, Bond L, Bonell C, Hardeman W, et al. Process evaluation of complex interventions: Medical Research Council guidance. BMJ. 2015;350:h1258. doi:10.1136/bmj.h1258. 20. Loudon K, Treweek S, Sullivan F, Donnan P, Thorpe KE, Zwarenstein M. The PRECIS-2 tool: designing trials that are fit for purpose. BMJ. 2015;350:h1247. doi:10.1136/bmj.h2147. 21. Foy R, Ovretveit J, Shekelle PG, Pronovost PJ, Taylor SL, Dy S, et al. The role of theory in research to develop and evaluate the implementation of patient safety practices. BMJ Qual Saf. 2011;20(5):453–9. doi:10.1136/bmjqs. 2010.047993. Received: 27 April 2016 Accepted: 27 November 2016 Bryant et al. Trials (2017) 18:40 Bryant et al. Trials (2017) 18:40 Page 13 of 13 2015/07/summary-responses-seeking-informed-consent-simple-efficient- trials-2015-v-1-0-final.pdf. Accessed 8 Dec 2016. 24. Stevens J, Taber DR, Murray DM, Ward DS. Advances and controversies in the design of obesity prevention trials. Obesity (Silver Spring). 2007;15(9): 2163–70. doi:10.1038/oby.2007.257. 46. Hartley S, Carder P, Foy R, Heudtlass P, Glidewell L, Ingleson E, et al. Optimising participation and generalisability: the use of opt-out recruitmen for an implementation trial in primary care [abstract]. Trials. 2015;16 Suppl 2 O76. 25. Willis TA, George J, Hunt C, Roberts KP, Evans CE, Brown RE, et al. Combating child obesity: impact of HENRY on parenting and family lifestyle. Pediatr Obes. 2014;9(5):339–50. doi:10.1111/j.2047-6310.2013.00183.x. 47. Carter BR, Hood K. Balance algorithm for cluster randomized trials. BMC Med Res Methodol. 2008;8:65. doi:10.1186/1471-2288-8-65. 26. Hunt C, Rudolf M. Tackling childhood obesity with HENRY: a handbook for community practitioners. London: Unite/Community Practitioners’ and Health Visitors’ Association; 2008. 48. Department for Communities and Local Government. The English Index of Multiple Deprivation (IMD) 2015 – guidance. London: Department for Communities and Local Government; 2015. https://www.gov.uk/ government/uploads/system/uploads/attachment_data/file/464430/English_ Index_of_Multiple_Deprivation_2015_-_Guidance.pdf. Accessed 8 Dec 2016. 27. Rudolf MCJ, Hunt C, George J, Hajibagheri K, Blair M. HENRY: development, pilot and long-term evaluation of a programme to help practitioners work more effectively with parents of babies and pre-school children to prevent childhood obesity. Child Care Health Dev. 2010;36(6):850–7. doi:10.1111/j. 1365-2214.2010.01116.x. 49. International Conference on Harmonisation E9 Expert Working Group. ICH Harmonised Tripartite Guideline: statistical principles for clinical trials. Stat Med. 1999;18:1905–42. 28. Willis TA, Potrata B, Hunt C, Rudolf MC. Training community practitioners to work more effectively with parents to prevent childhood obesity: the impact of HENRY upon children’s centres and their staff. J Hum Nutr Diet. 2012;25(5):460–8. doi:10.1111/j.1365-277X.2012.01247.x. 50. Dumka LE, Stoerzinger HD, Jackson KM, Roosa MW. Examination of the cross-cultural and cross-language equivalence of the Parenting Self-Agency Measure. Fam Relat. 1996;45(2):216–22. doi:10.2307/585293. 29. Bumbarger B, Perkins D. After randomised trials: issues related to dissemination of evidence-based interventions. J Child Serv. 2008;3(2):55–64. doi:10.1108/17466660200800012. 51. Willis TA, Roberts KPJ, Berry TM, Bryant M, Rudolf MCJ. The impact of HENRY on parenting and family lifestyle: a national service evaluation of a preschool obesity prevention programme. Public Health. 2016;136:101–8. doi:10.1016/j.puhe.2016.04.006. 30. Mian ND. Little children with big worries: addressing the needs of young, anxious children and the problem of parent engagement. Clin Child Fam Psychol Rev. 2014;17(1):85–96. doi:10.1007/s10567-013-0152-0. 52. Golan M, Weizman A. Bryant et al. Trials (2017) 18:40 Reliability and validity of the Family Eating and Activity Habits Questionnaire. Eur J Clin Nutr. 1998;52(10):771–7. 31. Chiolero A, Paradis G, Paccaud F. The pseudo-high-risk prevention strategy. Int J Epidemiol. 2015;44(5):1469–73. doi:10.1093/ije/dyv102. 53. Hammond J, Nelson M, Chinn S, Rona RJ. Validation of a food frequency questionnaire for assessing dietary intake in a study of coronary heart disease risk factors in children. Eur J Clin Nutr. 1993;47(4):242–50. 32. Reyno SM, McGrath PJ. Predictors of parent training efficacy for child externalizing behavior problems – a meta-analytic review. J Child Psychol Psychiatry. 2006;47(1):99–111. doi:10.1111/j.1469-7610.2005.01544.x. 54. Drummond M, O’Brien B, Stoddart GL, Torrance GW. Methods for the economic evaluation of health care programmes. 2nd ed. Oxford: Oxford University Press; 1997. 33. Mah JWT, Johnston C. Parental social cognitions: considerations in the acceptability of and engagement in behavioral parent training. Clin Child Fam Psychol Rev. 2008;11(4):218–36. doi:10.1007/s10567-008-0038-8. 55. Hayes RJ, Moulton LH. Cluster randomised trials. Boca Raton: Chapman & Hall/CRC Press; 2009. 34. Arai L, Stapley S, Roberts H. ‘Did not attends’ in children 0–10: a scoping review. Child Care Health Dev. 2014;40(6):797–805. doi:10.1111/cch.12111. 56. White IR, Royston P, Wood AM. Multiple imputation using chained equations: issues and guidance for practice. Stat Med. 2011;30(4):377–99. doi:10.1002/sim.4067. 35. Lindsay G, Cullen MA. Evaluation of the Parenting Early Intervention Programme: a short report to inform local commissioning processes. Research Report DFE-RR121(b). London: Department for Education; 2011. 57. Fairclough DL, editor. Design and analysis of quality of life studies in clinical trials. 2nd ed. Boca Raton: Chapman & Hall/CRC Press; 2010. 36. Atherton H, Sawmynaden P, Meyer B, Car J. Email for the coordination of healthcare appointments and attendance reminders. Cochrane Database Syst Rev. 2012;(8):CD007981. doi: 10.1002/14651858.CD007981.pub2 58. Health Research Authority. Information for participants at the end of a study: Guidance for Researchers/Sponsors/ Chief Investigators/Principal Investigators. 2015. 37. Car J, Gurol-Urganci I, de Jongh T, Vodopivec-Jamsek V, Atun R. Mobile phone messaging reminders for attendance at healthcare appointments. Cochrane Database of Syst Rev. 2012;(7):CD007458. doi: 10.1002/14651858. CD007458.pub2 38. Craig P, Dieppe P, Macintyre S, Michie S, Nazareth I, Petticrew M. Developing and evaluating complex interventions: new Medical Research Council guidance. BMJ. 2008;337:a1655. 39. Nock MK, Ferriter C. Parent management of attendance and adherence in child and adolescent therapy: a conceptual and empirical review. Clin Child Fam Psychol Rev. 2005;8(2):149–66. 39. Nock MK, Ferriter C. Bryant et al. Trials (2017) 18:40 Parent management of attendance and adherence in child and adolescent therapy: a conceptual and empirical review. Clin Child Fam Psychol Rev. 2005;8(2):149–66. 40. McKay MM, Nudelman R, McCadam K, Gonzales J. Evaluating a social work engagement approach to involving inner-city children and their families in mental health care. Res Soc Work Pract. 1996;6(4):462–72. doi:10.1177/ 104973159600600404. 41. Szapocznik J, Perez-Vidal A, Brickman AL, Foote FH, Santisteban D, Hervis O, et al. Engaging adolescent drug abusers and their families in treatment: a strategic structural systems approach. J Consult Clin Psychol. 1988;56(4):552–7. doi:10.1037/0022-006X.56.4.552. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: References 22. Michie S, Abraham C, Eccles MP, Francis JJ, Hardeman W, Johnston M. Strengthening evaluation and implementation by specifying components of behaviour change interventions: a study protocol. Implement Sci. 2011;6:10. doi:10.1186/1748-5908-6-10. 1. Lifestyles Statistics Team, Health and Social Care Information Centre, Department of Health. National Child Measurement Programme: England 2014/15 school year. London: NHS Digital; 2015. 2. D’Adamo E, Caprio S. Type 2 Diabetes in youth: epidemiology and pathophysiology. Diabetes Care. 2011;34 Suppl 2:S161–5. doi:10.2337/dc11- s212. 23. Foy R, Sales A, Wensing M, Aarons GA, Flottorp S, Kent B, et al. Implementation science: a reappraisal of our journal mission and scope. Implement Sci. 2015;10:51. Page 13 of 13 Page 13 of 13 Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: 42. McCabe K, Yeh M, Lau A, Argote CB. Parent-child interaction therapy for Mexican Americans: results of a pilot randomized clinical trial at follow-up. Behav Ther. 2012;43(3):606–18. doi:10.1016/j.beth.2011.11.001. 42. McCabe K, Yeh M, Lau A, Argote CB. Parent-child interaction therapy for Mexican Americans: results of a pilot randomized clinical trial at follow-up. Behav Ther. 2012;43(3):606–18. doi:10.1016/j.beth.2011.11.001. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: • We accept pre-submission inquiries 43. Damschroder LJ, Aron DC, Keith RE, Kirsh SR, Alexander JA, Lowery JC. Fostering implementation of health services research findings into practice: a consolidated framework for advancing implementation science. Implement Sci. 2009;4:50. doi:10.1186/1748-5908-4-50. 43. Damschroder LJ, Aron DC, Keith RE, Kirsh SR, Alexander JA, Lowery JC. Fostering implementation of health services research findings into practice: a consolidated framework for advancing implementation science. Implement Sci. 2009;4:50. doi:10.1186/1748-5908-4-50. 44. Michie S, van Stralen MM, West R. The behaviour change wheel: a new method for characterising and designing behaviour change interventions. Implement Sci. 2011;6:42. doi:10.1186/1748-5908-6-42. 44. Michie S, van Stralen MM, West R. The behaviour change wheel: a new method for characterising and designing behaviour change interventions. Implement Sci. 2011;6:42. doi:10.1186/1748-5908-6-42. 45. NHS Health Research Authority. Seeking informed consent for simple and efficient trials in the NHS draft guidance: summary of responses. London: NHS Health Research Authority; 2015. http://www.hra.nhs.uk/documents/ 45. NHS Health Research Authority. Seeking informed consent for simple and efficient trials in the NHS draft guidance: summary of responses. London: NHS Health Research Authority; 2015. http://www.hra.nhs.uk/documents/
https://openalex.org/W2960426240	https://archive.lstmed.ac.uk/11249/1/20190719_Feasey.pdf	English	null	Ascertaining the burden of invasive Salmonella disease in hospitalised febrile children aged under four years in Blantyre, Malawi	PLoS neglected tropical diseases	2,019	cc-by	9,678	OPEN ACCESS Citation: Msefula CL, Olgemoeller F, Jambo N, Segula D, Van Tan T, Nyirenda TS, et al. (2019) Ascertaining the burden of invasive Salmonella disease in hospitalised febrile children aged under four years in Blantyre, Malawi. PLoS Negl Trop Dis 13(7): e0007539. https://doi.org/10.1371/journal. pntd.0007539 Ascertaining the burden of invasive Salmonella disease in hospitalised febrile children aged under four years in Blantyre, Malawi Chisomo L. MsefulaID1,2☯, Franziska OlgemoellerID2,3☯, Ndaru Jambo1,2,4, Dalitso Segula2,5, Trinh Van Tan6, Tonney S. Nyirenda1,2, Wilfred NediID2, Neil Kennedy3,7, Matthew Graham6, Marc Y. R. Henrion2,8, Stephen Baker9, Nicholas Feasey2,8, Melita Gordon2,4, Robert S. Heyderman2,10 1 Pathology Department, College of Medicine, University of Malawi, Blantyre, Malawi, 2 Malawi-Liverpool- Wellcome Trust Clinical Research Programme, College of Medicine, University of Malawi, Blantyre, Malawi, 3 Department of Paediatrics, Queen Elizabeth Central Hospital, College of Medicine, University of Malawi, Blantyre, Malawi, 4 University of Liverpool, Liverpool, United Kingdom, 5 Department of Internal Medicine, Queen Elizabeth Central Hospital, College of Medicine, University of Malawi, Blantyre, Malawi, 6 The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam, 7 Centre for Medical Education, Queens University, Belfast, United Kingdom, 8 Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 9 The Department of Medicine, The University of Cambridge, Cambridge, United Kingdom, 10 Division of Infection & Immunity, University College London, London, England, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯These authors contributed equally to this work. cmsefula@medcol.mw Abstract T h id f i d i b S h Af i H ti t f th b d f ☯These authors contributed equally to this work. * cmsefula@medcol.mw Abstract T h id f i d i b S h RESEARCH ARTICLE Abstract T phoid fe er Typhoid fever is endemic across sub-Saharan Africa. However, estimates of the burden of typhoid are undermined by insufficient blood volumes and lack of sensitivity of blood culture. Here, we aimed to address this limitation by exploiting pre-enrichment culture followed by PCR, alongside routine blood culture to improve typhoid case detection. We carried out a prospective diagnostic cohort study and enrolled children (aged 0–4 years) with non-specific febrile disease admitted to a tertiary hospital in Blantyre, Malawi from August 2014 to July 2016. Blood was collected for culture (BC) and real-time PCR after a pre-enrichment culture in tryptone soy broth and ox-bile. DNA was subjected to PCR for invA (Pan-Salmonella), staG (S. Typhi), and fliC (S. Typhimurium) genes. A positive PCR was defined as invA plus either staG or fliC (CT<29). IgM and IgG ELISA against four S. Typhi antigens was also per- formed. In total, 643 children (median age 1.3 years) with nonspecific febrile disease were enrolled; 31 (4.8%) were BC positive for Salmonella (n = 13 S. Typhi, n = 16 S. Typhimur- ium, and n = 2 S. Enteritidis). Pre-enrichment culture of blood followed by PCR identified a further 8 S. Typhi and 15 S. Typhimurium positive children. IgM and IgG titres to the S. Typhi antigen STY1498 (haemolysin) were significantly higher in children that were PCR positive but blood culture negative compared to febrile children with all other non-typhoid ill- nesses. The addition of pre-enrichment culture and PCR increased the case ascertainment of invasive Salmonella disease in children by 62–94%. These data support recent burden estimates that highlight the insensitivity of blood cultures and support the targeting of pre- school children for typhoid vaccine prevention in Africa. Blood culture with real-time PCR Editor: Travis J. Bourret, University of Colorado Health Sciences Center, UNITED STATES Received: January 24, 2019 Accepted: June 10, 2019 Published: July 17, 2019 Editor: Travis J. Bourret, University of Colorado Health Sciences Center, UNITED STATES Received: January 24, 2019 Accepted: June 10, 2019 Published: July 17, 2019 Copyright: © 2019 Msefula et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Ascertaining the burden of invasive Salmonella disease following pre-enrichment should be used to further refine estimates of vaccine effectiveness in typhoid vaccine trials. following pre-enrichment should be used to further refine estimates of vaccine effectiveness in typhoid vaccine trials. Competing interests: The authors have declared that no competing interests exist. Abstract T phoid fe er Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was funded through a Strategic Award for the MLW Clinical Research Programme from the Wellcome Trust UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 16 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 Author summary There are increasing reports of typhoid fever epidemics in sub-Saharan Africa frequently affecting young adults and children aged between 5 and 16 years. In Asia where typhoid is hyperendemic, children aged 0 to 4 years also have a high burden of typhoid fever. Diag- nosis of typhoid in young children is particularly a challenge because collection of ade- quate blood sample for testing is not always possible and the Salmonella bacterial load is low. Established methods of diagnosis such as blood culture and serology have low sensi- tivity. This study has used a combination of blood culture and pre-enrichment culture fol- lowed by PCR to improve ascertainment of the burden of both nontyphoidal Salmonella disease and typhoid fever in Malawian children, aged 0 to 4 years. We found that diagnosis with blood culture and pre-enrichment culture followed by PCR together added 94% more of nontyphoidal Salmonella and 62% more of Salmonella Typhi than blood culture alone. Where blood culture was negative but Salmonella-specific PCR was positive we have validated our results using Haemolysin (STY1498)-based serology. There are ongo- ing typhoid vaccine efficacy trials in Africa and Asia. The findings from this study will inform future estimates of vaccine effectiveness. Pre-enrichment A modified pre-enrichment procedure for Salmonella was adopted [28]. A maximum of 2 mL of blood was added to 8 mL of tryptone soy broth mixed with 3% Ox-gall powder (TSB/Ox- gall) and incubated overnight at 37˚C. Blood samples with <2 mL were added to 8 mL TSB/ Ox-gall medium, topped up to 10 mL with sterile distilled water. Ascertaining the burden of invasive Salmonella disease real-time PCR with a pre-enrichment culture step alongside standard blood culture to improve case ascertainment of invasive Salmonella disease in children aged under 4 years in Malawi. real-time PCR with a pre-enrichment culture step alongside standard blood culture to improve case ascertainment of invasive Salmonella disease in children aged under 4 years in Malawi. Ethics statement The College of Medicine Research Ethics Committee approved the study, approval number P.08/13/1445. Written informed consent was sought from the parents or guardians before enrolment of participants into the study. Participant enrolment Recruitment of participants into the study was nested within the pre-existing sentinel surveil- lance for bacterial infections at Queen Elizabeth Central Hospital (QECH) and the Malawi- Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi. Children with non-specific febrile illness (temperature 38˚C or a history of fever) aged between 0 and 4 years old presenting to QECH were recruited. QECH is the largest government hospital in Malawi, providing free healthcare to the district of Blantyre with a population of approxi- mately 1.3 million and referrals from the Southern region [27]. Recruitment was from August 2014 to July 2016 covering three dry seasons and two rainy seasons. 5 mL blood was drawn from each child; 2 mL for culture, 2 mL for real-time PCR and 1 mL for serology. At six weeks in convalescence further 1 mL of blood was drawn and plasma was extracted for serology. Unless invasive bacterial infection was strongly suspected, children with a positive RDT or malaria blood film were not enrolled into the study. Blood culture processing Blood cultures were processed using the BacT/Alert automated system (Biomerieux, France), at the Malawi-Liverpool-Wellcome Trust Clinical Research Laboratories [1]. Bacteria were iso- lated and identified using previously described standard microbiological procedures [1]. A minimum of four days, including sub-culturing, biochemical testing and latex agglutination testing, was necessary to generate a definitive report of the diagnosis. Introduction Both Salmonella Typhi and nontyphoidal Salmonellae remain prominent contributors to the large burden of bloodstream infection (BSI) in sub-Saharan Africa (sSA) [1–4]. Until recently nontyphoidal serovars Salmonella Typhimurium and Salmonella Enteritidis were the most prevalent in sSA, mainly affecting young children and HIV-infected adults [5–7]. Outbreaks of typhoid fever are now being reported across sSA [1, 8–11], largely caused by a multidrug resis- tant S. Typhi genotype 4.3.1 [12–14]. In Malawi where surveillance for bloodstream infections has been conducted for over 20 years, S. Typhi has become one of the commonest blood cul- ture isolates amongst hospitalized febrile adults and children [1, 15]. In this context, ineffec- tive, commonly available antimicrobials and inadequate diagnostic tools with poor sensitivity and results turn-around time, hamper the identification, management and control of both iNTS and typhoid fever. Blood culture identifies 40 to 80% of cases of invasive Salmonella disease [16]. Limitations of culture arise due to low numbers of bacteria in blood, requiring large volume samples that are not feasible from young children [17]. Prior antimicrobial use may further compromise the diagnostic yield. Currently available, commercial serological tests detecting antibody against S. Typhi antigens have limited sensitivity and poor specificity [18–20]. A number of PCR methods for diagnosis of Salmonella have been developed. The inclusion of a culture step prior to nucleic acid amplification has been suggested to increase sensitivity [21, 22], but this has not been evaluated in the field. Studies from countries in Asia with endemic typhoid suggest a considerable disease burden in young children, including children aged under 4 years [3, 23–25]. If this is also the case in sSA, this could influence the age at which vaccination might be implemented within the National vaccination expanded programme on immunisation [26]. In this study, we adopted PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 2 / 16 DNA extraction and real-time PCR Pre-enriched blood samples were centrifugated in a macrocentrifuge at 3000 x g and the super- natant pipetted out leaving approximately 200 μl to resuspend the pellets. DNA from blood, including bacterial genomic DNA, was extracted following the UltraClean BloodSpin Kit (MO BIO Laboratories CA USA) extraction protocol. DNA was eluted in a final volume of 100 μl and stored at -20˚C. A 40-cycle real-time PCR was performed using a mastermix of Platinum Quantitative UDG reagents on an ABI 7500. Three primer pairs (Table 1) designed using Primer Quest, were added in monoplex reactions (S1 Table) to detect the presence of either S. Typhi or S. Typhimurium. A pan-Salmonella primer pair targeting invA was designed using sequences from S. Typhimurium ST313 D23580 (Accession FN424405) and S. Typhi CT18 (Accession AL513382). A primer pair specifically identifying S. Typhimurium was designed PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 3 / 16 Thirdly, a receiver operating characteristic (ROC) curve was constructed to assess the cut-off value that maximises sensitivity and specificity of the PCR amplification, considering the blood culture result as a true positive. S. Typhi serology Serology for S. Typhi (Table 2), with previously identified serodiagnostic antigens [29, 30] and S. Typhi Vi polysaccharide antigen [31], was conducted to validate the PCR amplification data in blood culture negative samples. Sera from febrile children acutely ill and plasma at six weeks in convalescence were analysed. Archived serum samples from healthy Malawian children aged 0 to 4 years (median age 10 months) were included in the analysis as controls [32]. IgM and IgG titres in acute and convalescent plasma samples and in healthy control sera were mea- sured using a previously described ELISA with 3 purified protein antigens STY1498 (Haemoly- sin), STY1479 (a possible ATP binding protein), STY1886 (cytolethal distending toxin subunit B homolog) and S. Typhi Vi polysaccharide antigen. Table 1. Primer and probe sequences. Primer Sequence Source invA Forward AGCGTACTGGAAAGGGAAAG This study Probe FAM-TTACGGTTCCTTTGACGGTGCGAT-BHQ1 Reverse CACCGAAATACCGCCAATAAAG staG Forward CCGACCAAGTTCCAGATCAA This study Probe VIC-TGGCCAGTAATAATGTCGGGACGA-BHQ1 Reverse GTTGGTTAGTAGCGAGGTGTT fliC Forward TGCTGATTTGACAGAGGCTAAA This study Probe FAM-TGTTACCGGCACAGCATCTGTTGT-BHQ1 Reverse TCGCCTACCTTAACTGCTAAAC https://doi.org/10.1371/journal.pntd.0007539.t001 Table 2. Salmonella Typhi antigens for ELISA confirmation of blood culture negative/ PCR positive samples. Antigen ID Annotation STY1498 Haemolysin STY1479 P ibl ATP bi di t i against the flagellin gene fliC and S. Typhi was detected using a primer pair targeting the fim- brial gene staG. Table 1. Primer and probe sequences. Primer Sequence Source invA Forward AGCGTACTGGAAAGGGAAAG This study Probe FAM-TTACGGTTCCTTTGACGGTGCGAT-BHQ1 Reverse CACCGAAATACCGCCAATAAAG staG Forward CCGACCAAGTTCCAGATCAA This study Probe VIC-TGGCCAGTAATAATGTCGGGACGA-BHQ1 Reverse GTTGGTTAGTAGCGAGGTGTT fliC Forward TGCTGATTTGACAGAGGCTAAA This study Probe FAM-TGTTACCGGCACAGCATCTGTTGT-BHQ1 Reverse TCGCCTACCTTAACTGCTAAAC https://doi.org/10.1371/journal.pntd.0007539.t001 Table 1. Primer and probe sequences. against the flagellin gene fliC and S. Typhi was detected using a primer pair targeting the fim- brial gene staG. Establishing a cycle threshold cut-off Establishing a cycle threshold cut-off A positive PCR result was assigned to a sample only if the pan-Salmonella (invA) amplification was simultaneously positive with either one of the serovar-specific amplifications (fliC or staG) within a specified cycle threshold (CT) cut-off. The CT-value cut-off was determined using a combination of three approaches. Firstly, the primer pairs were tested against DNA extracted from serially diluted cultures of S. Typhi, S. Typhimurium, Staphylococcus aureus, E. coli, Kleb- siella pneumoniae, Micrococcus spp., and Bacillus spp (S2 Table). Five replicates were prepared for each organism. A cut-off was set to exclude all CT-values observed for non-specific target amplifications. Secondly, the limit of target detection was established for the three primer pairs by running the real-time PCR assay on five replicates of serially diluted cultures of S. Typhi and S. Typhimurium. Thirdly, a receiver operating characteristic (ROC) curve was constructed to assess the cut-off value that maximises sensitivity and specificity of the PCR amplification, considering the blood culture result as a true positive. against the flagellin gene fliC and S. Typhi was detected using a primer pair targeting the fim- brial gene staG. Establishing a cycle threshold cut-off A positive PCR result was assigned to a sample only if the pan-Salmonella (invA) amplification was simultaneously positive with either one of the serovar-specific amplifications (fliC or staG) within a specified cycle threshold (CT) cut-off. The CT-value cut-off was determined using a combination of three approaches. Firstly, the primer pairs were tested against DNA extracted from serially diluted cultures of S. Typhi, S. Typhimurium, Staphylococcus aureus, E. coli, Kleb- siella pneumoniae, Micrococcus spp., and Bacillus spp (S2 Table). Five replicates were prepared for each organism. A cut-off was set to exclude all CT-values observed for non-specific target amplifications. Secondly, the limit of target detection was established for the three primer pairs by running the real-time PCR assay on five replicates of serially diluted cultures of S. Typhi and S. Typhimurium. Thirdly, a receiver operating characteristic (ROC) curve was constructed to assess the cut-off value that maximises sensitivity and specificity of the PCR amplification, considering the blood culture result as a true positive. Table 1. Primer and probe sequences. Primer Sequence Source invA Forward AGCGTACTGGAAAGGGAAAG This study Probe FAM-TTACGGTTCCTTTGACGGTGCGAT-BHQ1 Reverse CACCGAAATACCGCCAATAAAG staG Forward CCGACCAAGTTCCAGATCAA This study Probe VIC-TGGCCAGTAATAATGTCGGGACGA-BHQ1 Reverse GTTGGTTAGTAGCGAGGTGTT fliC Forward TGCTGATTTGACAGAGGCTAAA This study Probe FAM-TGTTACCGGCACAGCATCTGTTGT-BHQ1 Reverse TCGCCTACCTTAACTGCTAAAC https://doi.org/10.1371/journal.pntd.0007539.t001 Ascertaining the burden of invasive Salmonella disease Ascertaining the burden of invasive Salmonella disease against the flagellin gene fliC and S. Typhi was detected using a primer pair targeting the fim- brial gene staG. Establishing a cycle threshold cut-off A positive PCR result was assigned to a sample only if the pan-Salmonella (invA) amplification was simultaneously positive with either one of the serovar-specific amplifications (fliC or staG) within a specified cycle threshold (CT) cut-off. The CT-value cut-off was determined using a combination of three approaches. Firstly, the primer pairs were tested against DNA extracted from serially diluted cultures of S. Typhi, S. Typhimurium, Staphylococcus aureus, E. coli, Kleb- siella pneumoniae, Micrococcus spp., and Bacillus spp (S2 Table). Five replicates were prepared for each organism. A cut-off was set to exclude all CT-values observed for non-specific target amplifications. Secondly, the limit of target detection was established for the three primer pairs by running the real-time PCR assay on five replicates of serially diluted cultures of S. Typhi and S. Typhimurium. Ascertaining the burden of invasive Salmonella disease Statistical analysis Fisher’s exact test and Chi-squared test were used to determine whether clinical characteristics (fever, vomiting and diarrhoea), hospital admission and reported prior use of antibiotics were related to detection of Salmonella by both culture and PCR. Statistical significance was defined as P < 0.05, although exact p-values were reported. Pearson’s correlation coefficient was used to analyse the association between blood sample volume and cycle threshold value on PCR. Anti- body (IgG and IgM) titre differences between S. Typhi positive and negative groups were log- transformed and tested using two-tailed two-sample t-tests and we also looked for differential clustering between these 2 groups using multidimensional scaling and principal component analysis. Analyses were performed using IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp, GraphPad prism version 7.0 and R v3.5.1. Study population A total of 661 children aged less than 4 years (median age 15.9 months; range 1–48 months; 357 males), with non-specific febrile disease were recruited at QECH over the study period. Eighteen children were excluded from the analysis; 5 had no blood culture results, 4 had no PCR sample, and 9 had inconclusive PCR results on re-testing (Fig 1). The baseline characteristics of the 643 children in these analyses are presented in Table 3. Real-time PCR and CT-value cut-off Real-time PCR and CT-value cut-off Real-time PCR amplifications were generated in 323 pre-enriched blood samples for the pan-Sal- monella primers, in 234 for the S. Typhimurium specific primers, and for 168 of the S. Typhi spe- cific primers, and the remaining 320 samples were negative. The CT values ranged from 8.643 to 39.857 for the pan-Salmonella primers (median = 33.713, interquartile range (IQR) 30.476– 35.662), from 10.855 to 39.9997 for the S. Typhimurium specific primers (median = 34.765, IQR 32.237–36.066), and 9.849 to 38.868 for the S. Typhi specific primers (median = 33.978, IQR 31.872–36.708). A plot of the CT-values generated bimodal distributions for each of the three amplifications (Fig 2). A cut-off was set at CT = 29 because non-specific PCR amplifications gen- erated in validation experiments clustered above a CT value of 29 (Fig 3). An ROC curve showed that a CT value of 29 cut-off maximised sensitivity and specificity of the respective PCR primer pairs to identify S. Typhi (invA: Sensitivity = 84.6%, Specificity = 92.9%; staG: Sensitivity = 76.9%, Specificity = 98.7%) and S. Typhimurium (invA: Sensitivity = 68.8%, Specificity = 92.8%; fliC: Sensitivity = 68.8%, Specificity = 97.0%) (S1 Fig and S3 Table) S. Typhi serology Serology for S. Typhi (Table 2), with previously identified serodiagnostic antigens [29, 30] and S. Typhi Vi polysaccharide antigen [31], was conducted to validate the PCR amplification data in blood culture negative samples. Sera from febrile children acutely ill and plasma at six weeks in convalescence were analysed. Archived serum samples from healthy Malawian children aged 0 to 4 years (median age 10 months) were included in the analysis as controls [32]. IgM and IgG titres in acute and convalescent plasma samples and in healthy control sera were mea- sured using a previously described ELISA with 3 purified protein antigens STY1498 (Haemoly- sin), STY1479 (a possible ATP binding protein), STY1886 (cytolethal distending toxin subunit B homolog) and S. Typhi Vi polysaccharide antigen. Table 2. Salmonella Typhi antigens for ELISA confirmation of blood culture negative/ PCR positive samples. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 4 / 16 Blood culture and PCR diagnosis A positive blood culture was recorded in 15.4% (99/643) of the recruited children. Nine patho- gens were isolated including the Salmonella enterica serovars Typhi, Typhimurium and Enteri- tidis (Table 4). S. Typhimurium was the major NTS serovar with 88.9% (16/18) of NTS isolates. Thirteen of the 643 children had an S. Typhi infection; all were older than one year of age and 84.6% (11/13) of them were between 2 and 4 years old. Contaminated blood culture growth was present in 8.9% (57/643) of the study participants. Real-time PCR with pre-enrich- ment added an additional 8 S. Typhi infections and 15 additional S. Typhimurium infections. Three S. Typhi and 5 S. Typhimurium cases had a positive blood culture but were negative by real-time PCR (Table 5). In the study population, the combination of blood culture and real- time PCR with pre-enrichment detected S. Typhimurium and S. Typhi positivity frequencies of 4.8% (31/643) and 3.3% (21/643) respectively. PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 5 / 16 of invasive Salmonella disease Ascertaining the burden of invasive Salmonella disease Clinical characteristics, antimicrobial use and Salmonella detection Non-specific clinical presentations including fever, vomiting and diarrhoea were common and there was no association between clinical signs or symptoms and detection of either S. Typhi or S. Typhimurium infection (P range 0.27 to 0.79). However, hospital admission was more fre- quent in children with S. Typhimurium infection (23/31; 74.1%) than those with typhoid fever (8/21; 38.0%) (P = 0.02). Reported prior-antibiotic use (288/642; 44.9%) was not significantly associated with lack of growth of either S. Typhi (P = 0.22) or nontyphoid Salmonella (P = 0.93). Fig 1. Flowchart of participant recruitment. Numbers of children screened, recruited and processed on PCR, blood culture and serology are provided. https://doi.org/10.1371/journal.pntd.0007539.g001 en screened, recruited and processed on PCR, blood Clinical characteristics, antimicrobial use and Salmonella detection Non-specific clinical presentations including fever, vomiting and diarrhoea were common and there was no association between clinical signs or symptoms and detection of either S. Typhi or S. Typhimurium infection (P range 0.27 to 0.79). However, hospital admission was more fre- quent in children with S. Typhimurium infection (23/31; 74.1%) than those with typhoid fever (8/21; 38.0%) (P = 0.02). Reported prior-antibiotic use (288/642; 44.9%) was not significantly associated with lack of growth of either S. Typhi (P = 0.22) or nontyphoid Salmonella (P = 0.93). Real-time PCR and sample volume Ascertaining the burden of invasive Salmonella disease the volume of the pre-enriched blood sample for the pan-primer (P = 0.35, r = .05), S. Typhi specific primer (P = 0.59, r = .04), and S. Typhimurium specific primer (P = 0.13, r = .10) (S2 Fig). Typhoid IgG and IgM confirmation of PCR diagnostics Serum was sampled from 445 children at the time of illness; 142 provided additional plasma sample six weeks after the admission date. All the 445 febrile children (regardless of presence or absence of S. Typhi infection) had significantly elevated IgM and IgG titres against all four antigens, compared to healthy controls (n = 61) (P range <0.0001 to 0.0394) (S3 Fig). IgM and IgG responses in acute typhoid infection, confirmed by both blood culture and PCR (or blood Table 3. Characteristics of 643 children 4 years. Characteristic Children 4yrs n (%) Number 643 Sex, male 351 (54.6) Age, months, median [IQR] 15.8 [8.9–28] 1–8.9 165 (25.5) 9–16.9 172 (26.7) 17–24.9 114 (17.7) 25–48 192 (29.7) Vomiting 261/641 (40.7) Diarrhoea 227/641 (35.4) Malaria positive by RDT or Blood Film Microscopy 20/640 (3.1) Recruitment by season, wet 360 (56.0) Hospital admission 252/639 (39.4) Prior reported antibiotic use 288/642 (44.9) https://doi.org/10.1371/journal.pntd.0007539.t003 Fig 2. Bimodal distribution of CT-values for primers invA, fliC and staG. Graph shows the distribution of CT- values for the salmonella pan primer and S.Typhi and S.Typhiurium specific primers with a clear distinction between PCR positives and negatives. https://doi.org/10.1371/journal.pntd.0007539.g002 Ascertaining the burden of invasive Salmonella disease Table 3. Characteristics of 643 children 4 years. Characteristic Children 4yrs n (%) Number 643 Sex, male 351 (54.6) Age, months, median [IQR] 15.8 [8.9–28] 1–8.9 165 (25.5) 9–16.9 172 (26.7) 17–24.9 114 (17.7) 25–48 192 (29.7) Vomiting 261/641 (40.7) Diarrhoea 227/641 (35.4) Malaria positive by RDT or Blood Film Microscopy 20/640 (3.1) Recruitment by season, wet 360 (56.0) Hospital admission 252/639 (39.4) Prior reported antibiotic use 288/642 (44.9) https://doi.org/10.1371/journal.pntd.0007539.t003 the volume of the pre-enriched blood sample for the pan-primer (P = 0.35, r = .05), S. Typhi specific primer (P = 0.59, r = .04), and S. Typhimurium specific primer (P = 0.13, r = .10) (S2 Fig). Real-time PCR and sample volume Blood sample volumes were recorded in 619 of the 643 recruited children. The targeted vol- ume of blood (2 mL) for real-time PCR was achieved in 100 /619 (16.2%) children. The mini- mum volume of blood sample collected for PCR after venesection for blood culture was 100μl (median = 1,200μl, IQR 1,000μl– 1,725μl). There was no correlation between the CT value and PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 6 / 16 T h id G d fi i f C di i Typhoid IgG and IgM confirmation of PCR diagnostics yp g g g Serum was sampled from 445 children at the time of illness; 142 provided additional plasma sample six weeks after the admission date. All the 445 febrile children (regardless of presence or absence of S. Typhi infection) had significantly elevated IgM and IgG titres against all four antigens, compared to healthy controls (n = 61) (P range <0.0001 to 0.0394) (S3 Fig). IgM and IgG responses in acute typhoid infection, confirmed by both blood culture and PCR (or blood Fig 2. Bimodal distribution of CT-values for primers invA, fliC and staG. Graph shows the distribution of CT- values for the salmonella pan primer and S.Typhi and S.Typhiurium specific primers with a clear distinction between PCR positives and negatives. https://doi.org/10.1371/journal.pntd.0007539.g002 Fig 2. Bimodal distribution of CT-values for primers invA, fliC and staG. Graph shows the distribution of CT- values for the salmonella pan primer and S.Typhi and S.Typhiurium specific primers with a clear distinction between PCR positives and negatives. Fig 2. Bimodal distribution of CT-values for primers invA, fliC and staG. Graph shows the distribution of CT- values for the salmonella pan primer and S.Typhi and S.Typhiurium specific primers with a clear distinction between PCR positives and negatives. https://doi.org/10.1371/journal.pntd.0007539.g002 7 / 16 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 Ascertaining the burden of invasive Salmonella disease Fig 3. CT cut-off set to demarcate non-specific amplification signals for primers invA (a), fliC (b) and staG (c). Graph shows CT-values from serial dilutions of contaminants; Micrococci (mc), Bacillus (bac), and pathogens; S. Typhi (sty), S. Typhimurium (stm), S. aureus (sta), Klebsiella (klb), and E. coli (eco). https://doi.org/10.1371/journal.pntd.0007539.g003 Table 4. Blood culture growth observed in the study population. Blood culture growth n (%) Pathogens: E. coli 3 (0.5) Enterobacter spp 1 (0.2) Pantoea spp 1 (0.2) Pseudomonas aeruginosa 1 (0.2) Salmonella Enteritidis 2 (0.3) Salmonella Typhi 13 (2.0) Salmonella Typhimurium 16 (2.5) Staphylococcus aureus 3 (0.5) Streptococcus pyogenes 2 (0.3) Contaminants: α-haemolytic Streptococci 4 (0.6) Bacillus spp. 10 (1.5) Coagulase negative Staphylococcus 22 (3.4) Diphtheroids 4 (0.6) Micrococcus spp 17 (2.6) No growth 544 (84.6) Total 643 (100) https://doi.org/10.1371/journal.pntd.0007539.t004 Ascertaining the burden of invasive Salmonella disease Fig 3. CT cut-off set to demarcate non-specific amplification signals for primers invA (a), fliC (b) and staG (c). The groups are bc+PCR+ (cases that had S. Typhi infection confirmed by both blood culture and PCR (or blood culture alone), in acute infection [age 14 to 45, median 37 months], and in convalescence [age 14 to 45, median 33 months]), PCR+ (S. Typhi infection confirmed by PCR only, in cute infection [age 8 to 46, median 22 months] and in convalescence [age 22 to 46, median 40 months]), Negative (febrile but negative for typhoid on blood culture and PCR, in acute infection [age 1 to 48, median 13.5 months] and in convalescence [age 1 to 45, median 16 months]), and Control (afebrile healthy controls [age 0 to 52.5, median 10.2 months]). https://doi.org/10.1371/journal.pntd.0007539.g004 https://doi.org/10.1371/journal.pntd.0007539.g004 culture alone) (n = 10) against STY1498 were significantly elevated in comparison to responses in febrile children with all other non-typhoid illnesses (n = 428) (IgM, P = 0.0172; IgG, P = 0.0001) (Figs 4 and 5). The concentration of IgG against STY1498 in convalescent plasma from children who previously had a negative blood culture but were PCR amplification posi- tive for S. Typhi were significantly higher than in children with non-typhoid illnesses (P< 0.0001) (Fig 4). There was no significant difference in IgM or IgG titres (P> = 0.05) against STY1479, STY1886 or Vi between children with typhoid infection and those with other ill- nesses (Figs 4 and 5). Further, neither multidimensional scaling nor principal component analysis, when run on both IgM and IgG titres for all four antigens, was able to clearly separate Salmonella positive from negative cases on blood culture and / or PCR (S4 Fig). Discussion Ascertaining the burden of invasive Salmonella disease Fig 4. Distribution of IgG antibody titres during acute infection (A, C, E, G) and in convalescence (B, D, F, H). IgG antibody titres were measured against four antigens (STY1498 in A & B, STY1479 in C & D, STY1886 in E & F, & Vi in G & H) across distinct groups of children. The groups are bc+PCR+ (cases that had S. Typhi infection confirmed by both blood culture and PCR (or blood culture alone), in acute infection [age 14 to 45, median 37 months], and in convalescence [age 14 to 45, median 33 months]), PCR+ (S. Typhi infection confirmed by PCR only, in cute infection [age 8 to 46, median 22 months] and in convalescence [age 22 to 46, median 40 months]), Negative (febrile but negative for typhoid on blood culture and PCR, in acute infection [age 1 to 48, median 13.5 months] and in convalescence [age 1 to 45, median 16 months]), and Control (afebrile healthy controls [age 0 to 52.5, median 10.2 months]). https://doi.org/10.1371/journal.pntd.0007539.g004 Fig 4. Distribution of IgG antibody titres during acute infection (A, C, E, G) and in convalescence (B, D, F, H). IgG antibody titres were measured against four antigens (STY1498 in A & B, STY1479 in C & D, STY1886 in E & F, & Vi in G & H) across distinct groups of children. The groups are bc+PCR+ (cases that had S. Typhi infection confirmed by both blood culture and PCR (or blood culture alone), in acute infection [age 14 to 45, median 37 months], and in convalescence [age 14 to 45, median 33 months]), PCR+ (S. Typhi infection confirmed by PCR only, in cute infection [age 8 to 46, median 22 months] and in convalescence [age 22 to 46, median 40 months]), Negative (febrile but negative for typhoid on blood culture and PCR, in acute infection [age 1 to 48, median 13.5 months] and in convalescence [age 1 to 45, median 16 months]), and Control (afebrile healthy controls [age 0 to 52.5, median 10.2 months]). https://doi.org/10.1371/journal.pntd.0007539.g004 Fig 4. Distribution of IgG antibody titres during acute infection (A, C, E, G) and in convalescence (B, D, F, H). IgG antibody titres were measured against four antigens (STY1498 in A & B, STY1479 in C & D, STY1886 in E & F, & Vi in G & H) across distinct groups of children. Graph shows CT-values from serial dilutions of contaminants; Micrococci (mc), Bacillus (bac), and pathogens; S. Typhi (sty), S. Typhimurium (stm), S. aureus (sta), Klebsiella (klb), and E. coli (eco). htt //d i /10 1371/j l td 0007539 003 Fig 3. CT cut-off set to demarcate non-specific amplification signals for primers invA (a), fliC (b) and staG (c). Graph shows CT-values from serial dilutions of contaminants; Micrococci (mc), Bacillus (bac), and pathogens; S. Typhi (sty), S. Typhimurium (stm), S. aureus (sta), Klebsiella (klb), and E. coli (eco). https://doi.org/10.1371/journal.pntd.0007539.g003 https://doi.org/10.1371/journal.pntd.0007539.g003 Table 4. Blood culture growth observed in the study population. Blood culture growth n (%) Pathogens: E. coli 3 (0.5) Enterobacter spp 1 (0.2) Pantoea spp 1 (0.2) Pseudomonas aeruginosa 1 (0.2) Salmonella Enteritidis 2 (0.3) Salmonella Typhi 13 (2.0) Salmonella Typhimurium 16 (2.5) Staphylococcus aureus 3 (0.5) Streptococcus pyogenes 2 (0.3) Contaminants: α-haemolytic Streptococci 4 (0.6) Bacillus spp. 10 (1.5) Coagulase negative Staphylococcus 22 (3.4) Diphtheroids 4 (0.6) Micrococcus spp 17 (2.6) No growth 544 (84.6) Total 643 (100) https://doi.org/10.1371/journal.pntd.0007539.t004 Table 5. PCR performance in relation to Blood culture. Blood culture Sensitivity (%) Specificity (%) PPV (%) NPV (%) + - S.Typhi PCR + 10 8 76.9 98.7 55.6 99.5 - 3 622 S. Typhimurium PCR + 11 15 68.8 97.6 42.3 99.2 - 5 612 https://doi.org/10.1371/journal.pntd.0007539.t005 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 8 / 16 Table 4. Blood culture growth observed in the study population. Blood culture growth n (%) Pathogens: E. coli 3 (0.5) Enterobacter spp 1 (0.2) Pantoea spp 1 (0.2) Pseudomonas aeruginosa 1 (0.2) Salmonella Enteritidis 2 (0.3) Salmonella Typhi 13 (2.0) Salmonella Typhimurium 16 (2.5) Staphylococcus aureus 3 (0.5) Streptococcus pyogenes 2 (0.3) Contaminants: α-haemolytic Streptococci 4 (0.6) Bacillus spp. 10 (1.5) Coagulase negative Staphylococcus 22 (3.4) Diphtheroids 4 (0.6) Micrococcus spp 17 (2.6) No growth 544 (84.6) Total 643 (100) https://doi.org/10.1371/journal.pntd.0007539.t004 Table 5. PCR performance in relation to Blood culture. Table 5. PCR performance in relation to Blood culture. Table 5. PCR performance in relation to Blood culture. 8 / 16 Ascertaining the burden of invasive Salmonella disease OS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 9 / 16 Ascertaining the burden of invasive Salmonella disease 9 / 16 Discussion The precise epidemiology of invasive Salmonella infections remains elusive in many resource- limited settings [33, 34]. We have utilised a combination of blood culture and real-time PCR with pre-enrichment to improve ascertainment of S. Typhi and S. Typhimurium bacteraemia in young children by 62% and 94% respectively. In sub-Saharan Africa, including Malawi, invasive nontyphoidal Salmonellae (iNTS) has been widely reported in children less than five years old [5, 6], leading to preventive strategies being targeted towards this age group [35]. In the context of multiple reports of declining inci- dence of iNTS in the region [1, 36], our findings suggest that the residual burden of this high mortality bloodstream infection may be greater than supposed. In contrast to iNTS, the con- ventional understanding has been that typhoid fever is a disease of older children and adults [25, 37, 38]. Our data suggests a considerable hidden burden in children 0–4 years who present with non-specific clinical features. We speculate that an even greater number of cases are pre- senting to community health centres and receiving only partially effective antibiotics. The performance of both real-time PCR with culture pre-enrichment and blood culture is likely to have been limited by small volumes of blood available from young children and prior antibiotic use [39]. Nonetheless, real-time PCR with pre-enrichment did identify additional cases to blood culture with few false negatives. Reported prior use of antibiotics, which is expected to impact negatively both real-time PCR with culture pre-enrichment and blood cul- ture, was found not to have affected detection of invasive salmonella infection. However, the unreliable nature of reported use of antibiotics [40, 41] may mean that our findings may still be an underestimate of invasive Salmonellosis in young children. To validate case ascertainment by pre-enrichment PCR in the context of a negative blood culture, we have used antibody-based diagnostics [42, 43], which are not dependent on bacte- rial concentration in the blood but can vary between populations [44]. In this study IgM and IgG antibody responses to S. Typhi antigens Vi and CdtB (STY1886) in Malawian children were not serodiagnostic as previously reported in Vietnamese [30] and Bangladeshi [31] popu- lations. Only IgG and IgM responses to STY1498 (haemolysin gene, hlyE) separated S. A check list of items relevant to this report of a prospective diagnostic cohort study. (DOC) S1 Fig. Receiver operating curve characteristic for the pan-primer and S. Typhi specific primer in (a) and for the pan-primer and S. Typhimurium specific primer in (b). Gener- ated from CT-values plotted against the blood culture ‘reference standard results’. (TIF) S2 Fig. Distribution of CT-values according to volume of blood sample pre-enriched for PCR. Graphs show distribution according to (a) pan-primer invA, (b) S. Typhi specific primer staG, (c) S. Typhimurium specific primer fliC. Red triangle = PCR positive for S. Typhi, Blue rectangle = PCR positive for S. Typhimurium, and Black circle = PCR negative. (TIF) S3 Fig. IgM and IgG antibody responses to four antigens (STY1886, STY1479, STY1498 and VI) in febrile children and healthy controls. The graph shows significantly higher IgM (blue) and IgG (red) levels in febrile children (n = 445) than IgM (clear) and IgG (purple) lev- els in healthy community controls (n = 61). (TIF) S4 Fig. Multidimensional scaling and Principal component analysis of IgM and IgG responses to all four antigens (STY1886, STY1479, STY1498 and VI). (1) is IgM and IgG responses where blood culture and PCR were positive for Salmonella. (3) is IgG and IgM responses where blood culture was positive for Salmonella and PCR was negative. (4) is IgG and IgM responses where PCR was positive for Salmonella and blood culture was negative. (7) illness due to other infections. The serodiagnostic capacity of STY1498 in Malawian children was evident in sera from active infection, as described for Vietnamese population [30], and also in convalescent plasma screening. STY1498 serology also validated the blood culture nega- tive/ PCR negative results. In conclusion, the combination of real-time PCR with culture pre-enrichment with blood culture improved case ascertainment among children aged between 0 and 4 years. These data highlight the hidden burden of invasive Salmonellosis in young children and support the tar- geting of pre-school children for typhoid vaccine prevention. The recent roll-out of a typhoid conjugate vaccine trial in Malawi and subsequent implementation will likely avert a greater burden of disease than previously reported. However, impact assessment of the vaccine may be affected by poor sensitivity of blood culture which is the primary endpoint for the vaccine trial. Discussion Typhi cases confirmed by blood culture and PCR with culture pre-enrichment, from those with PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 10 / 16 Ascertaining the burden of invasive Salmonella disease Ascertaining the burden of invasive Salmonella disease \| PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 11 / 16 Future vaccine trial designs should consider using a combination of blood culture and real-time PCR with culture pre-enrichment as part of the evaluation of the full impact of the intervention. Supporting information S1 STROBE. A check list of items relevant to this report of a prospective diagnostic cohort study. (DOC) Supporting information S1 STROBE. A check list of items relevant to this report of a prospective diagnostic cohort study. (DOC) S1 Fig. Receiver operating curve characteristic for the pan-primer and S. Typhi specific primer in (a) and for the pan-primer and S. Typhimurium specific primer in (b). Gener- ated from CT-values plotted against the blood culture ‘reference standard results’. (TIF) S2 Fig. Distribution of CT-values according to volume of blood sample pre-enriched for PCR. Graphs show distribution according to (a) pan-primer invA, (b) S. Typhi specific primer staG, (c) S. Typhimurium specific primer fliC. Red triangle = PCR positive for S. Typhi, Blue rectangle = PCR positive for S. Typhimurium, and Black circle = PCR negative. (TIF) S3 Fig. IgM and IgG antibody responses to four antigens (STY1886, STY1479, STY1498 and VI) in febrile children and healthy controls. The graph shows significantly higher IgM (blue) and IgG (red) levels in febrile children (n = 445) than IgM (clear) and IgG (purple) lev- els in healthy community controls (n = 61). (TIF) S4 Fig. Multidimensional scaling and Principal component analysis of IgM and IgG responses to all four antigens (STY1886, STY1479, STY1498 and VI). (1) is IgM and IgG responses where blood culture and PCR were positive for Salmonella. (3) is IgG and IgM responses where blood culture was positive for Salmonella and PCR was negative. (4) is IgG and IgM responses where PCR was positive for Salmonella and blood culture was negative. (7) S1 Fig. Receiver operating curve characteristic for the pan-primer and S. Typhi specific primer in (a) and for the pan-primer and S. Typhimurium specific primer in (b). Gener- ated from CT-values plotted against the blood culture ‘reference standard results’. (TIF) S2 Fig. Distribution of CT-values according to volume of blood sample pre-enriched for PCR. Graphs show distribution according to (a) pan-primer invA, (b) S. Typhi specific primer staG, (c) S. Typhimurium specific primer fliC. Red triangle = PCR positive for S. Typhi, Blue rectangle = PCR positive for S. Typhimurium, and Black circle = PCR negative. (TIF) S2 Fig. Ascertaining the burden of invasive Salmonella disease Fig 5. Distribution of IgM antibody titres during acute infection (A, C, E, G) and in convalescence (B, D, F, H). IgM antibodies were measured against four antigens (STY1498 in A & B, STY1479 in C & D, STY1886 in E & F & Vi in G &H) across distinct groups of children. The groups are bc+PCR+ (cases that had S. Typhi infection confirmed by both blood culture and PCR (or blood culture alone), in acute infection [age 14 to 45, median 37 months], and in convalescence [age 14 to 45, median 33 months]), PCR+ (S. Typhi infection confirmed by PCR only, in cute infection [age 8 to 46, median 22 months] and in convalescence [age 22 to 46, median 40 months]), Negative (febrile but negative for typhoid on blood culture and PCR, in acute infection [age 1 to 48, median 13.5 months] and in convalescence [age 1 to 45, median 16 months]), and Control (afebrile healthy controls [age 0 to 52.5, median 10.2 months]). https://doi.org/10.1371/journal.pntd.0007539.g005 illness due to other infections. The serodiagnostic capacity of STY1498 in Malawian children was evident in sera from active infection, as described for Vietnamese population [30], and also in convalescent plasma screening. STY1498 serology also validated the blood culture nega- tive/ PCR negative results. illness due to other infections. The serodiagnostic capacity of STY1498 in Malawian children was evident in sera from active infection, as described for Vietnamese population [30], and also in convalescent plasma screening. STY1498 serology also validated the blood culture nega- tive/ PCR negative results. In conclusion, the combination of real-time PCR with culture pre-enrichment with blood culture improved case ascertainment among children aged between 0 and 4 years. These data highlight the hidden burden of invasive Salmonellosis in young children and support the tar- geting of pre-school children for typhoid vaccine prevention. The recent roll-out of a typhoid conjugate vaccine trial in Malawi and subsequent implementation will likely avert a greater burden of disease than previously reported. However, impact assessment of the vaccine may be affected by poor sensitivity of blood culture which is the primary endpoint for the vaccine trial. Future vaccine trial designs should consider using a combination of blood culture and real-time PCR with culture pre-enrichment as part of the evaluation of the full impact of the intervention. Supporting information S1 STROBE. Ascertaining the burden of invasive Salmonella disease is IgG and IgM responses where both blood culture and PCR were negative for Salmonella. Ctrl are healthy controls. (TIF) S1 Table. Concentrations of primers and probes for the PCR reaction. (PDF) S2 Table. Bacterial colony forming units per mL. Concentrations of bacteria serially diluted and DNA extracted to assess PCR non-specific amplification. (PDF) S3 Table. Area under the curve for the receiver operating curve. The pan-primer and S. Typhi primer in (a) and the pan-primer and S. Typhimurium primer in (b). (PDF) Acknowledgments We would like to thank all the children, parents and guardians who participated in this study. We would like to thank Dr Sharon Tennant for the expert advice. Author Contributions Conceptualization: Chisomo L. Msefula, Robert S. Heyderman. Formal analysis: Chisomo L. Msefula, Franziska Olgemoeller, Matthew Graham, Marc Y. R. Henrion, Stephen Baker, Nicholas Feasey, Melita Gordon, Robert S. Heyderman. Funding acquisition: Robert S. Heyderman. Investigation: Chisomo L. Msefula, Franziska Olgemoeller, Ndaru Jambo, Dalitso Segula, Trinh Van Tan, Tonney S. Nyirenda, Wilfred Nedi, Robert S. Heyderman. Methodology: Chisomo L. Msefula, Franziska Olgemoeller, Ndaru Jambo, Dalitso Segula, Tonney S. Nyirenda, Stephen Baker, Nicholas Feasey, Melita Gordon, Robert S. Heyderman. Project administration: Chisomo L. Msefula. Supervision: Chisomo L. Msefula, Robert S. Heyderman. Writing – original draft: Chisomo L. Msefula. Writing – review & editing: Chisomo L. Msefula, Franziska Olgemoeller, Ndaru Jambo, Dalitso Segula, Trinh Van Tan, Tonney S. Nyirenda, Wilfred Nedi, Neil Kennedy, Marc Y. R. Henrion, Stephen Baker, Nicholas Feasey, Melita Gordon, Robert S. Heyderman. References 1. Feasey NA, Masesa C, Jassi C, Faragher EB, Mallewa J, Mallewa M, et al. Three Epidemics of Invasive Multidrug-Resistant Salmonella Bloodstream Infection in Blantyre, Malawi, 1998–2014. Clin Infect Dis. 2015; 61 Suppl 4:S363–71. 2. Keddy KH, Sooka A, Smith AM, Musekiwa A, Tau NP, Klugman KP, et al. Typhoid Fever in South Africa in an Endemic HIV Setting. PLoS One. 2016; 11(10):e0164939. https://doi.org/10.1371/journal.pone. 0164939 PMID: 27780232 3. Antillon M, Warren JL, Crawford FW, Weinberger DM, Kurum E, Pak GD, et al. The burden of typhoid fever in low- and middle-income countries: A meta-regression approach. PLoS neglected tropical dis- Author Contributions Investigation: Chisomo L. Msefula, Franziska Olgemoeller, Ndaru Jambo, Dalitso Segula, Trinh Van Tan, Tonney S. Nyirenda, Wilfred Nedi, Robert S. Heyderman. Investigation: Chisomo L. Msefula, Franziska Olgemoeller, Ndaru Jambo, Dalitso Segula, Trinh Van Tan, Tonney S. Nyirenda, Wilfred Nedi, Robert S. Heyderman. W W R W W R 1 2 3 Writing – review & editing: Chisomo L. Msefula, Franziska Olgemoeller, Ndaru Jambo, Dalitso Segula, Trinh Van Tan, Tonney S. Nyirenda, Wilfred Nedi, Neil Kennedy, Marc Y. R. Henrion, Stephen Baker, Nicholas Feasey, Melita Gordon, Robert S. Heyderman. itso Segula, Trinh V Henrion, Stephen Ba rences Distribution of CT-values according to volume of blood sample pre-enriched for PCR. Graphs show distribution according to (a) pan-primer invA, (b) S. Typhi specific primer staG, (c) S. Typhimurium specific primer fliC. Red triangle = PCR positive for S. Typhi, Blue rectangle = PCR positive for S. Typhimurium, and Black circle = PCR negative. (TIF) S3 Fig. IgM and IgG antibody responses to four antigens (STY1886, STY1479, STY1498 and VI) in febrile children and healthy controls. The graph shows significantly higher IgM (blue) and IgG (red) levels in febrile children (n = 445) than IgM (clear) and IgG (purple) lev- els in healthy community controls (n = 61). (TIF) S4 Fig. Multidimensional scaling and Principal component analysis of IgM and IgG responses to all four antigens (STY1886, STY1479, STY1498 and VI). (1) is IgM and IgG responses where blood culture and PCR were positive for Salmonella. (3) is IgG and IgM responses where blood culture was positive for Salmonella and PCR was negative. (4) is IgG and IgM responses where PCR was positive for Salmonella and blood culture was negative. (7) PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 12 / 16 Ascertaining the burden of invasive Salmonella disease 4. Marks F, von Kalckreuth V, Aaby P, Adu-Sarkodie Y, El Tayeb MA, Ali M, et al. Incidence of invasive salmonella disease in sub-Saharan Africa: a multicentre population-based surveillance study. Lancet Glob Health. 2017; 5(3):e310–e23. https://doi.org/10.1016/S2214-109X(17)30022-0 PMID: 28193398 5. Gordon MA, Graham SM, Walsh AL, Wilson L, Phiri A, Molyneux E, et al. Epidemics of invasive Salmo- nella enterica serovar enteritidis and S. enterica Serovar typhimurium infection associated with multi- drug resistance among adults and children in Malawi. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2008; 46(7):963–9. 6. Graham SM, Walsh AL, Molyneux EM, Phiri AJ, Molyneux ME. Clinical presentation of non-typhoidal Salmonella bacteraemia in Malawian children. Trans R Soc Trop Med Hyg. 2000; 94(3):310–4. https:// doi.org/10.1016/s0035-9203(00)90337-7 PMID: 10975008 7. Msefula CL, Kingsley RA, Gordon MA, Molyneux E, Molyneux ME, Maclennan CA, et al. Genotypic homogeneity of multidrug resistant s. Typhimurium infecting distinct adult and childhood susceptibility groups in Blantyre, Malawi. PLoS One. 2012; 7(7):e42085. https://doi.org/10.1371/journal.pone. 0042085 PMID: 22848711 8. Kabwama SN, Bulage L, Nsubuga F, Pande G, Oguttu DW, Mafigiri R, et al. A large and persistent out- break of typhoid fever caused by consuming contaminated water and street-vended beverages: Kam- pala, Uganda, January—June 2015. BMC Public Health. 2017; 17(1):23. https://doi.org/10.1186/ s12889-016-4002-0 PMID: 28056940 9. Sejvar J, Lutterloh E, Naiene J, Likaka A, Manda R, Nygren B, et al. Neurologic manifestations associ- ated with an outbreak of typhoid fever, Malawi—Mozambique, 2009: an epidemiologic investigation. PLoS One. 2012; 7(12):e46099. https://doi.org/10.1371/journal.pone.0046099 PMID: 23226492 10. Mogasale V, Maskery B, Ochiai RL, Lee JS, Mogasale VV, Ramani E, et al. Burden of typhoid fever in low-income and middle-income countries: a systematic, literature-based update with risk-factor adjust- ment. Lancet Glob Health. 2014; 2(10):e570–80. https://doi.org/10.1016/S2214-109X(14)70301-8 PMID: 25304633 11. Hendriksen RS, Leekitcharoenphon P, Lukjancenko O, Lukwesa-Musyani C, Tambatamba B, Mwaba J, et al. Genomic signature of multidrug-resistant Salmonella enterica serovar typhi isolates related to a massive outbreak in Zambia between 2010 and 2012. J Clin Microbiol. 2015; 53(1):262–72. https://doi. org/10.1128/JCM.02026-14 PMID: 25392358 edy N, Dube Q, et al. Mat phoid Fever in Blantyre, M , Feasey NA, et al. Phylog monella Typhi identifies int 9. https://doi.org/10.1038/ 12. Pitzer VE, Feasey NA, Msefula C, Mallewa J, Kennedy N, Dube Q, et al. Mathematical Modeling to Assess the Drivers of the Recent Emergence of Typhoid Fever in Blantyre, Malawi. Clin Infect Dis. References 1. Feasey NA, Masesa C, Jassi C, Faragher EB, Mallewa J, Mallewa M, et al. Three Epidemics of Invasive Multidrug-Resistant Salmonella Bloodstream Infection in Blantyre, Malawi, 1998–2014. Clin Infect Dis. 2015; 61 Suppl 4:S363–71. 2. Keddy KH, Sooka A, Smith AM, Musekiwa A, Tau NP, Klugman KP, et al. Typhoid Fever in South Africa in an Endemic HIV Setting. PLoS One. 2016; 11(10):e0164939. https://doi.org/10.1371/journal.pone. 0164939 PMID: 27780232 2. Keddy KH, Sooka A, Smith AM, Musekiwa A, Tau NP, Klugman KP, et al. Typhoid Fever in South Africa in an Endemic HIV Setting. PLoS One. 2016; 11(10):e0164939. https://doi.org/10.1371/journal.pone. 0164939 PMID: 27780232 3. Antillon M, Warren JL, Crawford FW, Weinberger DM, Kurum E, Pak GD, et al. The burden of typhoid fever in low- and middle-income countries: A meta-regression approach. PLoS neglected tropical dis- eases. 2017; 11(2):e0005376. https://doi.org/10.1371/journal.pntd.0005376 PMID: 28241011 3. Antillon M, Warren JL, Crawford FW, Weinberger DM, Kurum E, Pak GD, et al. The burden of typhoid fever in low- and middle-income countries: A meta-regression approach. PLoS neglected tropical dis- eases. 2017; 11(2):e0005376. https://doi.org/10.1371/journal.pntd.0005376 PMID: 28241011 13 / 16 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 Ascertaining the burden of invasive Salmonella disease 22. Zhou L, Pollard AJ. A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples. BMC Infect Dis. 2012; 12:164. https://doi.org/10.1186/ 1471-2334-12-164 PMID: 22839649 23. Sinha A, Sazawal S, Kumar R, Sood S, Reddaiah VP, Singh B, et al. Typhoid fever in children aged less than 5 years. Lancet. 1999; 354(9180):734–7. https://doi.org/10.1016/S0140-6736(98)09001-1 PMID: 10475185 24. Siddiqui FJ, Rabbani F, Hasan R, Nizami SQ, Bhutta ZA. Typhoid fever in children: some epidemiologi- cal considerations from Karachi, Pakistan. Int J Infect Dis. 2006; 10(3):215–22. https://doi.org/10.1016/ j.ijid.2005.03.010 PMID: 16431148 25. Brooks WA, Hossain A, Goswami D, Nahar K, Alam K, Ahmed N, et al. Bacteremic typhoid fever in chil- dren in an urban slum, Bangladesh. Emerg Infect Dis. 2005; 11(2):326–9. https://doi.org/10.3201/ eid1102.040422 PMID: 15752457 26. World Health O. Typhoid vaccines: WHO position paper, March 2018—Recommendations. Vaccine. 2019; 37(2):214–6. https://doi.org/10.1016/j.vaccine.2018.04.022 PMID: 29661581 27. Feasey NA, Gaskell K, Wong V, Msefula C, Selemani G, Kumwenda S, et al. Rapid emergence of multi- drug resistant, h58-lineage salmonella typhi in Blantyre, Malawi. PLoS neglected tropical diseases. 2015; 9(4):e0003748. https://doi.org/10.1371/journal.pntd.0003748 PMID: 25909750 28. Zhou L, Pollard AJ. A fast and highly sensitive blood culture PCR method for clinical detection of Salmo- nella enterica serovar Typhi. Ann Clin Microbiol Antimicrob. 2010; 9:14. https://doi.org/10.1186/1476- 0711-9-14 PMID: 20403166 29. Charles RC, Sultana T, Alam MM, Yu Y, Wu-Freeman Y, Bufano MK, et al. Identification of immuno- genic Salmonella enterica serotype Typhi antigens expressed in chronic biliary carriers of S. Typhi in Kathmandu, Nepal. PLoS Negl Trop Dis. 2013; 7(8):e2335. https://doi.org/10.1371/journal.pntd. 0002335 PMID: 23936575 30. Liang L, Juarez S, Nga TV, Dunstan S, Nakajima-Sasaki R, Davies DH, et al. Immune profiling with a Salmonella Typhi antigen microarray identifies new diagnostic biomarkers of human typhoid. Sci Rep. 2013; 3:1043. https://doi.org/10.1038/srep01043 PMID: 23304434 31. Tran Vu Thieu N, Trinh Van T, Tran Tuan A, Klemm EJ, Nguyen Ngoc Minh C, Voong Vinh P, et al. An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever. J Infect. 2017; 75(2):104–14. https://doi.org/10.1016/j.jinf.2017.05.007 PMID: 28551371 32. Nyirenda TS, Gilchrist JJ, Feasey NA, Glennie SJ, Bar-Zeev N, Gordon MA, et al. Sequential acquisi- tion of T cells and antibodies to nontyphoidal Salmonella in Malawian children. J Infect Dis. 2014; 210 (1):56–64. https://doi.org/10.1093/infdis/jiu045 PMID: 24443544 33. Feasey NA, Dougan G, Kingsley RA, Heyderman RS, Gordon MA. 2015; 61 Suppl 4:S251–8. 13. Wong VK, Baker S, Pickard DJ, Parkhill J, Page AJ, Feasey NA, et al. Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events. Nat Genet. 2015; 47(6):632–9. https://doi.org/10.1038/ng.3281 PMID: 25961941 2015; 47(6):632–9. https://d ngo DM, Mwituria J, Muyodi ant Salmonella enterica sero Microbiol. 2010; 48(6):2171– Keddy KH, Angulo FJ, Crum Emerg Infect Dis. 2015; 21( 14. Kariuki S, Revathi G, Kiiru J, Mengo DM, Mwituria J, Muyodi J, et al. Typhoid in Kenya is associated with a dominant multidrug-resistant Salmonella enterica serovar Typhi haplotype that is also wide- spread in Southeast Asia. J Clin Microbiol. 2010; 48(6):2171–6. https://doi.org/10.1128/JCM.01983-09 PMID: 20392916 15. Ao TT, Feasey NA, Gordon MA, Keddy KH, Angulo FJ, Crump JA. Global burden of invasive nontyphoi- dal Salmonella disease, 2010(1). Emerg Infect Dis. 2015; 21(6). 16. Andrews JR, Ryan ET. Diagnostics for invasive Salmonella infections: Current challenges and future directions. Vaccine. 2015; 33 Suppl 3:C8–15. 17. Parry CM, Wijedoru L, Arjyal A, Baker S. The utility of diagnostic tests for enteric fever in endemic loca- tions. Expert Rev Anti Infect Ther. 2011; 9(6):711–25. https://doi.org/10.1586/eri.11.47 PMID: 21692675 8. Lim PL, Tam FC, Cheong YM, Jegathesan M. One-step 2-minute test to detect typhoid-specific antibod- ies based on particle separation in tubes. J Clin Microbiol. 1998; 36(8):2271–8. PMID: 9666004 19. Prakash P, Sen MR, Mishra OP, Gulati AK, Shukla BN, Nath G. Dot enzyme immunoassay (Typhidot) in diagnosis of typhoid fever in children. J Trop Pediatr. 2007; 53(3):216–7. https://doi.org/10.1093/ tropej/fmm008 PMID: 17387102 20. Wasihun AG, Wlekidan LN, Gebremariam SA, Welderufael AL, Muthupandian S, Haile TD, et al. Diag- nosis and Treatment of Typhoid Fever and Associated Prevailing Drug Resistance in Northern Ethiopia. Int J Infect Dis. 2015; 35:96–102. https://doi.org/10.1016/j.ijid.2015.04.014 PMID: 25931197 21. Zhou L, Jones C, Gibani MM, Dobinson H, Thomaides-Brears H, Shrestha S, et al. Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples. PLoS One. 2016; 11(3):e0150576. https://doi.org/10.1371/journal.pone.0150576 PMID: 26930553 14 / 16 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa. Lancet. 2012; 379(9835):2489–99. https://doi.org/10.1016/S0140-6736(11)61752-2 PMID: 22587967 34. Wain J, Hendriksen RS, Mikoleit ML, Keddy KH, Ochiai RL. Typhoid fever. Lancet. 2015; 385 (9973):1136–45. https://doi.org/10.1016/S0140-6736(13)62708-7 PMID: 25458731 35. Tennant SM, MacLennan CA, Simon R, Martin LB, Khan MI. Nontyphoidal salmonella disease: Current status of vaccine research and development. Vaccine. 2016; 34(26):2907–10. https://doi.org/10.1016/j. vaccine.2016.03.072 PMID: 27032517 36. Mackenzie G, Ceesay SJ, Hill PC, Walther M, Bojang KA, Satoguina J, et al. A decline in the incidence of invasive non-typhoidal Salmonella infection in The Gambia temporally associated with a decline in malaria infection. PLoS One. 2010; 5(5):e10568. https://doi.org/10.1371/journal.pone.0010568 PMID: 20485496 37. Ferreccio C, Levine MM, Manterola A, Rodriguez G, Rivara I, Prenzel I, et al. Benign bacteremia caused by Salmonella typhi and paratyphi in children younger than 2 years. J Pediatr. 1984; 104(6):899–901. https://doi.org/10.1016/s0022-3476(84)80492-8 PMID: 6427437 38. Mahle WT, Levine MM. Salmonella typhi infection in children younger than five years of age. Pediatr Infect Dis J. 1993; 12(8):627–31. PMID: 8414773 39. Antillon M, Saad NJ, Baker S, Pollard AJ, Pitzer VE. The Relationship Between Blood Sample Volume and Diagnostic Sensitivity of Blood Culture for Typhoid and Paratyphoid Fever: A Systematic Review and Meta-Analysis. J Infect Dis. 2018; 218(suppl_4):S255–S67. https://doi.org/10.1093/infdis/jiy471 PMID: 30307563 40. Norris P, Ng LF, Kershaw V, Hanna F, Wong A, Talekar M, et al. Knowledge and reported use of antibi- otics amongst immigrant ethnic groups in New Zealand. J Immigr Minor Health. 2010; 12(1):107–12. https://doi.org/10.1007/s10903-008-9224-5 PMID: 19139990 41. Demore B, Le Govic D, Thilly N, Boivin JM, Pulcini C. Reliability of self-reported recent antibiotic use among the general population: a cross-sectional study. Clin Microbiol Infect. 2017; 23(7):486 e7– e12. 15 / 16 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 Ascertaining the burden of invasive Salmonella disease 42. Baker S, Favorov M, Dougan G. Searching for the elusive typhoid diagnostic. BMC Infect Dis. 2010; 10:45. https://doi.org/10.1186/1471-2334-10-45 PMID: 20205702 43. Ktsoyan Z, Ghazaryan K, Manukyan G, Martirosyan A, Mnatsakanyan A, Arakelova K, et al. Inflamma- tory Responses to Salmonella Infections Are Serotype-Specific. Int J Bacteriol. 2013; 2013:168179. https://doi.org/10.1155/2013/168179 PMID: 26904722 44. Pulickal AS, Gautam S, Clutterbuck EA, Thorson S, Basynat B, Adhikari N, et al. Kinetics of the natural, humoral immune response to Salmonella enterica serovar Typhi in Kathmandu, Nepal. Clin Vaccine Immunol. 2009; 16(10):1413–9. https://doi.org/10.1128/CVI.00245-09 PMID: 19710294 42. Baker S, Favorov M, Dougan G. Searching for the elusive typhoid diagnostic. BMC Infect Dis. 2010; 10:45. https://doi.org/10.1186/1471-2334-10-45 PMID: 20205702 43. Ktsoyan Z, Ghazaryan K, Manukyan G, Martirosyan A, Mnatsakanyan A, Arakelova K, et al. Inflamma- tory Responses to Salmonella Infections Are Serotype-Specific. Int J Bacteriol. 2013; 2013:168179. https://doi.org/10.1155/2013/168179 PMID: 26904722 44. Pulickal AS, Gautam S, Clutterbuck EA, Thorson S, Basynat B, Adhikari N, et al. Kinetics of the natural, humoral immune response to Salmonella enterica serovar Typhi in Kathmandu, Nepal. Clin Vaccine Immunol. 2009; 16(10):1413–9. https://doi.org/10.1128/CVI.00245-09 PMID: 19710294 44. Pulickal AS, Gautam S, Clutterbuck EA, Thorson S, Basynat B, Adhikari N, et al. Kinetics of the natural, humoral immune response to Salmonella enterica serovar Typhi in Kathmandu, Nepal. Clin Vaccine Immunol. 2009; 16(10):1413–9. https://doi.org/10.1128/CVI.00245-09 PMID: 19710294 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 16 / 16 PLOS Neglected Tropical Diseases \| https://doi.org/10.1371/journal.pntd.0007539 July 17, 2019 16 / 16
W4232381631.txt	https://zenodo.org/records/2262537/files/article.pdf	de		Zur Lehr von den Zuständen der Materie	Zeitschrift für Chemie und Industrie der Kolloide	1,909	public-domain	2,973	117 Zur Lehre yon den Zustiinden der Materie. Von P. P. von W e i m a r n , St. Petersburg. Zweiter Tell. wegen der geringeren Umwandiungsgeschwindig§ 24. Reakfionsgeschwindigkeit und Assoziation. keit der schon zusammengetroffenen reagierenElnige Erg~inzungen zum Massenwirkungs- den Molekularkomplexe. (Wir haben im § 21 gesetz. Ueber ,,unbestimmte" chemtsche gesehen, dat~ die Aggregation der Molektile zu Komprexen ihre Reaktionsfahigkeit herabsetzt.) Verbindungen, 04). (Fortsetzung) In den vorausgehenden Paragraphen war mit genfigender Ausfiihrlichkeit ausgefiihrt worden, inwi'eweit die Assoziation der Reaktionskomponenten auf die Geschwindigkeit der chemischen Prozesse, als deren Resultat iiuBerst feink6rnige kristallinische Niederschl/ige entstehen, einwirkt. Aus dem in diesen Paragraphen Dargelegten geht vollstiindig Mar hervor, dat~ die Verlangsamung der Reaktionsgeschwindigkeit nicht nur durch die Eigenartigkeit der Niederschlagsform (Existenz von halb durchl/issigen Zellen usw.) bedingt ist, sondern auch durch die geringere Beweglichkeit der assoziierten Molekfilkomplexe, und ebenso auch durch die Verringerung der Umwandlungsgeschwindigkeit der schon zusammengestot~enen Komplexe der reagierenden Molekfile. Natfirlich muB auch bei Reaktionen im homogenen Medium, ohne Bildung yon Niederschl~igen fester K6rper, im Falle einer Anwendung yon stark assoziierten L6sungen ebenfalls eine Verlangsamung der Reaktionsgeschwindigkeit eintreten. Nehmen wir zur Untersucbung eine Reaktion des einfachsten Typus, und zwar Al-k A~ ~ A'I q-A'~ und mOge die Reaktion p r a k t i s c h vollst~ndig in der yon den beiden Pfeilen bezeichneten Richtung verlaufen. Wenn die Molekfile A1 und A2 nicht assoziiert sind, so haben wit ffir die Reaktionsgeschwindigkeit: Vo = K. C1 C2 (I). M6ge ietzt dieselbe Reaktion in einem anderen L6sungsmittel verlaufen, das, o h n e irgendwelche Nebenreaktionen herv o r z u r u f e n, die Substanzen A1 und A~. stark assoziiert. Dann wird augenscheinlich bei densetben Volumkonzentrationen C1 und C2 die Reaktion mit geringerer Geschwindigkeit verlaufen als im ersten Fall, erstens wegen der geringeren H/iufigkeit des Zusammentreffens der stark assoziierten Komplexe, und zweitens ~04) Mitgeteilt in der Russisehen ChemischenGesellschaR in der Sitzung am 1. (13.) November 1907. Siehe Koll.-Zeitschr. 2, 329--330 (1908). Wir woUen die Reaktion zwischen den assoziierten Reaktionskomponenten als eine selbst~indige Reaktion mit neuen reagierenden Molekiilen betrachten, wobei aber die Menge der get6sten Substanzen gleichbleibt, d. h. entsprechend den Konzentrationen C1 und C2. M6ge der durchschnittliche Assoziationsgrad, d. h. die durchschnittliche Zahl der Molekiile in den reagierenden Molekularkomplexen ffir den K6rper A1 gleich a i sein und ffir den K6rper A2 gleich a 2. Dann sind augenscheinlich die Volumkonzentrationen der reagierenden Komplexe [Alal] und [A~a2] entsprechend C1 al und C2, und die Reaktionskonstante oder tier a2 Affinitiitskoeffizient wird wegen Verminderung der Reaktionsfiihigkeit bei den assoziierten Komplexen kleiner sein als im vorhergehenden Falle. Er wird also gleich K sein. e Wenn wit dieses in Betracht ziehen und das Massenwirkungsgesetz bei unserem Fall anwenden, so erhalten wit: ; K v- ~ .c1.c~ (II) ata~ Bei a I = a s = ~ = I erhalten wir den gew6hnlichen Ausdruck fiir das Oesetz yon G u l d b e r g und W a a g e . Es muff bemerkt werden, dab a 1 und a s variable Gr6t~en sind, und deshalb ist es notwendig, genau die Gesetze der Verminderung des Assoziationsgrades beim Sinken der Konzentration der reagierenden L6sungen zu kennen, was aber meiner Ansicht nach beim gegenwiirtigen Stand der Frage unm6glich ist. Es ist leicht einzusehen, dab die Formel 1, streng genommen, nut f/.ir ideale F~ille Bedeutung hat, denn Assoziation findet in allen L6sungen yon nur irgend betrachtlichen Konzentrationen start. Die vorziigliche Ueberein- 118 stimmung der Versuche aber mit der Formel I kann dadurch erkliirt werden, daiS in den bis jetzt untersuchten Systemen der Assoziationsglad klein war, d. h. ul, u 2 und Q vom Werte Eins wenig verschieden waren. Anders ausgedrfickt heiflt es, daft stark assoziierte Systeme noch nicht untersucht wurden. Aus diesem Orunde muff die Formel I1 als allgemeiner angesehen werden als Formel I. Wir haben oben gesehen (ira weiteren Verlauf meiner Darlegungen werde ich noch Oeiegenheit haben, auf diesen Punkt zurfickzukommen), daft zwischen wahren nicht assoziierten L6sungen, assoziierten L6sungen, Solen und groben Suspensionen ein auflerordenttich stetiger Uebergang vorhanden ist. Es muff folglich auch der Charakter des Reaktionsverlaufs in molekularheterogenen und aggregiertheterogenen (ultramikro- und mikroheterogenen) Systemen mit derselben Stetigkeit sich ~indern. Das Massenwirkungsgesetz erfordert, wie es auch aus seiner kinetischen Begrfindung hervorgeht, die Anwesenheit einer fortschreitenden Bewegung der reagierenden Teilehen. W~tre diese Bewegung klein und wfirden die Teiichen bei der gegebenen Konzentration nur sehr selten zusammentreffen, so wfirde die Reaktion, wie grois auch die Affinitiit sein m6ge, nur mit einer aui~erordentlichen Langsamkeit vor sich geheu, denn die chemisehe Affinitiit iibt ihre Wirkungen nut dann aus, wenn die Entfernung zwisehen den Teilchen sehr klein ist. (Die Versuehe von D o n n y und Mareska, vonRaoultundPiktetfiberden Verlauf der Reaktionen bei sehr tiefen Temperaturen [ - - 8 0 0 ; 125 °] bestiitigen dieses vortrefflich. Deshatb mut~ der Ausdruck fiir die Reaktionsgeschwindigkeit, falls die M6glichkeit der engen Berfihrung der reagierenden Teilchen vollstiiudig ausgeschlossen ist, dem Werte Null gleichkommen. Streng genommen muff gesagt werden, wenn man berfieksichtigt, dafl auch bei den /iuiserst schwer verdampfenden und sehwerl6slichen K6rpern eine gewisse, wenn auch iiuiSerst geringe Dampfdichte und L6slichkeit anzunehmen ist, dait die ReaMionsgesehwindigkeit selbst bei Reaktionen zwischen groben Suspensionen der schwerst 16slichen K6rper doch nicht gleieh Null ist. Aus diesem Grunde ist sowohl bei sehr tiefer Temperatur als auch bei sehr starker Assoziation die Reaktionsgeschwindigkeit p r a k t i s e h gleich Null. Es ist dabei gar nicht notwendig, daiS gl und ~2 gleich unendlieh seien, wie es der mathematische Sinn der Formel II erfordert, denn wie durch ultramikroskopische Untersuchungen festgestellt wurde, besitzen die Ultramikronen schon eine kleine Beweglichkeit. W/iren uns die Dimensionen der Molekfile genau bekannt und h~itten wir schon quantitative Untersuchungen fiber die Aenderung der Reaktionsf/ihigkeit bei Vergr6Berung der Anzahl der Molekfile in den Komplexen, so wfirde die Formel K V= • .Cl.C~_ ('1 u.a leicht auf alle Reaktionsf/ille verallgemeinert werden k6nnen, sowohl auf die Reaktionsfiille zwischen wahren L6sungen und Solen, als auch zwischen Solen, unter der Bedingung, dab bei solchen Reaktionen Produkte gebildet werden, die im Reaktionsmedium 16slieh sind. Bei Abwesenheit der letzten Bedingung kann die Formel II nur auf die ersten Reaktionsmomente angewendet werden, denn die Bildung von Sehutzhiillen u. a. wird die Reaktion verz6gern und ihr weiterer Verlauf wird yon Faktoren abh~ingen, die in der Formel II nicht enthalten sind. Es ist zweifellos, dai~ eine solche allgemeine Formet fiir Reaktionsgeschwindigkeiten gegeben werden kann, die u n b e d i n g t a l l e Reaktionsfiilie umfaflt, sowohl im homogenen als aueh im heterogenen Medium. Von dieser Formel aus wird man durch Ausschaltung tier entsprechenden Faktoren zur Formel I gelangen k•nnen. Jedoch hat eine solehe allgemeine Formel keinen praktischen Sinn, denn sie wird sehr kompliziert sein. Es muis also als festgesteltt angesehen werden, dais d i e O e s c h w i n d i g k e i t und der Charakter des Reaktionsverlaufs zwischen molekular-heterogenen und aggregiert-heterogenen S y s t e m e n sich mit autierordentlieher Stetigkeit ver~tndert. leh mul~ h i e r b e i b e m e r k e n , dais d e r U n t e r s c h i e d zwischen eiuer ~iuiserst f e i n z e r t e i l t e n k r i s t a l l i n i s c h e n S u b s t a n z u n d d e r s e l b e n S u b s t a n z im w a h r h a f t g e l 6 s t e n Z u s t a n d e im S i n n e der R e a k t i o n s f ~ i h i g k e i t b e d e u t e n d g e r i n g e r ist ats d e r U n t e r s e h i e d z w i s c h e n grol3en K r i s t a l l e n u n d d e r s e l b e n k r i s t a l l i n i s e h e n S u b s t a n z im iiuflerst rein zerteilten Zustande. Zum Schluis dieser Paragraphen will ich noeh die , , u n b e s t i m m t e n chemisehen Verbindungen" erw~hnen. 119 Meiner Ansicht nach ist der Begriff von ,,unbestimmten chemischen Verbindungen" einer der unrichtigsten Begriffe der modernen Chemie. In der Chemie k6nnen wie in jeder anderen wissenschaftlichen Disziplin ,, u n d e f i n i e r b a r e" (bei den gegenwitrtigen Hilfsmethoden der Forschung) Fragen vorhanden sein, aber keinesfalls , , u n b e s t i m m t e " . Die Anwendung des Terminus ,,unbestimmt" kann um so weniger gerechffertigt werden, da eine durchaus richtige Bezeichnung ffir diese Systeme existiert, und zwar - - ,,Systeme, die den s t 6 c h i o m e t r i s c h e n G e s e t z e n nicht g e h o r e h e n " . Es fragt sich jetzt nun, ob solche Systeme wirklich chemische Verb i n d u n g e n sind, die den s t 6 c h i o m e t r i schen G e s e t z e n nicht g e h o r c h e n , oder ob diese Systeme ein feines Gemisch m e h r e r e r c h e m i s c h e r , den s t 6 c h i o m e t r i s c h e n Gesetzen folgenden Verbindungen darst ell e n, das sich entweder im dynamischen Gteichgewicht oder im Zustande einer langsamen Umwandlung befindet. In den vorangehenden Paragraphen (siehe auch § 25) haben wit schon eine ganze Reihe yon Verbindungen kennen gelernt, die gew6hnlich als ,,unbestimmte" Verbindungen bezeichnet werden. Wit haben auch gesehen, dab diese Verbindungen durchaus der in § 22 angeffihrten Definition entsprechen, d. h. da~ sie feine Gemische von chemischen, den st6chiometrischen Gesetzen gehorchenden Verbindungen darstellen. Hier muff ich noch hinzuffigen, dab auf Grund meiner Untersuchungen fiber die L6sungsprozesse (die Resultate dieser Untersuchungen werden in einer besonderen Abhandlung ver6ffentlicht werden) hervorgeht, da~ fltissige und feste L6sungen ebensolche Gemische sind, aber noch feinerer Art, d.h. dab die Substanzen in diesen Gemischen sich sehon in molekularer Zerteilung befinden. Auf Grund pers6nlicher Beobachtungen kann also meiner Ansicht nach auf die oben gestellte Frage nur eine Antwort gegeben werden: ,,Unbestimmte chemische Verbindungen existieren in W i r k l i c h k e i t nicht~°s)- D i e i e n i g e n S y s t e m e , d i e a l s u n b e s t i m m t e Verbindungen bezeichnet werden, sind feine Gemische mehrerer den st6chiometrischen Gesetzen geh o r c h e n d e r V e r b i n d u n g e n . D i e s e Verb i n d u n g e n k 6 n n e n sich in Form k l e i n s t e r Molekfilaggregateoderinmolekularer 10,) Ueber physikalischeAdsorption. Siehe § 27. Zerteilung befinden. Sehrhiiufigbefinden sich d i e als u n b e s t i m m t e chemische Verbindungen bezeichneten S y s t e m e im Z u s t a n d e n o c h u n v o l l endeter chemischer Umwandlungen, d. h. in dynamischem Zustand. Ich will reich vorlaufig mit dem in den vorangehenden Paragraphen fiber, unbestimmte" Verbindungen Gesagten begnfigen, denn ich hoffe sp~iter in anderen Abhandlungen diese Frage ausffihrlicher zu betrachten. Einige Typen der ,unbestimmten" Verbitidungen werden wir fibrigens noch im Laufe dieser Darlegungen kennen lernen. § 95. Ueber den Reaktionsverlauf zwischen kristallinischen Pulvern und wahren L6sungen. Auf Seite 255 (4) habe ich zur anschaulichen Darstellung des Reaktionsmechanismus in stark assoziierten L6sungen, die Frage fiber den Reaktionsverlauf zwischen kristaUinischen Pulvern und wahren L6sungen kurz erwiihnt. Dieser Paragraph ist der ausffihrlichen Darlegung dieser Frage gewidmet. Ich will bier den Fall betrachten, in dem die Korngr6Be des kristallinischen Putvers :~wischen makroskopischen undwinzigsten mikroskopischen Dimensionen schwankt. Durch die Aenderungen der Gr6t3e der K6rner innerhalb der bezeichneten Grenzen erleiden die Erscheinungen, von denen im vorliegenden Paragraphen die Rede sein wird, in ihrem Wesen keine Verlinderung, nur der MaBstab der Erscheinungen iindert sich. Es muB daher auch der Forscher beim Uebergang der K6rner yon makroskopischen zu mikroskopischen Dimensionen die Beobachtung mit dem unbewaffneten Auge verlassen und zum Mikroskop greifen. Nehmen wir an, wir h~tten z. B. ein kristallinisches Pulver einer Substanz X. Die S~ittigungskonzentration dieser Substanz irn gewiihlten Reaktionsmedium wlirea Orammolekfile. Nehmen wir einen anderen KOrper Y in Form einer L6sung. Die K6rper X und Y seien so gew~ihlt, dab bei ihrer Zusammenwirkung ein neuer K6rper Z entstfinde, der im gewiihlten Reaktionsmedium praktisch unl6slich sei. Beim Eintragen des pulverisierten K6rpers X in eine L6sung der Substanz Y (es seien b Grammolekfile Y in der L6sung) k6nnen, bedingt dutch das gegenseitige Verh~iltnis yon a und b, drei verschiedene F~lle eintreten 10~). 10b) Bei der Betrachtung der osmotischen Erscheinungen vemachlllssige ich, urn die Auseinanderlegung nicht zu komplizieren, den hydrostatischenDruck. Dies 120 Ich will alle diese F/ille einzeln betrachten. I. a ~ > b ; II. a < ~ b ; III. a - - b . Also bei gewissen Konzentrations-Verh~iltnissen a und b und unter der Bedingung, dais a anfangs grbt~er war als b, kann der Kristall innerhalb der ZeUe bestehen bleiben. I. a ~ b . Beim Eintragen des Kristalls des K6rpers X in Dies wird dann z. B. eintreffen, wenn der Kristall die L6sung des K6rpers Y /iberzieht sich der nicht zu klein ist und a nut um weniges grSt~er Kristall mit einer Hfille des K6rpers Z, die nur ist als b. Unter diesen Bedingungen ist die das L6sungsmittel durchl~itSt. Das L6sungsmittel Menge des L6sungsmittels, die in die Hfille eingewird, sobald es unter irgend einen Tell der drungen und eine ges~ttigte L6sung gebildet hat, gerade ausreichend, um die Konzentration Hiille, die aus dem Niederschlag Z besteht, der ttufleren L6sung auf den Wert a zu bringen. eingedrungen ist, s e h r schnell eine gewisse Ist aber die LOsung des K6rpers Y bedeutend Menge des K6rpers X aufl6sen. Zwischen der schw~icher als die gestittigte L6sung des K6rpers Hiille und der Oberfl~che des Kristalls X entsteht eine diesem Kristall gegenfiber ges/ittigte L~sung X, so wird der eingetragene k l e i n e Kristall v o n d e r Konzentration yon a Orammolekfilen. sich frfiher aufl6sen bevor die L6sungen isosDa nach den Bedingungen a )> b, so wird eine motisch geworden, deshalb wird auch die GleichOsmose eintreten. Die Hiille wird sich in Form heit der osmotischen Drucke bei einer geringeren eines Sackes auseinanderziehen. Dieser Sack Konzentration als a bestehen. Wenn also die Zellwt~nde wegen der Konnimmt sehr oft wegen der ungleichen Diehte [infolge der Bildung yon Str6men] des umge- zentrationserniedrigung infolge der Bildung des benden Mediums sehr eigenartige Formen an. Niederschlages Z vor dem Eintritt eines osmoDer Sack wird platzen, sobald die Orenzen der tischen Gleichgewichtes zwischen den L6sungen eine grob kristaltinische Struktur nicht erreichen Elastizit/it der Hfitle iiberschritten sind. Dutch die gebildete Oeffnung im Sack wird (s. oben)106) und ihre Halbdurchltissigkeit nicht verlieren, so mu• die Reaktion zum Stillstand die Berfihrungsfl~lche der reagierenden L6sungen bloiSgelegt. Infolge der Reaktion wird sie yon kommen, obgleich reagierende Molektile noch neuem mit einer Hfille fiberzogen und die Osmose vorhanden sind. In Wirklichkeit trifft dies auch zu, aber der beginnt von neuem. Reaktionsstillstand h6rt auf, sobald die ZellenAuf diese Weise wird der Sack sich periodisch auseinanderziehen, platzen und wieder geflickt wtinde infolge mechanischer Ursachen platzen oder grobk6rniger werden infolge eines Umwerden. kristallisierungsprozesses, der in den ZellenWann wird dieser Prozel~ ein Ende linden? Kann dieser ProzeiS aufh6ren, solange in der yore wtinden, wenn auch sehr langsam, vor sich geht. 107) Bei der Betrachtung des Falls I wurde vorSack eingeschlossenen L6sung noch der Kristall des KSrpers X sich befindet? Infolge der Osmose ausgesetzt, dat~ die Konzentration der L6sung wird die /iutSere L6sung augenscheinlich immer des K6rpers Y derartig ist, dab sie infolge der konzentrierter werden, und die innere wird eine Osmose nicht so stark anwachsen kann, dab eine konstante Konzentration so lange bewahren, so- Ausscheidung (Kristallisation) des K6rpers Y eintritt. Ich habe eine solche Kristallisation lange der Kristall nicht aufgel6st ist. Die Osmose kann deshalb vor der Aufl6sung des Kristalls nicht betrachtet, die in Wirklichkeit manchmai beim Falle I eintrifft, da diese Kristallisation sich aufh6ren, wenn die Konzentration der ~ulSeren aus der Beschreibung der Erscheinungen des L6sung die Stittigungskonzentration der inneren II. Falls, zu deren Darlegung ich jetzt fibergehen erreicht, oder dann, wenn die Konzentration will, ergeben wird. beider L6sungen nach Aufl6sung des eingetragenen Kristalls eine CorSl~e erreicht, die x06) Vgl. auch O. Btitschli: ,Untersuchungen tiber kleiner ist als a. Stmkturen usw. Leipzig, 1998, Abschnitt 7: ,Unteran kohlensauren Kalk". S. 110--t30. ist durchaus zul~tssig, wenn man mit kleinen Votumina suchungen ~¢r) Vgl. aus 1. c. der klassischen Arbeit yon der L6sungen arbeitet. Weiterhin fasse ich aus entsprechenden Gdinden und gem~l] allgemeiner An- O. Biitschli, Abschnitt 11 : ,Einige Versuche fiber sehauung L6sungen, die die gleiche Zahl Qramm- Niederschlagsmembranen a und hauptsachlich AbMolekflle enlhalten, als isosmotisch auf, obschon letzteres schni/t 12: , Einige Bemerkungen fiber die Waoensxrukmr kristallinischer Bildangen ira ~.Jegensatz zu fen aurca Veffahren mir pers6nlich nicht ganz einwandkei er- Gerinnung oder Eintrocknung entstehenden echten scheint. Denn die Krtifte, die verschiedene KOrper in gel6stem Zustande erhalien, k0nnen nicht immer ihrer Wabens/rukturen'. S. 138-147-Fortsetzung( folgt.) lniensit~t nach gteich sein.
https://openalex.org/W3215958238	https://riviste.fupress.net/index.php/caryologia/article/download/1122/1017	English	null	Influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats	Caryologia	2,021	cc-by	4,681	Yuri A. Kirillov1, Maria A. Kozlova1, Lyudmila A. Makartseva1, Igor A. Chernov2, Evgeniya V. Shtemplevskaya1, David A. Areshidze1,* 1 Federal State Budgetary Scientific Institution «Research Institute of Human Morphol- ogy» Published: December 21, 2021 Abstract. The features of the diurnal dynamics of the area of rat hepatocyte nuclei and their ploidy were studied under conditions of a standart (fixed) light regime and con- stant illumination, as well as under chronic exposure to alcohol in the mentioned light regimes. It has been shown that exposure to alcohol and exposure to constant illumi- nation separately lead to a change in the amplitude-phase characteristics of the circa- dian rhythm of the nucleus area, while the combined effect of these factors leads to a complete destruction of the rhythm, which indicates a violation of adaptation process- es. An increase in the average ploidy of hepatocyte nuclei in chronic alcohol intoxica- tion is also shown, while in animals kept under constant illumination without drinking alcohol, the values of this parameter decrease, which indicates a successful course of the adaptation process. The conducted research indicates that the results of karyomet- ric and ploidometric analysis characterize the degree of influence of alcohol intoxica- tion and changes in the light regime on the liver of rats, reflecting the rate of efficiency of adaptation to these factors. Copyright: © 2021 Yuri A. Kirillov, Maria A. Kozlova, Lyudmila A. Makartseva, Igor A. Chernov, Evgeniya V. Shtem- plevskaya, David A. Areshidze. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Key words: caryometry, ploidy, hepatocyte, circadian, morphometry. Competing Interests: The Author(s) declare(s) no conflict of interest. Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 45-51, 2021 ISSN 0008-7114 (print) \| ISSN 2165-5391 (online) \| DOI: 10.36253/caryologia-1122 Received: October 26, 2020 2 FSBEI HE Tyumen State Medical University of the Ministry of Health of Russia Corresponding author. E-mail: labcelpat@mail.ru Accepted: June 01, 2021 Accepted: June 01, 2021 Firenze University Press www.fupress.com/caryologia Firenze University Press www.fupress.com/caryologia Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Yuri A. Kirillov, Maria A. Kozlova, Lyudmila A. Makartseva, Igor A. Chernov, Evgeniya V. Shtemplevs- kaya, David A. Areshidze (2021) Influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats. Caryologia 74(3): 45-51. doi: 10.36253/caryologia-1122 Yuri A. Kirillov1, Maria A. Kozlova1, Lyudmila A. Makartseva1, Igor A. Chernov2, Evgeniya V. Shtemplevskaya1, David A. Areshidze1, INTRODUCTION The liver is the main organ of metabolism of various exogenous and endogenous chemical compounds, while the main functionally active liver cells (hepatocytes) are among the first to be exposed to these factors. Dam- age and death of these cells renders to the disability of the liver to perform its functions (Aizava et al., 2020). One of the mechanisms for maintaining the structural and functional integrity of the liver is cellular regeneration, which occurs due to mitotic and amitotic division of hepatocytes (Gilgenkrantz et al., 2018; Clemens et al., 2019). Mass death of hepatocytes by necrosis and/or apoptosis activates the pro- cesses that trigger the entry of “resting” hepatocytes into the cell-division cycle to restore the original cell mass and maintain the cellular homeostasis Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 45-51, 2021 ISSN 0008-7114 (print) \| ISSN 2165-5391 (online) \| DOI: 10.36253/caryologia-1122 Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 45-51, 2021 ISSN 0008-7114 (print) \| ISSN 2165-5391 (online) \| DOI: 10.36253/caryologia-1122 46 Yuri A. Kirillov et al. Yuri A. Kirillov et al. of the organ. The liver always has a reserve of hepato- cytes with polyploid nuclei, constantly ready to divide. Thus, an increase in the level of ploidy of hepatocytes is one of the main compensatory-adaptive reactions of the liver, ensuring the preservation of function of the organ. It is known that in mammals in the prenatal and at the early stages of postnatal ontogenesis, diploid hepato- cytes prevail, then their polyploidization occurs, and the proportion of polyploid hepatocytes can reach 80% of the total number of cells. In addition, it was found that ploidy of hepatocytes increases with aging, after hepa- tectomy, under the influence of a number of unfavorable factors, but at the same time ploidy decreases, for exam- ple, in hepatocellular carcinomas (Duncan, 2013; Zhang et al., 2019; Donne et al., 2020). Exposure to endogenous or exogenous desynchro- nizing factors leads to disorganization of circadian rhythmicity (Roennenberg et al., 2017). In the case of prolonged or regular exposure to desynchronizing fac- tors, for example, constant lighting at night, desyn- chronosis develops, which is a pathological condition characterized by a mismatch of rhythms in phase, the loss of their mutual synchronization or their destruction (Beauvalet et al., 2017; Walker et al., 2020). INTRODUCTION One of the organs, the normal rhythm of the func- tioning of which is very important for maintaining homeostasis, is the liver (Trefts et al., 2017). In the regu- lation and realization of plastic and energy metabolism, the coordination of rhythmic processes in the liver with the rhythms of processes in other organs and systems of the organism plays a fundamental role. Moreover, most of these processes demonstrate the daily rhythm (Tahara et al., 2016). Another, very important and informative approach to determining the functional state of the liver, as well as diagnosing various kinds of diseases of this organ is karyometry. Karyometric analysis is used to assess the intensity of dystrophic, inflammatory, reparative pro- cesses in chronic viral hepatitis, liver fibrosis, hepatocel- lular carcinoma, etc (El-Sokkary et al., 2005;Esperandim et al., 2010; Makovsky P et al., 2018).h In most cases, the rhythm of metabolic processes arises and is maintained due to dynamic interactions between the molecular clock of the organism and exter- nal zeitgebers, such as, for example, light (main CR synchronizer) and nutrition (secondary synchronizer) (Stubblefield et al., 2016; Ding et al., 2018).h Thus, the approaches of the study of the liver in dif- ferent morphofunctional states can be associated with the karyometric and ploidometric assessment of hepato- cytes. The data obtained using these methods will make it possible to assess the morphofunctional state of the liver more accurately and, in accordance with this, to solve the problems of prognosis. i The disruption of circadian rhythmicity in the form of a shift in biorhythms or desynchronosis in the liver entails the development of pathological conditions and diseases such as cholestasis, fatty hepatosis, impaired biotransformation of toxic and medicinal substances, hepatitis, cirrhosis and liver tumors (Tong et al., 2013; DeBruyne et al., 2014). An indicator of functional chang- es in hepatocytes is the modification of their morphologi- cal structure, which has a wide range of variations, from subtle ultrastructural transformations to cell death. (Li et al., 2020). The linear dimensions of hepatocytes and their nuclei, nuclear-cytoplasmic ratio, and ploidy of hepato- cytes are the significant parameters for assessing the morphofunctional state of the liver (Junatas et al., 2016). Rhythmicity of functioning is peculiar for living systems at every level of organization. Biosystems have rhythms with different periodicity, however, diurnal, or circadian rhythms (CR) are the most significant for mammals (Gillette, 2013; McKenna et al., 2018). INTRODUCTION Circadian system of mammals includes central circadian rhythm generators (suprachiasmatic nuclei of the hypothalamus (SCN), pineal gland), which are connected with peripheral pacemakers – morphologi- cal structures in organs and tissues. It is endogenous and is determined genetically (genes Per1, Per2, Cry, etc.), however, it has significant plasticity and can be modulated by the action of external zeitgebers (time givers), the most important of which is light (Tahara et al., 2017). The significant reason of desorganization of bio- rhythms in the modern world is the disturbance of natural light regime, known as light pollution. Due to a number of social reasons (prolonged interaction with digital technique, overtime and shift work, transmeridian flights, etc.), a person is currently exposed to abundant exposure to artificial lighting in the dark, which leads to a shift in the circadian rhythms of the organism, or to the development of desynchronosis (Lunn et al., 2017).l Separate biorhythms of physiological processes in various systems form a strongly coordinated ensemble, the chronostructure of the organism. The presence of a rhythmic structure of biological processes ensures the necessary order of their course, coherence, maintenance of the functioning of systems of organism at an optimal level (Roennenberg et al., 2016). Another factor that influences CR is alcohol con- sumption. In a study of the effect of alcohol on rhythms in mammals, two areas of interest are distinguished. The first one considers the chrono-effecter action of alcohol, i.e. how the effects of alcohol change depending on the Influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats Influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats 47 liminary experiment was carried out for 3 days in indi- vidual cages with free access to both liquids.t time of day in which it was consumed. The second area of interest is chronergic, with wider approach, explor- ing mainly the effect of alcohol on biorhythms of other parameters of organism (Wasielewski et al., 2001). Alco- hol abuse and alcoholic disease are associated with wide- spread disturbances in CR (Rosenwasser, 2015; Davis et al., 2018). It is shown that disturbances in circadian homeostasis make liver and intestines more suscepti- ble to alcohol toxicity. Studies in human alcoholics have shown altered expression of circadian genes. Animals g Microscopy of histological preparations was per- formed using a Nikon Eclipse 80I digital microscope with use of a Nikon DI-FI digital camera (Japan). For micros- copy, eyepieces ×10, ×15, lenses ×4, ×10, ×20, ×40, ×100 were used. From each studied preparation, 10 digital images of randomly selected visual fields were taken at a magnification of ×400, ×1000, with the use of which kary- ometry were subsequently carried out, the daily dynam- ics of the nucleus was determined, estimated by their area. In morphometric studies, the ImageJ program (USA) with the appropriate plug-ins was used to determine the cross- sectional area of hepatocyte nuclei (Broeke et al., 2015). The measurements were carried out in micrometers after preliminary geometric calibration on an object-microme- ter scale digitized with the same magnification. fi The study was conducted on 160 male Wistar rats at age of 6 months, weighing 300±20 g. Animals were tak- en from the Stolbovaya nursery (the “Stolbovaya” affili- ate of the FSBIS “Scientific Center for Biomedical Tech- nologies of the Federal Medical and Biological Agency). INTRODUCTION Anyway, alcohol has a significant chronotoxic effect, which causes desynchronosis (Huang et al., 2010; Filiano et al., 2013; Martínez-Salvador J. et al., 2018.) Euthanasia was carried out three weeks after the start of the experiment in a carbon dioxide chamber equipped with a device for the upper gas supply (100% CO2) at 9.00, 15.00, 21.00 and 3.00. The chamber volume was filled with gas at a rate of 20% per minute to avoid dyspnea and pain in animals. After sacrifice, the liver was removed for morphological examination. All animal experiments were performed in according to the compli- ance with EC Directive 86/609/EEC and with the Rus- sian law regulating experiments on animals.hif The liver was fixed in 10% neutral buffered forma- lin with further passage through alcohols of increasing concentration (50°, 60°, 70°, 80°, and 96°) and xylol, fol- lowed by pouring into Histomix histological medium (BioVitrum, Russia). When conducting studies of organs embedded in paraffin, serial sections with a thickness of 5-6 μm were prepared. Histological sections were made on the rotor microtome MPS-2 (USSR). Hematoxylin- eosin staining was carried out according to the stand- ard technique. Stained sections were put in a BioMount mounting medium (BioVitrum, Russia). We considered it important to study the daily dynamics of the area of hepatocyte nuclei and their ploidy under normal light conditions and under constant illumination, as well as in combination of these condi- tions with experimental chronic alcohol intoxication. Design of experiment Rats were divided into 4 equal groups. The experi- ment lasted 3 weeks for every group.i 1st group (control): animals of the first group served as control. The individuals were housed in plastic cages with free access to water under the conditions of a fixed light regime “light-dark” (10:14 hours). i For ploidometry, paraffin sections were stained with methylene-green - pyronin G, with following process- ing of sections with RNA-ase. The hepatocyte ploidy was calculated in units of ploidy relative to the optical den- sity of the staining results of diploid nuclei of small lym- phocytes. 2nd group: animals of the second group were kept under the same conditions, but instead of water, a 15% ethanol ad libitum solution was offered daily as a drink. f 3rd group: animals of the third group were kept under the same conditions as the animals of the first group, except for the light regime, which represented constant lighting (“light-light”). Micromorphometry of only mononuclear interphase hepatocytes without signs of pathological changes was carried out. 4th group: animals of the fourth group were kept under conditions of constant lighting and got 15% etha- nol ad libitum solution as a drink instead of water.h The criterion for the selection of rats in the 2 and 4 group, along with the absence of visible deviations in the state and behavior, was the initial preference for a 15% solution of ethyl alcohol to a tap water. For this, a pre- Methods of statistical processing The obtained data were analyzed using the Graph- Pad Prism 6.0 program by calculating average values, 48 Yuri A. Kirillov et al. Considering the results of measuring the ploidy of mononuclear hepatocytes, it was found that the stud- ied samples contain diploid, tetraploid and octoploid cells. The average ploidy of the studied hepatocytes is 4.47±2.12n in the first group, 5.02±2.18n in the second, 4.04±2.16n in the third, and 5.18±2.14n in the fourth. Analysis of ploidy distribution of hepatocyte nuclei revealed significant intergroup differences (Table 1). 30 35 40 45 50 55 I group II group III group IV group 9 hours 15 hours 21 hours 3 hours Figure 1. Daily rhythm of the cross-sectional area of hepatocyte nuclei of rats. Figure 2. Results of cosinor analysis of circadian rhythmicity of area of nuclei of hepatocytes of rats. 30 35 40 45 50 55 I group II group III group IV group 9 hours 15 hours 21 hours 3 hours Figure 1. Daily rhythm of the cross-sectional area of hepatocyte nuclei of rats. standard deviation, and arithmetic mean error. The numerical rows characterizing the diurnal fluctuations of the studied physiological rhythms of animals were subjected to mathematical processing, on the basis of which group chronograms were drawn. We studied the form of chronograms and calculated daily average val- ues. Statistical differences in studied parameters were determined using t-student test. A p value <0.05 was considered statistically significant. i For the statistical estimation of the amplitude and acrophase of CRs, cosinor analysis was performed, which is an international, recognized method for the unified study of biological rhythms using the CosinorEl- lipse2006-1.1 program. The presence of a reliable circa- dian rhythm was determined, as well as its acrophase and amplitude. Acrophase is a measure of the peak time of the total rhythmic variability over a 24-hour period. The amplitude corresponds to half the total rhythmic variability in the cycle. Acrophase is expressed in hours; amplitude values are expressed in the same units as the studied variables (Cornelissen, 2014). Figure 1. Daily rhythm of the cross-sectional area of hepatocyte nuclei of rats. Figure 1. Daily rhythm of the cross-sectional area of hepatocyte nuclei of rats. Figure 2. Results of cosinor analysis of circadian rhythmicity of area of nuclei of hepatocytes of rats. RESULTS Considering the results of karyometry, we found that the cross-sectional area of hepatocyte nuclei of rats of the first three groups, which amounted to 41.79±8.13 μm2, 42.65±4.80 μm2, and 42.72±5.63 μm2, respectively, did not differ significantly from each other, but the sig- nificant decrease in this parameter up to 35.50±3.01 μm2 in hepatocytes of animals of the fourth group was found.h The daily rhythm of the cross-sectional area of the hepatocyte nuclei of rats of 2-4 groups significantly dif- fered from the control (Fig. 1). In particular, the maxi- mum of area of nuclei in control group is noted at 15:00 with acute decrease to minimum at evening and night- time – 21:00 and 3:00. In the second group the rhythm is less pronounced, a maximum at 15:00 is noted. In the third group, the maximum values are noted at 9:00 with a gradual decrease to a minimum at 3:00. In the fourth group, daily fluctuations in the area of hepatocyte nuclei are unreliable. Figure 2. Results of cosinor analysis of circadian rhythmicity of area of nuclei of hepatocytes of rats. Considering the results of measuring the ploidy of mononuclear hepatocytes, it was found that the stud- ied samples contain diploid, tetraploid and octoploid cells. The average ploidy of the studied hepatocytes is 4.47±2.12n in the first group, 5.02±2.18n in the second, 4.04±2.16n in the third, and 5.18±2.14n in the fourth. The results of the cosinor analysis of diurnal chang- es in the area of the nucleus indicate the presence of a reliable CR of this process in the first three groups and its destruction in the fourth group. Therewith, acro- phases of rhythms in groups 1 and 3 are noted in the daytime - at 1221 and 1136, with an amplitude of 10.03 μm2 and 4.60 μm2, respectively, and the acrophase of the rhythm in the second group shifts by 1802 with an amplitude of 3.37 μm2 (Fig. 2). Analysis of ploidy distribution of hepatocyte nuclei revealed significant intergroup differences (Table 1). In particular, in groups in which animals were exposed to chronic alcohol intoxication, the number of diploid hepatocytes significantly decreases, but at the same time, the proportion of octoploid cells in the sec- ond group significantly increases, as well as and the pro- portion of tetraploid cells in the fourth group. RESULTS Influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats 49 Figure 4. Liver of rat of IV group, methylene-green - pyronin G, ×400. Figure 3. Liver of rat of control group, methylene-green - pyronin G, ×400. Figure 3. Liver of rat of control group, methylene-green - pyronin G, ×400. Figure 4. Liver of rat of IV group, methylene-green - pyronin G, ×400. Table 1. Distribution of hepatocyte nuclei in rat liver depending on ploidy. Group Ploidy of nuclei of hepatocytes 2n, % 4n, % 8n, % 1st group (n=40) 23.98±3.51 52.1±4.60 23.23±2.20 2nd group (n=40) 14.15±2.02 * 53.47±5.18 32.38±3.21* 3rd group (n=40) 34.0±4.81 * 45.9±3.95 * 20.1±1.89* 4th group (n=40) 13.70±2.84 * 79.6±5.18 * 6.70±0.81* Note: hereinafter: (P≤0.05); (P≤0.005); **(P≤0.0005) - statistical significance of differences in comparison with the control group. Table 1. Distribution of hepatocyte nuclei in rat liver depending on ploidy. Table 1. Distribution of hepatocyte nuclei in rat liver depending on ploidy. REFERENCES Huang MC, Ho CW, Chen CH, Liu SC, Chen CC, Leu SJ. 2010. Reduced expression of circadian clock genes in male alcoholic patients. Alcohol Clin Exp Res. 34(11):1899-904. Aizawa S, Brar G, Tsukamoto H. 2020. Cell Death and Liver Disease. Gut Liver. 14(1):20-29. Beauvalet, J. C., Quiles, C. L., de Oliveira, M. A. B., Ilgen- fritz, C. A. V., Hidalgo, M. P. L., & Tonon, A. C. 2017. Social jetlag in health and behavioral research: a sys- tematic review. ChronoPhysiology and Therapy, 7, 19-31. Junatas KL, Tonar Z, Kubíková T, Liška V, Pálek R, Mik P, Králíčková M, Witter K. 2017. Stereological analysis of size and density of hepatocytes in the porcine liver. J Anat. 230(4):575-588. Lazzeri E, Angelotti ML, Conte C, Anders HJ, Romagna- ni P. 2019. Surviving Acute Organ Failure: Cell Poly- ploidization and Progenitor Proliferation. Trends Mol Med. 25(5):366-381. Broeke J., Pérez J. M. M., Pascau J. 2015. Image process- ing with ImageJ. – Packt Publishing Ltd., 259 p. Clemens MM, McGill MR, Apte U. 2019. Mechanisms and biomarkers of liver regeneration after drug- induced liver injury. Adv Pharmacol. 85:241-262. h Li W, Li L, Hui L. 2020. Cell Plasticity in Liver Regenera- tion. Trends Cell Biol. 30(4):329-338. j y Cornelissen G. 2014. Cosinor-based rhythmometry. The- or Biol Med Model. 11:16. Lunn RM, Blask DE, Coogan AN, Figueiro MG, Gorman MR, Hall JE, Hansen J, Nelson RJ, Panda S, Smolen- sky MH, Stevens RG, Turek FW, Vermeulen R, Car- reón T, Caruso CC, Lawson CC, Thayer KA, Twery MJ, Ewens AD, Garner SC, Schwingl PJ, Boyd WA. 2017. Health consequences of electric lighting prac- tices in the modern world: A report on the Nation- al Toxicology Program’s workshop on shift work at night, artificial light at night, and circadian disrup- tion. Sci Total Environ. 607-608:1073-1084. Davis BT 4th, Voigt RM, Shaikh M, Forsyth CB, Kes- havarzian A. 2018. Circadian Mechanisms in Alcohol Use Disorder and Tissue Injury. Alcohol Clin Exp Res. 42(4):668-677.h DeBruyne JP, Weaver DR, Dallmann R. 2014. The hepatic circadian clock modulates xenobiotic metabolism in mice. J Biol Rhythms. 29(4):277-87. Ding G, Gong Y, Eckel-Mahan KL, Sun Z. 2018. Central Circadian Clock Regulates Energy Metabolism. Adv Exp Med Biol. 1090:79-103. Makovicky P, Tumova E, Volek Z, Arnone JM, Samasca G, Makovicky P. 2018. The relationship between hepatocytes and small bowel after early and short food restriction: What the results show in morphom- etry. COMPLIANCE WITH ETHICS GUIDELINES All the experimental protocols were performed in accordance to ethical guidelines approved by the Research and Ethics Committee of Scientific Center for Biology of Cells and Applied Biotechnology of the Mos- cow State Regional University, Moscow, Russian Federa- tion prior executing the experiments. Experiments were performed as per “Directive 2010/63/EU of the European Parliament for animal use for scientific purpose” and “NIH Guidelines for the Care and Use of Laboratory Animals”. Filiano AN, Millender-Swain T, Johnson R Jr, Young ME, Gamble KL, Bailey SM. 2013. Chronic ethanol con- sumption disrupts the core molecular clock and diur- nal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus. PLoS One. 8(8):e71684. Gilgenkrantz H, Collin de l’Hortet A. 2018. Understand- ing Liver Regeneration: From Mechanisms to Regen- erative Medicine. Am J Pathol. 188(6):1316-1327. Gillette MU. 2013. Introduction to biological timing in health and disease. Prog Mol Biol Transl Sci. 119:XI- XIV. ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS Financial support for this study was carried out by Research Institute of Human morphology. Esperandim VR, da Silva Ferreira D, Saraiva J, Silva ML, Costa ES, Pereira AC, Bastos JK, de Albuquerque S. 2010. Reduction of parasitism tissue by treatment of mice chronically infected with Trypanosoma cruzi with lignano lactones. Parasitol Res. 107(3):525-30. DISCUSSION AND CONCLUSION occurs due to tetra- and octaploid nuclei, which, accord- ing to a number of authors, indicates the development of hepatocyte hypertrophy against the background of an increase in nuclear ploidy (Miyaoka et al., 2012; Zhou et al., 2016). It has been suggested that the polyploid state functions as a growth suppressor, limiting the prolifera- tion of most of cells and causing compensatory-adaptive reactions in the form of diploid cell hypertrophy. The conducted study allowed us to establish that both the violation of the light regime and the effect of ethanol, individually and jointly, have a significant effect on the studied parameters.hi The alcoholic intoxication at fixed light regime causes the decrease in proportion of diploid cells with a simulta- neous increase in the proportion of octaploid cells. In turn, the nature of the circadian rhythm of the size of the cell nuclei indicates that constant illumina- tion and ethanol, acting separately, cam use a rear- rangement of the CR, but the combined action of these parameters leads to the destruction of the circadian rhythm, which indicates a disruption of adaptation pro- cesses in animals of this group (Maruani et al., 2018; Matkarimov, 2020)i Changing of the normal light regime to constant light leads to a change in the nature of the ploidy distri- bution of hepatocytes. The increase in proportion of dip- loid hepatocytes indicates a successful course of adapta- tion processes in the organ, apparently due to the divi- sion of cells of higher ploidy, the proportion of which has decreased (Nagy et al., 2001; Yelchaninov et al., 2011; Lazzeri et al., 2019).h So, the conducted study testifies that the results of caryometric and ploidometric studies characterize the degree of influence of alcohol intoxication and changes in the light regime on the liver of rats, representing the degree of effectiveness of adaptation to these factors. The alcoholic intoxication in conditions of constant lighting lead to decrease in size of nuclei and increase in proportion of tetraploid hepatocytes.h The increase in general ploidy in groups 2 and 4 (i.e. in those where animals were exposed to alcohol) 50 Yuri A. Kirillov et al. Yuri A. Kirillov et al. El-Sokkary GH, Abdel-Rahman GH, Kamel ES. 2005. Melatonin protects against lead-induced hepatic and renal toxicity in male rats. Toxicology. 213(1-2):25-33. REFERENCES Bratisl Lek Listy. 119(3):156-159. Donne R, Saroul-Aïnama M, Cordier P, Celton-Morizur S, Desdouets C. 2020. Polyploidy in liver develop- ment, homeostasis and disease. Nat Rev Gastroenter- ol Hepatol. 17(7):391-405. Matkarimov R. 2020. Questions of temporary adaptation of weightlifters to different climatic and geographical conditions. Euras. J of Sport Sc. 1(1):18-22. p Duncan AW. 2013. Aneuploidy, polyploidy and ploidy reversal in the liver. Semin Cell Dev Biol. 24(4):347-56. Influence of chronic alcoholic intoxication and lighting regime on karyometric and ploidometric parameters of hepatocytes of rats 51 Zhang S, Lin YH, Tarlow B, Zhu H. 2019. The origins and functions of hepatic polyploidy. Cell Cycle. 18(12):1302-1315. Martínez-Salvador J, Ruiz-Torner A, Blasco-Serra A, Martínez-Soriano F, Valverde-Navarro AA. 2018. Morphologic variations in the pineal gland of the albino rat after a chronic alcoholisation process. Tis- sue Cell. 51:24-31. h Zhou D, Wang Y, Chen L, Jia L, Yuan J, Sun M, Zhang W, Wang P, Zuo J, Xu Z, Luan J. 2016. Evolving roles of circadian rhythms in liver homeostasis and pathol- ogy. Oncotarget. 7(8):8625-39. Maruani J. 2018. The neurobiology of adaptation to sea- sons: relevance and correlations in bipolar disorders. Chronobiol. Int. 35(10):1335-1353. McKenna H, van der Horst GTJ, Reiss I, Martin D. 2018. Clinical chronobiology: a timely consideration in critical care medicine. Crit Care. 22(1):124. Miyaoka Y, Ebato K, Kato H, Arakawa S, Shimizu S, Miyajima A. 2012. Hypertrophy and unconventional cell division of hepatocytes underlie liver regenera- tion. Curr Biol. 22(13):1166-75. Nagy P, Teramoto T, Factor VM, Sanchez A, Schnur J, Paku S, Thorgeirsson SS. 2001. Reconstitution of liver mass via cellular hypertrophy in the rat. Hepatology. 33(2):339-45. Roenneberg T, Merrow M. 2016. The Circadian Clock and Human Health. Curr Biol. 26(10):R432-43. Roenneberg T, Pilz LK, Zerbini G, Winnebeck EC. 2019. Chronotype and Social Jetlag: A (Self-) Critical Review. Biology (Basel). 8(3):54. Rosenwasser AM. 2015. Chronobiology of ethanol: ani- mal models. Alcohol. 49(4):311-9. i Stubblefield, J.J., Green, C.B. 2016. Mammalian Circadian Clocks and Metabolism: Navigating Nutritional Chal- lenges in a Rhythmic World. In Circadian Clocks: Role in Health and Disease (pp. 153-174). Springer, New York, NY.h Tahara Y, Aoyama S, Shibata S. 2017. The mammalian circadian clock and its entrainment by stress and exercise. J Physiol Sci. 67(1):1-10. Tahara Y, Shibata S. 2016. Circadian rhythms of liver physiology and disease: experimental and clinical evidence. Nat Rev Gastroenterol Hepatol. 13(4):217- 26. Tong X, Yin L. 2013. REFERENCES Circadian rhythms in liver physiol- ogy and liver diseases. Compr Physiol. 3(2):917-40. th Trefts E, Gannon M, Wasserman DH. 2017. The liver. Curr Biol. 27(21):R1147-R1151. Walker WH 2nd, Walton JC, DeVries AC, Nelson RJ. 2020. Circadian rhythm disruption and mental health. Transl Psychiatry. 10(1):28. Wasielewski JA, Holloway FA. 2001. Alcohol’s interac- tions with circadian rhythms. A focus on body tem- perature. Alcohol Res Health. 25(2):94-100. Yelchaninov AV, Bolshakova GB. 2011. Size of hepato- cytes and their nuclei in the regenerating fetal liver of rats // Russian medical and biological bulletin named after academician IP Pavlov. 2(2):128-180.
https://openalex.org/W4375932860	https://eprints.gla.ac.uk/295171/2/295171.pdf	English	null	An ultrathin and flexible terahertz electromagnetically induced transparency-like metasurface based on asymmetric resonators	EPJ. Applied metamaterials	2,023	cc-by	3,973	Received: 18 November 2022 / Accepted: 24 March 2023 Received: 18 November 2022 / Accepted: 24 March 2023 Abstract. Terahertz (THz) electromagnetically induced transparency-like (EIT-like) metasurfaces have been extensively explored and frequently used for sensing, switching, slow light, and enhanced nonlinear effects. Reducing radiation and non-radiation losses in EIT-like systems contributes to increased electromagnetic (EM) ﬁeld conﬁnement, higher transmission peak magnitude, and Q-factor. This can be accomplished by the use of proper dielectric properties and engineering novel designs. Therefore, we fabricated a THz EIT-like metasurface based on asymmetric metallic resonators on an ultra-thin and ﬂexible dielectric substrate. Because the quadruple mode is stimulated in a closed loop, an anti-parallel surface current forms, producing a transparency window with a transmission peak magnitude of 0.8 at 1.96 THz. To control the growing trend of EIT-like resonance, the structure was designed with four asymmetry levels. The effect of the substrate on the resonance response was also explored, and we demonstrated experimentally how the ultra-thin substrate and the metasurface asymmetric novel pattern contributed to higher transmission and lower loss. Keywords: Terahertz / metasurface / EIT-like resonance Keywords: Terahertz / metasurface / EIT-like resonance EPJ Appl. Metamat. 10, 4 (2023) © S. Nourinovin et al., published by EDP Sciences, 2023 https://doi.org/10.1051/epjam/2023001 Available online at: j d Available online at: epjam.edp-open.org RESEARCH ARTICLE * e-mail: s.nourinovin@qmul.ac.uk This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. EPJ Appl. Metamat. 10, 4 (2023) ibuted under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), ricted use, distribution, and reproduction in any medium, provided the original work is properly cited. EPJ Appl. Metamat. 10, 4 (2023) © S. Nourinovin et al., published by EDP Sciences, 2023 https://doi.org/10.1051/epjam/2023001 An ultrathin and ﬂexible terahertz electromagnetically induced transparency-like metasurface based on asymmetric resonators Shohreh Nourinovin1,*, Sae June Park1, Qammer H. Abbasi2,3, and Akram Alomainy1 1 School of Electronic Engineering and Computer Science, Queen Mary University of London, London E1 4FZ, UK 2 James Watt School of Engineering, University of Glasgow, Glasgow, G12 8QQ, UK 3 School of Electronic Engineering and Computer Science, Queen Mary University of London, London E1 4FZ, UK Received: 18 November 2022 / Accepted: 24 March 2023 1 Introduction The combination of Fano and EIT-like structures with nonlinear and phase-changing media opens up new possibilities for switching and electro- optics applications as well [7,21]. used to simulate and optimise the metasurface resonance response. Along the x, y, and z axes, the electric, magnetic, and open (add space) boundary conditions are used. Several processing procedures were used to create the metasurface sensor. A polyimide layer was spin-coated and baked on a silicon wafer and then a bilayer of photoresist was spin-coated onto the polyimide. Metasurface was patterned and built using maskless laser lithography and conventional developing process, respectively. A Ti and Au adhesion layer was deposited using a sputter coater. Finally, the wafer was submerged in hydroﬂuoric acid to remove the SiO2 layer from the polyimide. Figure 2 depicts the fabricated metasurface with 6.3 6.3 mm in size and an array of 90 90 unit cells. For the measurements, THz-TDS in transmission mode was used to analyze the transmission spectra. It has a coherent synchronized source and detector that provides instant amplitude measurement of the THz waves. The measurments performed in a setup using four F/2 parabolic mirrors in a conventional optical arrangement. To eliminate absorption from atmospheric water vapour, the THz beam path was purged with dry air. Samples were placed at the focal plane of the beam, normal to the beam axis, with a beam diameter of 3 mm. The frequency resolution was 10 GHz. The operating frequency is kept below 3 THz which is in adaption with THz-TDS and also for avoidance of the higher noise and water absorption in upper frequency ranges. [ ] In order to develop THz Fano and EIT-like meta- surfaces that properly meet the requirement in mentioned applications, we need to reduce the radiation and non- radiation losses in the system. This can be achieved by correct dielectric characteristics and engineering novel design to increase the EM ﬁeld conﬁnement and obtain a higher transmission window. Thus, we created a ﬂexible EIT-like metasurface based on asymmetric SRRs on an ultra-thin polyimide. Because the quadruple mode is stimulated in a closed loop, an anti-parallel surface current develops, and by reducing radiation losses, a transparency window with a transmission peak magnitude of 0.8 develops at 1.96 THz. 1 Introduction 10, 4 (2023) 2 Fig. 1. Schematic representation of the designed metasurface’s unit cells (a) top view of the unit cell with the circle center of O and the following parameters: l1 = 39 mm, l2 = 36 mm, l3 = 25.45 mm, l4 = 29.45 mm, g = 4 mm, n = 8 mm, p = 14 mm, d = 20 mm, w = 44 mm, (b) side view of the unit cell with following parameters: a = b = 70 mm, c = 0.2 mm, e = 5 mm. Fig. 2. Display of the metasurface’s ﬂexibility and image under an optical microscope. Fig. 1. Schematic representation of the designed metasurface’s unit cells (a) top view of the unit cell with the circle center of O and the following parameters: l1 = 39 mm, l2 = 36 mm, l3 = 25.45 mm, l4 = 29.45 mm, g = 4 mm, n = 8 mm, p = 14 mm, d = 20 mm, w = 44 mm, (b) side view of the unit cell with following parameters: a = b = 70 mm, c = 0.2 mm, e = 5 mm. Fig. 2. Display of the metasurface’s ﬂexibility and image under an optical microscope. Fig. 1. Schematic representation of the designed metasurface’s unit cells (a) top view of the unit cell with the circle center of O and the following parameters: l1 = 39 mm, l2 = 36 mm, l3 = 25.45 mm, l4 = 29.45 mm, g = 4 mm, n = 8 mm, p = 14 mm, d = 20 mm, w = 44 mm, (b) side view of the unit cell with following parameters: a = b = 70 mm, c = 0.2 mm, e = 5 mm. Fig. 2. Display of the metasurface’s ﬂexibility and image under an optical microscope. result, even small perturbations can cause signiﬁcant variations in their resonance spectra. This feature makes Fano and EIT-like resonances extremely appealing for a wide range of applications. They have been widely used for biological sensing where changes in the refractive index of biological samples can be manifested in the resonance frequency shift and transmission magnitude [18,19]. In the same manner, the sensitivity to local displacement or deformation of Fano and EIT-like structures can also be used to detect physical quantities such as displacement, temperature, or pressure by affecting their coupled oscillator properties [20]. 1 Introduction The structure was built with four asymmetry levels to tune the intensifying trend of EIT-like resonance, as well as the analysis of surface current, transmission peak magnitude, and FWHM parameters. A mechanical delay stage provides a time delay between two split laser beams. A pump beam illuminates the low- temperature grown GaAs (LT-GaAs) photoconductive antenna to produce picoseconds THz waves which are then collimated and focused into the metasurface. The trans- mitted THz pulses are detected using electro-optic (EO) sampling technique by measuring THz-ﬁeld induced birefringence of EO crystal with one of the split laser beams called probe beam. Time-domain THz response of the metasurface can be recorded using lock-in detection technique and then analysed in frequency-domain by performing fast Fourier transform (FFT). 2 Simulation and experimental methods The unit cell of the designed metasurface is made of asymmetric gold split ring resonators (SRRs) on a dielectric substrate, displayed in Figure 1. The material of the dielectric layer is polyimide with a thickness of 5 mm, a permittivity of 2.9, and a loss tangent of 0.05 at 1 THz. THz waves are incident and interact with the gold metallic pattern before passing through the dielectric layer. The caption of Figure 1 shows the precise dimensions of the unit cell geometries. The asymmetry value between the coupled resonators, d, will be employed to tune the EIT-like resonance. The CST microwave studio software, which is based on a ﬁnite difference time domain (FDTD) solver, is The unit cell of the designed metasurface is made of asymmetric gold split ring resonators (SRRs) on a dielectric substrate, displayed in Figure 1. The material of the dielectric layer is polyimide with a thickness of 5 mm, a permittivity of 2.9, and a loss tangent of 0.05 at 1 THz. THz waves are incident and interact with the gold metallic pattern before passing through the dielectric layer. The caption of Figure 1 shows the precise dimensions of the unit cell geometries. The asymmetry value between the coupled resonators, d, will be employed to tune the EIT-like resonance. The CST microwave studio software, which is based on a ﬁnite difference time domain (FDTD) solver, is 1 Introduction surface plasmon. It is now possible to build a wide range of spoof surface plasmons [8] under the metamaterial paradigm. Surface plasmons (SPs) are electronic oscillations that occur at the interface of materials with opposing permittivity, notably metals and dielectrics. At optical and infrared frequencies, EM waves can link to surface plasmons to form a propagating wave at metal-dielectric contacts, known as surface plasmon polariton (SPP). SPP waves can be excited in a variety of ways, including prism- coupled, grating-coupled, waveguide-coupled, and optical ﬁber [1]. Surface plasmon resonance (SPR) is the phenomenon of EM wave coupling to an SPP wave. Regardless of technique, SPRs amplify and conﬁne EM ﬁeld and have been widely used in photonic devices for a variety of purposes, most notably biosensing [2–7]. Many metasurface applications rely on the creation of high Q-factor metasurfaces. We are primarily concerned with radiation losses in THz resonant-based metasurfaces, which should be decreased to increase the Q-factor. There are higher-order plasmonic modes, such as Fano and EIT- like resonance-based metasurfaces, that can satisfy this criterion. Fano resonances were ﬁrst found in quantum physics to explain asymmetrically structured atom and molecule ionisation [9]. The theory of Fano resonance was recently applied to the ﬁelds of photonics and metamaterials [10]. Exciting Fano and EIT-like resonances by breaking the symmetry of coupled resonators was ﬁrst demonstrated in the microwave regime [10] and later in THz [11,12]. After that many research works studied these structures in THz metasurfaces [13–15]. By using an oblique incidence, they can also be induced in symmetric resonators [16,17]. [ ] Metals are good conductors at THz frequencies (below plasma frequency) and may often be treated as simply perfect conductors; this prevents them from sustaining SPPs. To take advantage of SPPs’ capacity in the THz regime, one feasible option is to use metals with textured surfaces that enable SPP-like modes, known as spoof [ ] Fano and EIT-like resonance-based metasurfaces give a substantial ampliﬁcation of EM ﬁelds in their vicinity, as well as a greater Q-factor. Because they are caused by the interference of oscillators, they are naturally sensitive to changes in geometry or the immediate environment. As a This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. S. Nourinovin et al.: EPJ Appl. Metamat. 3 The transmission spectra of the structure in two cases of symmetric and asymmetric SRRs with asymmetry degree of 24 mm is presented in Figure 3a. While the symmetric S. Nourinovin et al.: EPJ Appl. Metamat. 10, 4 (2023) 3 structure shows a non-EIT-like resonance behaviour, the asymmetry structure with asymmetry degree of 24 mm causes an EIT-like resonance at 1.96 THz with a transmission peak magnitude of 0.8. The concept of forming the EIT-like resonance can be understood based on the characteristics of the atomic system. In the same manner, the photonic structure must consist of two resonance elements: “bright,” which is strongly coupled to the input light and has a short radiative lifespan, and “dark,” which is weakly coupled to the incident wave and has a long lifetime. When the resonance frequency of the bright mode equals the resonance frequency of the dark mode and their Q-factor contrasts signiﬁcantly, the Fano resonance is referred to as EIT-like resonance. In the absence of coupling, the incident wave’s energy is scattered into free space by the bright resonator. When the bright and dark resonators are coupled, the system’s EM response improves dramatically, and the energy received through the bright mode is transmitted to the dark mode at the resonance frequency of 1.96 THz. As a result, absorption and scattering losses are considerably reduced, and a transparency window forms which with increasing asym- metry rises more upward. Fig. 3. (a) Transmission spectra of the designed EIT-like metasurfaces without and with asymmetry degree of 24 mm. (b) Transmission spectra of the metasurface simulated with various dielectric substrate thickness with ﬁxed loss tangent of 0.05. (c) Transmission spectra of the metasurface simulated with various dielectric substrate loss tangent with ﬁxed thickness of 6 mm. Because of the short interaction length (radiation with metal) in THz resonant-based metasurfaces, an ohmic loss is frequently overlooked, and in the case of a dielectric substrate, related losses can be decreased by selecting a low-loss material. Therefore, we investigated the effect of substrate dielectric thickness and loss tangent on the THz spectrum of the designed metasurface. According to Figure 3b, narrower thickness results in better transmis- sion and narrower linewidth, which is consistent with predictions. Furthermore, the effect of the dielectric loss tangent, as shown in Figure 3c, displays a direct relationship with the magnitude of the transmission peak and the full width at half maximum (FWHM). References 1. J. Polo, T. Mackay, A. Lakhtakia, Electromagnetic surface waves: a modern perspective (Elsevier, 2013) 2. S.J. Park, Y.H. Ahn, Detection of polystyrene microplastic particles in water using surface-functionalized terahertz microﬂuidic metamaterials, Appl. Sci. 12, 7102 (2022) 3. S.J. Park, J. Cunningham, Effect of substrate etching on terahertz metamaterial resonances and its liquid sensing applications, Sensors 20, 3133 (2020) Fig. 5. Variation in FWHM and transmission peak magnitudes of designed metasurfaces with four degrees of asymmetry. 4. D. Li, F. Hu, H. Zhang, Z. Chen, G. Huang, F. Tang, S. Lin, Y. Zou, Y. Zhou, Identiﬁcation of early-stage cervical cancer tissue using metamaterial terahertz biosensor with two resonant absorption frequencies, IEEE J. Selected Topics Quantum Electr. 27, 1 (2021) In order to analyze the EIT-like effect, the current distribution of the structure is depicted at the transmission peak of 1.96 THz in Figures 4a–4d. In response to the activation of the quadrupole mode, a magnetic dipole arises between coupled resonators, resulting in anti-parallel surface currents in a closed loop for d = 16 mm. At this frequency, a transparency window forms, and radiation losses are inhibited. The magnetic dipole and surface current becomes stronger when the asymmetry degree increases gradually to 20 and 24 mm and ﬁnally at 28 mm, we observed the strongest surface circulation current. 5. A. Oueslati, A. Hlali, H. Zairi, Modeling of a metamaterial biosensor based on split ring resonators for cancer cells detection, in 2021 18th International Multi-Conference on Systems, Signals & Devices (SSD). (IEEE, 2021), pp. 392–396 y , g ( ) ( , ), pp 6. M. Zhu, L. Zhang, S. Ma, J. Wang, J. Su, A. Liu, Terahertz metamaterial designs for capturing and detecting circulating tumor cells, Mater. Res. Express 6, 045805 (2019) 7. L. Liu, T. Li, Z. Liu, F. Fan, H. Yuan, Z. Zhang, S. Chang, X. Zhang, Terahertz polarization sensing based on metasurface microsensor display anti-proliferation of tumor cells with aspirin, Biomed. Optics Express 11, 2416 (2020) We have calculated the transmission peak magnitude and FWHM of the transmission spectra of the four structures and the results are shown in Figure 5. It shows that with increasing the asymmetry degree, the transmis- sion peak rises and the linewidth gets wider. 3 As a result, selecting a dielectric substrate with the correct characteristics aids in providing a stronger transmission window peak. Then, for the ﬁnal fabrication, we used an ultrathin 6 mm polyimide with permittivity as low as 2.9 and a loss tangent of 0.05. This ultra-thin substrate provides mechanical support as well as lower loss. Another advantage of using this substrate is its ﬂexibility, which eliminates several barriers in practical applications [22]. [ ] When the asymmetry degree d is applied, the coupling between the bright and dark resonators occurs, breaking the resonance equilibrium in the nearby arms. Figures 4a– 4d depict the EM simulation and experimental transmis- sion spectra of the structures with four asymmetry values of 16, 20, 24, and 28 mm. Except for a minor discrepancy due to fabrication error, the simulations and observations of both the transmission magnitude and phase are in good agreement. Due to the coupling of the bright and dark modes, the peak of the EIT-like resonance occurs at 1.96 THz. As can be seen in Figures 4a–4d, as the asymmetry degree d grows from 16 to 28 mm, the coupling strength between the resonators steadily increases and the peak transmission intensiﬁes, resulting in a quadruple EIT-like mode with narrower linewidth and stronger transmission window [10]. Fig. 3. (a) Transmission spectra of the designed EIT-like metasurfaces without and with asymmetry degree of 24 mm. (b) Transmission spectra of the metasurface simulated with various dielectric substrate thickness with ﬁxed loss tangent of 0.05. (c) Transmission spectra of the metasurface simulated with various dielectric substrate loss tangent with ﬁxed thickness of 6 mm. S. Nourinovin et al.: EPJ Appl. Metamat. 10, 4 (2023) 4 The magnitude and phase of transmission spectra for the designed THz metasurface as measured (black circle) an ne) for (a) d = 16 mm, (b) d = 20 mm, (c) d = 24 mm and (d) d = 28 mm along with their corresponding physical nd surface current distribution represented in (e–h). Fig. 4. The magnitude and phase of transmission spectra for the designed THz metasurface as measured (black circle) and simulated (brown line) for (a) d = 16 mm, (b) d = 20 mm, (c) d = 24 mm and (d) d = 28 mm along with their corresponding physical asymmetry degree and surface current distribution represented in (e–h). S. Nourinovin et al.: EPJ Appl. Metamat. 10, 4 (2023) 5 Fig. 3 5. Variation in FWHM and transmission peak magnitudes of designed metasurfaces with four degrees of asymmetry. 0.8. The novel design and ultra-thin dielectric helped to reduce radiation losses, resulting in a higher transparent window peak magnitude with a narrower line width. This research was supported by the School of EECS at the Queen Mary University of London. References Then, although the design with an asymmetry value of 28 mm has a higher transmission peak magnitude than 24 mm, it suffers from a lower FWHM, and with considering a tradeoff, the metasurface with an asymmetry degree of 24 mm ﬁnalized. In comparison with the literature, the measured transmission peak magnitude is higher and FWHM is narrower [23], [24]. Reference [25] uses the etching technique combined with Fano resonance in a THz metasurface to suppress more losses and increasing the performance in liquid sensing application in which measured maximum 0.5 in transmission peak magnitude with wider line width for the bare structure. Another THz EIT-like metasurface introduced by [26] for polymer sensing reported transmission peak magnitude of less than 0.6 with wider line width. 8. J. Pendry, L. Martin-Moreno, F. Garcia-Vidal, Mimicking surface plasmons with structured surfaces, Science 305, 847 (2004) 9. U. Fano, Effects of conﬁguration interaction on intensities and phase shifts, Phys. Rev. 124, 1866 (1961) 10. V. Fedotov, M. Rose, S. Prosvirnin, N. Papasimakis, N. Zheludev, Sharp trapped-mode resonances in planar meta- materials with a broken structural symmetry, Phys. Rev. Lett. 99, 147401 (2007) 11. R. Singh, I.A. Al-Naib, M. Koch, W. Zhang, Sharp fano resonances in thz metamaterials, Opt. Express 19, 6312 (2011) 12. N. Liu, L. Langguth, T. Weiss, J. Kästel, M. Fleischhauer, T. Pfau, H. Giessen, Plasmonic analogue of electromagnetically induced transparency at the drude damping limit, Nat. Mater. 8, 758 (2009) 13. L. Zhu, H. Li, L. Dong, W. Zhou, M. Rong, X. Zhang, J. Guo, Dual-band electromagnetically induced transparency (eit) terahertz metamaterial sensor, Opt. Mater. Express 11, 2109 (2021) 14. T. Wang, T. Li, H. Yao, Y. Lu, X. Yan, M. Cao, L. Liang, M. Yang, J. Yao, High-sensitivity modulation of electromagnet- ically induced transparency analog in a thz asymmetric metasurface integrating perovskite and graphene, Photonics Res. 10, 2317 (2022) This research was supported by the School of EECS at the Queen Mary University of London. 4 Conclusion In conclusion, we demonstrated a ﬂexible THz metasurface biosensor made up of asymmetric resonators on an ultra- thin and ﬂexible dielectric polyimide that induces EIT-like resonance. For four asymmetry levels, we reported experimental proof of bright-dark mode coupling at 1.96 THz and transmission peak magnitude higher than 15. S. Wang, S. Wang, X. Zhao, J. Zhu, Q. Li, T. Chen, Excitation of electromagnetically induced transparency effect in asymmetrical planar terahertz toroidal dipole metasurfaces, J. Infrared Millimeter Terahertz Waves 42, 40 (2021) S. Nourinovin et al.: EPJ Appl. Metamat. 10, 4 (2023) 6 operations at terahertz frequencies, Nat. Commun. 9, 4056 (2018) 16. Z. Liao, S. Liu, H.F. Ma, C. Li, B. Jin, T.J. Cui, Electromagnetically induced transparency metamaterial based on spoof localized surface plasmons at terahertz frequencies, Sci. Rep. 6, 1 (2016) 22. H. Yao, H. Mei, W. Zhang, S. Zhong, X. Wang, Theoretical and experimental research on terahertz metamaterial sensor with ﬂexible substrate, IEEE Photon. J. 14, 3700109 (2021) 17. S. Han, L. Cong, H. Lin, B. Xiao, H. Yang, R. Singh, Tunable electromagnetically induced transparency in coupled three- dimensional split-ring-resonator metamaterials, Sci. Rep. 6, 27596 (2016) 23. M. Yang, L. Liang, Z. Zhang, Y. Xin, D. Wei, X. Song, H. Zhang, Y. Lu, M. Wang, M. Zhang et al., Electromag- netically induced transparency-like metamaterials for detection of lung cancer cells, Opt. Express 27, 19520 (2019) 18. S. Nourinovin, M.M. Rahman, M.P. Philpott, A. Alomainy, Terahertz characterisation of artiﬁcially cultured oral cancer with stem cell lines for healthcare applications, in 2022 IEEE International Symposium on Medical Measurements and Applications (MeMeA). (IEEE, 2022), pp. 1–5 24. X. Yan, M. Yang, Z. Zhang, L. Liang, D. Wei, M. Wang, M. Zhang, T. Wang, L. Liu, J. Xie et al., The terahertz electromagnetically induced transparency-like metamateri- als for sensitive biosensors in the detection of cancer cells, Biosens. Bioelectr. 126, 485 (2019) 19. S. Nourinovin, A. Alomainy, A terahertz electromagnetically induced transparency-like metamaterial for biosensing, in 2021 15th European Conference on Antennas and Propaga- tion (EuCAP). (IEEE, 2021), pp. 1–5 25. T. Lin, Y. Huang, S. Zhong, Y. Zhong, Z. Zhang, Q. Zeng, Y. Yu, Z. Peng, Field manipulation of electromagnetically induced transparency analogue in terahertz metamaterials for enhancing liquid sensing, Opt. Lasers Eng. 157, 107127 (2022) 20. B. Luk’yanchuk, N.I. Zheludev, S.A. Maier, N.J. Halas, P. Nordlander, H. Giessen, C.T. Chong, The fano resonance in plasmonic nanostructures and metamaterials, Nat. Mater. Cite this article as: Shohreh Nourinovin, Sae June Park, Qammer H. Abbasi, Akram Alomainy, An ultrathin and ﬂexible terahertz electromagnetically induced transparency-like metasurface based on asymmetric resonators, EPJ Appl. Metamat. 10, 4 (2023) 4 Conclusion 9, 707 (2010) ( ) 26. Y. Hu, X. Zhou, Q. Sun, G. Zeng, Y. Xiong, Sensitive detection of doped polymer thin ﬁlms using terahertz metamaterial based on analog of electromagnetically induced transparency, IEEE Sens. J. 23, 3431 (2023) 21. M. Manjappa, P. Pitchappa, N. Singh, N. Wang, N.I. Zheludev, C. Lee, R. Singh, Reconﬁgurable mems fano metasurfaces with multiple-input-output states for logic
https://openalex.org/W1973689693	http://cds.cern.ch/record/1483137/files/prd.87.e034007.pdf	English	null	Universality of multiparticle production in QCD at high energies	Physical review. D. Particles, fields, gravitation, and cosmology/Physical review. D, Particles, fields, gravitation, and cosmology	2,013	cc-by	14,038	I. INTRODUCTION These adjoint Wilson lines can be subsequently replaced by two fundamental Wilson lines by means of the identity Calculating cross sections for multiparticle (multijet) production processes can be theoretically challenging when a resummation of multiple interactions is needed, as is the case in large parton density environments such as the small-x regime accessible in high-energy nuclear col- lisions. The main complication comes from the fact that partons scatter coherently and cannot be regarded sepa- rately. In particular, it is very important to appropriately consider the color structure of the multiparticle states since color conservation plays an important role as a source of correlations. WabðxÞ ¼ 2 Tr½taUðxÞtbUyðxÞ; (1) (1) WabðxÞ ¼ 2 Tr½taUðxÞtbUyðxÞ; where WðxÞ stands for a Wilson line in the adjoint repre- sentation at a ﬁxed transverse coordinate, UðxÞ is the corresponding Wilson line in the fundamental representa- tion, and ta are color matrices in the fundamental repre- sentation as well. The color matrices can be removed using the Fierz identity ta ijta kl ¼ 1 2 iljk 1 2Nc ijkl. After these manipulations one is left with a sum of products of traces of Wilson lines in the fundamental representation only. The standard way of calculating such multiple- scattering processes is to consider the small-x gluons as an external ﬁeld where high-energy probes scatter in an eikonal way (see Ref. [1]). Under that framework, each parton traversing the ﬁeld contributes to the scattering amplitude with a Wilson line in the appropriate represen- tation (fundamental for quarks, adjoint for gluons) at a ﬁxed transverse coordinate. Provided they are put together in the correct order, the Wilson lines account for the color ﬂow of the process under consideration. After averaging (summing) over initial (ﬁnal) colors at the cross-section level, one is left with a product of traces involving Wilson lines in the fundamental representation and color matrices which are contracted with adjoint Wilson lines. In order to calculate measurable observables in this framework, including cross sections for multiparticle pro- duction, it is necessary to take a weighted average over the possible external ﬁeld conﬁgurations. This averaging pro- cess introduces nontrivial correlations between the ﬁelds entering the different Wilson lines, therefore giving rise to the aforementioned coherent scattering involving possibly all of the partons appearing in the process. Universality of multiparticle production in QCD at high energies , ( ) Fabio Dominguez Institut de Physique The´orique, CEA-Saclay, F-91191 Gif-sur-Yvette, France Cyrille Marquet Physics department, Theory Unit, CERN, CH-1211 Geneva, Switzerland and Departamento de F Universidade de Santiago de Compostela, 15782 Santiago de Compostel Cyrille Marquet Physics department, Theory Unit, CERN, CH-1211 Geneva, Switzerland and Departamento de Fı´sica de Partı´culas and IGFAE, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Anna M. Stasto Department of Physics, Pennsylvania State University, University Park, Pennsylvania 16802, USA; Brookhaven National Laboratory, RIKEN BNL Research Center, Building 510A, Upton, New York 11973, USA, and H. Niewodniczan´ski Institute of Nuclear Physics, Polish Academy of Sciences, Krako´w 31-342, Poland Anna M. Stasto Department of Physics, Pennsylvania State University, University Park, Pennsylvania 16802, USA; Brookhaven National Laboratory, RIKEN BNL Research Center, Building 510A, Upton, New York 11973, USA, and H. Niewodniczan´ski Institute of Nuclear Physics, Polish Academy of Sciences, Krako´w 31-342, Poland Bo-Wen Xiao Institute of Particle Physics, Central China Normal University, Wuhan 430079, China and Department of Physics, Pennsylvania State University, University Park, Pennsylvania 16802, USA (Received 5 October 2012; published 4 February 2013) Bo-Wen Xiao nstitute of Particle Physics, Central China Normal University, Wuhan 430079, China and Department of Physics, Pennsylvania State University, University Park, Pennsylvania 16802, USA (Received 5 October 2012; published 4 February 2013) By studying the color structure of multiparticle production processes in p þ A-type (dilute-dense) collisions, we ﬁnd that higher-point functions beyond typical dipoles and quadrupoles, e.g., sextupoles, octupoles, etc., naturally appear in the cross sections, but are explicitly suppressed in the large-Nc limit. We evaluate the sextupole in the McLerran-Venugopalan model and ﬁnd that, in general, its analytical form cannot be written as combination of dipoles and quadrupoles. Within the color glass condensate framework, we present a proof that in the large-Nc limit, all multiparticle production processes in the collision of a dilute system off a dense system can, up to all orders in s, be described in terms of only dipoles and quadrupoles. PACS numbers: 24.85.+p, 12.38.Cy, 13.85.Hd DOI: 10.1103/PhysRevD.87.034007 DOI: 10.1103/PhysRevD.87.034007 Universality of multiparticle production in QCD at high energies PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) DOMINGUEZ et al. DOMINGUEZ et al. accounted for by perturbative considerations under the color glass condensate framework [2]. accounted for by perturbative considerations under the color glass condensate framework [2]. More complicated correlators naturally arise in the cal- culation of cross sections of processes with more particles in the ﬁnal state, but they are suppressed by powers of Nc as compared with terms with only dipole and quadrupole amplitudes. They are nevertheless independent from the dipole and quadrupole amplitudes and in principle should be evaluated on their own. A procedure to evaluate higher- point correlators—in a Gaussian model in the large-Nc limit—is described in the Appendix, and the explicit example of a correlator of six Wilson lines is evaluated explicitly. The small-x evolution of such correlators was recently studied analytically in Ref. [8] and numerically in Ref. [9]; the evaluation shown in the Appendix provides a suitable initial condition for such equations. Regardless of the model under consideration, for the speciﬁc calculation of such ﬁeld averages the complexity of the calculation of higher-point correlators increases accordingly with the number of Wilson lines involved. In the same way, the evolution equations derived pertur- batively become very cumbersome and not well-suited for numerical evaluations. The ﬁrst step in an attempt to simplify the treatment of such higher-point correlators is to consider the large-Nc limit, in which the average of a product of traces of Wilson lines reduces to a product of averages involving only one trace at a time. In other words, correlations involving ﬁelds coming from Wilson lines from different traces are suppressed by factors of Nc. The main purpose of this paper is to show that, in the large-Nc limit, all multiparticle production processes con- sidered under this framework can be described in terms of only dipoles and quadrupoles. We ﬁrst work out explicitly examples with three particles in the ﬁnal state before proceeding to prove the general statement by induction in the number of particles in the ﬁnal state. The inductive step is greatly simpliﬁed by the observation that it is not neces- sary to consider all the diagrams contributing to a given process; rather, one only needs to ﬁnd at least one diagram with a term given only by dipoles and quadrupoles which dominates in the large-Nc limit. PHYSICAL REVIEW D 87, 034007 (2013) Now, in this large-Nc limit it becomes necessary to appropriately count the factors of Nc coming from these multiple-scattering terms and keep only the leading terms in such an expansion. Once all the color matrices (funda- mental or adjoint) from vertex contributions have been removed by means of the proper color identities, the power of Nc associated to a given term is just the number of color traces involved. In principle, all sorts of correlations involving multiple Wilson lines at various transverse coordinates can appear when considering the cross section of a multiparticle pro- duction process, but when the large-Nc limit is taken only a few of those contributions are important. Taking into account the way that powers of Nc appear from the corre- lators, it is not difﬁcult to realize that the terms with simpler structure are the ones that contribute the most. For a given process, the number and identity (quark or gluon) of the particles in the ﬁnal state determines the maximum number of Wilson lines present in the multiple-scattering terms. The maximum power of Nc present in such terms will correspond to the conﬁguration in which all the Wilson lines can be grouped in as many traces as possible, therefore favoring terms with traces of only a few Wilson lines. I. INTRODUCTION The physics encoded in this ﬁeld average is inherently nonperturbative and therefore it is necessary to adopt a suitable model to be able to obtain quantitative results. Nevertheless, some features of the averaging process, such as the rapidity dependence in the low-x region, can be appropriately 034007-1 1550-7998=2013=87(3)=034007(16) 2013 American Physical Society II. GENERAL CONSIDERATIONS . PHYSICAL REVIEW D 87, 034007 (2013) FIG. 1. Diagram contributing to the process q ! qgg. The dotted line represents the multiple scattering with the target. (a) Sample diagram. (b) Sample diagram in the large-Nc limit. UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . PHYSICAL REVIEW D 87, 034007 (2013) FIG. 1. Diagram contributing to the process q ! qgg. The dotted line represents the multiple scattering with the target. (a) Sample diagram. (b) Sample diagram in the large-Nc limit. four Wilson lines (quadrupoles). The same will be true for any term with the maximal number of traces. general observations at the level of the amplitude based on color conservation. For a ﬁxed process, with a speciﬁc parton in the initial state and a deﬁnite particle content in the ﬁnal state, it is possible to determine the maximum number of color traces one can have in the description of the process in the leading order. Take a diagram for such a process at tree level in the large-Nc limit, replacing all gluon lines with double quark-antiquark lines which make the color ﬂow explicit, as in Fig. 1(b). As a consequence of color conservation each of the external fermion lines must be connected to another and, since we are considering only tree-level diagrams for the moment, there are no closed loops. From this observation we can see immediately that the maximal number of color traces at tree level is given by the total number of external fermion lines divided by two (with each external gluon contributing two fermion lines). Furthermore, one can see that this maximal number of traces is realized when one considers the square of a given diagram, while interference terms involving different diagrams on the amplitude and conjugate amplitude can possibly have less color traces. Diagrams like those in Fig. 1 are useful for visualizing the branching of the initial parton into the multiparticle ﬁnal state but do not contain all of the information needed to resolve the color structure of the different contributions to the cross section. For that purpose it is necessary to consider diagrams including the amplitude and the con- jugate amplitude where one can perform the necessary color sums and averages. As an example, let us consider the simple process of a quark splitting into a quark and a gluon which then scatters with a background ﬁeld. II. GENERAL CONSIDERATIONS For deﬁniteness’ sake, let us be more speciﬁc about the scenario which we are explicitly considering. We are inter- ested in processes where multiparticle production takes place in the presence of a (strong) background ﬁeld where a highly energetic initial parton (photon, quark, or gluon) undergoes multiple scatterings. This scenario is particu- larly well-suited for nuclear deep inelastic scattering (DIS) experiments and forward particle production in proton- nucleus collisions (using collinear factorization for the proton) where the high-density effects of the target are encoded in the background ﬁeld. For this particular case the situation simpliﬁes even more since the coherence times of the produced particles are long compared with the length of the target nucleus and therefore the multiple interaction with the dense system can be considered as instantaneous. The process is then regarded as an incoming high-energy parton which splits several times into a given ﬁnal multiparticle state, interacting at given time with the target which induces a color rotation in the whole multiparticle system [see Fig. 1(a)]. Of course, one has to consider the scattering with the target happening at all possible stages of the splitting process and sum all of these contributions, but for the counting of powers of Nc, which is of our interest, this does not make a difference. y As a ﬁrst guess, one could suggest that this Nc power counting implies that the leading Nc contribution always comes from a term which only includes color dipole amplitudes (traces of two Wilson lines) and therefore has a maximal number of color traces. This guess has been proven wrong since thorough studies of two-particle pro- duction processes [3–5] have shown that some processes do not admit a description in terms of only color dipoles, but that in addition color quadrupoles (traces of four Wilson lines) are involved as well in the large-Nc limit. This quadrupole amplitude cannot in general be written in terms of dipole amplitudes only and its small-x evolu- tion is determined by an independent equation as shown in Refs. [3,6,7]. It has also been shown that there is a direct relation between this quadrupole amplitude and the so-called Weisza¨cker-Williams unintegrated gluon distribution function [5]. The Nc power counting can only be done at the level of the cross section after one has already averaged (summed) over initial (ﬁnal) colors. Nevertheless, one can make some 034007-2 UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . DOMINGUEZ et al. DOMINGUEZ et al. DOMINGUEZ et al. the initial state. These particles in the initial state should be color connected on both sides of the diagram since one averages over initial colors at the level of the cross section, but such a connection is not explicit in this kind of diagram. the initial state. These particles in the initial state should be color connected on both sides of the diagram since one averages over initial colors at the level of the cross section, but such a connection is not explicit in this kind of diagram. FIG. 4. Graphical representation of the Fierz identity. p g If one is interested in the color structure only, then it is more convenient to draw one fermion loop for each color trace and gluon lines for each adjoint index contraction (including adjoint Wilson lines too). This can be easily achieved by folding the corresponding diagram with the amplitude and conjugate amplitude on itself in such a way that the color connections of the initial state are made explicit. Also, in order to be able to visualize in the diagram which are the Wilson lines entering the expression of the cross section, we will stretch the lines involved in the scattering (both in the amplitude and conjugate amplitude) in the horizontal direction, and use the other lines only to illustrate the color connections. For example, the diagram corresponding to Fig. 2(a) is shown in the left-hand side of Fig. 3. One can go one step further and replace all of the gluon lines by double fermion lines using the correspond- ing color identity—which has the graphical representation depicted in Fig. 4—in which case the example above takes the form shown in the right-hand side of Fig. 3. Let us focus on the ﬁrst term—which is the one surviving in the large-Nc limit—where one can identify right away two pieces which are color disconnected, each contributing a color trace to the cross section. One involves two Wilson lines, and therefore is a dipole, while the other involves four, and therefore is a quadrupole. crucial to identify the pieces corresponding to dipoles and quadrupoles, which we show separately in Fig. 5. It was also mentioned earlier that one should sum over all of the possibilities for the multiple interaction to take place in the amplitude and the conjugate amplitude. DOMINGUEZ et al. Consider the process in the example above but with the multiple interaction occurring in the conjugate amplitude before the splitting. Figure 6 illustrates how the different diagrams explained above look for this variation of the same process. For this particular case it is clear that the contribution to the cross section can be written in terms of two dipoles. This example illustrates a very important fact that will allow us to concentrate on a smaller number of diagrams: changing the position of the multiple scattering might change the number of Wilson lines appearing in the corresponding term in the cross section, but it does not change the number of color traces. It might happen that for some particular cases a color trace is left with no Wilson lines inside, giving a trivial factor of Nc, but what is important is that the counting of powers of Nc is the same for a given process regardless of the position of the multiple scattering with respect to the splittings. The last consideration to be noted before beginning a detailed explanation of particular cases is the effect of integrating out particles in the ﬁnal state. In order to consider inclusive processes it is sometimes necessary to integrate over the momenta of outgoing particles that are not explicitly measured. This happens at the leading order for processes in which one produces an additional fermion in the ﬁnal state without its corresponding antiparticle; for example, in single inclusive deep inelastic scattering where the incoming photon splits into a quark-antiquark pair, but only one of them is measured in the ﬁnal state. For all other processes this effect comes in at next-to-leading order and will be fundamental if we want our proof to be complete to all orders. The example above shows how this graphic approach allows us to easily recognize which kinds of correlators come into the expression for the cross section of a given process. One can also recognize that the combination of Wilson lines describing such a multiple scattering is given by Tr½U3Uy 4 tatbWac 1 Wycb 2 ¼ 1 2 Tr½U1Uy 2 U3Uy 4 Tr½Uy 1 U2 1 2Nc Tr½U3Uy 4 ; (2) (2) where Fierz identities were used to reach the right-hand side. II. GENERAL CONSIDERATIONS By putting the amplitude and the conjugate amplitude in the same diagram we obtain Fig. 2(a), where the dotted lines represent the moment of the scattering in both the ampli- tude and conjugate amplitude and the dashed line in the middle represents the ﬁnal state at t ¼ 1. Since the approach employed here makes explicit use of the eikonal approximation to account for the multiple scattering with the external ﬁeld, it is necessary to consider these diagrams in a coordinate representation where each particle has a deﬁnite transverse coordinate. All particles in the ﬁnal state which are to be detected, and therefore would have a ﬁxed transverse momentum, have different transverse coordi- nates on each side of the cut at t ¼ 1. When considered in the large-Nc limit, one can see in Fig. 2(b) how some of the fermion lines close into loops, which will contribute one color trace to the cross section, while other lines remain open due to the fact that they contain particles in Also illustrated in Fig. 1(b) is the fact that each fermion line appears at most twice at the moment of the multiple scattering, regardless of the exact position of the scattering with respect to the splittings. As a consequence, at most two fundamental Wilson lines are color connected at the amplitude level and therefore, when considering the am- plitude squared, the respective color traces in the cross section would have only two Wilson lines (dipoles) or FIG. 2. Diagrams with the amplitude and conjugate amplitude for the process q ! qg. (a) q ! qg. (b) q ! qg in the large-Nc limit. mplitude and conjugate amplitude for the process q ! qg. (a) q ! qg. (b) q ! qg in the large-Nc limit. 034007-3 034007-3 034007-3 FIG. 4. Graphical representation of the Fierz identity. PHYSICAL REVIEW D 87, 034007 (2013) A. SIDIS Semi-inclusive deep inelastic scattering (SIDIS) has been widely studied in the literature since it has been recognized as giving access to transverse momentum- dependent parton distribution functions. In the context of saturation physics, several studies have cemented the foun- dations of the formalism to treat deep inelastic scattering processes where the focus was mainly on the total cross section and form factors. Most of these studies are based on the dipole model approach [11], which shows directly a clear relation between the total cross section and the for- ward amplitude of a color dipole going through a back- ground color ﬁeld, but the same conclusions can be found from the setup explained in the previous sections after one integrates out all the particles in the ﬁnal state. For the particular case of SIDIS, it was explicitly shown in Refs. [12–14] that the only correlator needed in the expres- sion of the cross section is the dipole amplitude, and in Ref. [15] a direct connection to the transverse momentum- dependent quark distributions was made. From the point of view of the correlators we are inter- ested in, it is clear that the ones entering the cross sections of these more inclusive processes are either the same or simpler than the ones involved in the expression for the full exclusive process where all particles in the ﬁnal state are detected. This observation allows us to focus our attention to the fully exclusive processes only. DOMINGUEZ et al. Therefore integrat- ing over the transverse momentum of a particle in the ﬁnal state gives a delta function which identiﬁes the transverse coordinate of the particle in the amplitude with the one in the conjugate amplitude. Given the unitarity of the Wilson lines, this sort of manipulation will likely reduce the number of Wilson lines appearing in the corresponding contribution to the cross section of any particular diagram. The momenta of the particles of the ﬁnal state then enters through a Fourier transform of the respective coordinate in the amplitude and conjugate amplitude. Therefore integrat- ing over the transverse momentum of a particle in the ﬁnal state gives a delta function which identiﬁes the transverse coordinate of the particle in the amplitude with the one in the conjugate amplitude. Given the unitarity of the Wilson lines, this sort of manipulation will likely reduce the number of Wilson lines appearing in the corresponding contribution to the cross section of any particular diagram. The sum over color indices in the ﬁnal state guaranties that if a ﬁnal-state particle participates in the multiple scatter- ing both in the amplitude and the conjugate amplitude, both Wilson lines appear next to each other in the contri- bution to the cross section, and if they are placed at the same transverse coordinate then they exactly cancel out. This sort of cancellation between real and virtual contri- butions for ﬁnal-state interactions has been studied before, and it is well-known to be an immediate consequence of unitarity [10]; the only place where interactions with the unmeasured particle come in is in the interference terms between initial- and ﬁnal-state interactions. DOMINGUEZ et al. For more complicated processes the color algebra can be very cumbersome, and we will rely heavily on the graphic approach to be able to identify the leading Nc piece of the diagrams involved. In particular, it will be Under the eikonal approximation at work in this formal- ism, the multiple-scattering terms take the form of simple Wilson lines in the appropriate representation only when considering the process in transverse coordinate space. FIG. 3. Alternative representation for the process q ! qg. FIG. 3. Alternative representation for the process q ! qg. 034007-4 034007-4 UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . PHYSICAL REVIEW D 87, 034007 (2013) FIG. 5. Graphical representation of the two amplitudes to be used throughout the paper. (a) Dipole amplitude. (b) Quadrupole amplitude. the previously described setup, it is necessary to start our analysis with the simplest cases available. All of these cases have been previously studied in the literature; here we comment on the results and emphasize the features of the derivations which will be useful for the development of the argument for general cases. We start with processes with one particle in the ﬁnal state even though their color structure is better understood in the context of processes with two particles in the ﬁnal state. The reason for this is that the nontrivial contributions to these processes are obtained after integrating out one of the particles in the ﬁnal state. As explained in the previous section, this procedure yields simpler expressions where the correlators appearing in the cross section have less Wilson lines than the corresponding correlators for the processes with two ﬁnal-state particles. The choice to present the one-particle processes ﬁrst follows the thread of the main idea, where we intend to organize the processes in terms of the number of particles in the ﬁnal state; from there on the emphasis will be put on fully exclusive pro- cesses with less exclusive processes already accounted for by the observations of the previous section. FIG. 5. Graphical representation of the two amplitudes to be used throughout the paper. (a) Dipole amplitude. (b) Quadrupole amplitude. The momenta of the particles of the ﬁnal state then enters through a Fourier transform of the respective coordinate in the amplitude and conjugate amplitude. B. Single-hadron production in pA This process is of particular importance in the context of the study of cold-nuclear-matter effects. One of the ﬁrst measurements to show deviations from the purely additive scheme where a nucleus is considered as an uncorrelated ensemble of nucleons was the p? spectrum of produced hadrons in a proton(deuteron)-nucleus collision. Several measurements of the nuclear modiﬁcation factor for inclu- sive hadron production were performed, showing a clear enhancement at mid-rapidities and suppression at forward rapidities [16,17], which cannot be understood without coherent scattering involving several participants in the nucleus. y As a matter of fact, one can use induction to prove that, for any single inclusive processes in terms of any order in s, the scattering amplitudes only contain dipole amplitudes in the large-Nc limit. As discussed earlier, the above conclusion holds up to leading order and NLO for pA collisions. To obtain the contribution at next-to- next-to-leading order, one just needs to add one more gluon with one single coordinate among the dipoles at the previous order (NLO). Using the Fierz identity, one can easily prove that all the relevant graphs can be reduced to products of dipole amplitudes at next-to- next-to-leading order in the large-Nc limit. The proof for single inclusive DIS productions is identical. Therefore, we can conclude that all single inclusive processes can be universally described by the dipole amplitudes at the large-Nc limit. From the universality point of view, the large-Nc limit plays an important and indispensable role for the factorization proof. As shown in Ref. [25], higher-point functions—which are new objects—contribute to the cross sections as large-Nc corrections. Without the large-Nc limit, there is no uni- versality, and hence no factorization for single inclusive processes. In the framework described in this paper, the lowest- order contribution to this process is straightforward to calculate as the convolution of a quark distribution for the projectile with a dipole amplitude formed by the Wilson lines corresponding to the quark in the amplitude and the conjugate amplitude. At the partonic level this is simply the transverse momentum broadening of a quark going through a nucleus [18]. This level of the calculation has been proven to not be enough to describe the available data, and therefore it is necessary to consider additional contributions to the cross section coming mainly from gluon emissions, with the additional complication of hav- ing more particles in the ﬁnal state. DOMINGUEZ et al. DOMINGUEZ et al. [1,21,22]. The details of how this manipulation works out in the Wilson-line language will be postponed to the section on dijet production. process—in which one considers a virtual photon splitting into a quark-antiquark pair—the multiple scattering term at the amplitude level always appears as 1 Uðx1ÞUyðx2Þ, where x1 and x2 are the transverse positions of the quark and antiquark, respectively. Clearly, the square of this amplitude will have terms with traces of either zero, two, or four Wilson lines, but since one integrates over the momentum of one of the particles, two of the Wilson lines are at the same transverse coordinate and therefore cancel out in the term with four Wilson lines. j p The soft-gluon approximation implies a large rapidity gap between the measured hadron and the remnants of the proton in the forward region. In order to extend the region where the calculation is applicable, and in particular include hadrons in the forward region where the effects of saturation are expected to be stronger, it is necessary to consider the full vertex and allow the parent quark to have different transverse positions before and after the emission. This was ﬁrst done in Ref. [23], where the emission vertex was treated exactly but only the divergent pieces are kept after integrating over the momentum of one of the ﬁnal-state particles. These divergent pieces are shown to be absorbed by the parton distribution functions and fragmen- tation functions attached to the initial- and ﬁnal-state partons by considering the fully Dokshitzer–Gribov– Lipatov–Altarelli–Parisi-evolved distributions. In Ref. [24], an additional contribution to the cross section—formally of next-to-leading order (NLO) but nevertheless important to restore the correct high-pT limit—was calculated. The com- plete NLO calculation, including the calculation of all virtual terms and also the ﬁnite terms after integration over one of the outgoing momenta, was recently done in Ref. [25]. In all cases it can be seen that, in the large-Nc limit, only dipole amplitudes are needed in the full expression for the cross section. In terms of the small-x evolution at NLO, the same conclusion also holds [26]. In addition, it has been demonstrated in Ref. [26] that the dipole formalism employed in this calculation holds at next-to-leading order accuracy. DOMINGUEZ et al. We note that, by swapping the initial-state photon and the quark (or antiquark) in the ﬁnal state whose momentum is integrated out, the SIDIS process turns into photon þ hadron production in p þ A collisions (whether the photon is measured or not doesn’t matter because it does not multiply scatter with the gluons of the target). Therefore the only correlator needed to express the cross section is also the dipole amplitude. III. KNOWN CASES: ONE AND TWO PARTICLES IN THE FINAL STATE The lowest-order calculation of this process is very simple from the point of view of multiple scattering, which we are interested in here. It can be shown that for a DIS Since our goal is to prove by induction a statement that will be valid for all multiparticle production processes in FIG. 6. Diagrams for the interference term with interaction after the splitting in the amplitude and before the splitting in the conjugate amplitude. (a) Interference term. (b) Color structure. FIG. 6. Diagrams for the interference term with interaction after the splitting in the amplitude and before the splitting in the conjugate amplitude. (a) Interference term. (b) Color structure. 034007-5 PHYSICAL REVIEW D 87, 034007 (2013) C. Dijet production in DIS Heavy-Ion Collider measurement of di-hadron correlations in the forward region [29,30] is considered to be the strongest evidence to date of saturation. Heavy-Ion Collider measurement of di-hadron correlations in the forward region [29,30] is considered to be the strongest evidence to date of saturation. The process of dijet production in DIS is of particular interest to us since it is the simplest process where the quadrupole is needed for an accurate description of the multiple-scattering factors entering the expression for the cross section. It was carefully studied in Refs. [5,27], where its direct relation to the Weizsa¨cker-Williams gluon distribution function was also emphasized. Several studies concerning this kind of process are available in the literature, which include the role of quark distributions [31], general descriptions on how to calculate the cross sections in the presence of background ﬁelds [3,4,32,33], more phenomenological applications— including small-x evolution—which make direct contact with data [34–36], and factorization studies where a direct relationship is established between these observables and unintegrated gluon distributions [5]. Here we will just present a summary of the correlators appearing in the cross section for each of these processes and the speciﬁc form they take in the large-Nc limit. p It was already mentioned in Sec. III A and previously in Refs. [5,27,28] that, to leading order at the amplitude level, the corresponding multiple-scattering factor for a process including a photon splitting into a quark-antiquark pair is given by 1 Uðx1ÞUyðx2Þ, where x1 and x2 are the transverse positions of the quark and antiquark, respectively. Since for the dijet process one is interested in keeping the momentum variables for both particles explicit, the trans- verse coordinates in the amplitude and conjugate amplitude are different and therefore there is a term with four Wilson lines in the expression for the cross section. This quadrupole term takes the form hTr½Uðx1ÞUyðx2ÞUðx0 2ÞUyðx0 1Þi, and clearly corresponds to the contribution from the diagram where the interaction with the background ﬁeld occurs after the splitting in both the amplitude and conjugate amplitude. Figure 7 shows the two ways of graphically representing this process as indicated in Sec. II. The ﬁrst case to be considered is the quark-initiated pro- cess, where a quark from the projectile splits into a quark and a gluon that are both detected in the ﬁnal state. This is precisely the process chosen in Sec. C. Dijet production in DIS II as an illustration of the sort of analysis to be performed throughout this paper. There it was shown how all of the terms entering the cross section for that process at leading order in the large-Nc limit involve only the dipole and quadrupole correlators. Now we turn our attention to processes with gluons in the initial state. The ﬁrst one to be considered is the case where an initial gluon from the projectile splits into a quark- antiquark pair. The color algebra for this process in the large-Nc limit is particularly simple since the cross section can be written in terms of only dipole correlators; never- theless, it is important to look closely at some of the aspects of this process since it will allow us to draw general con- clusions about any process with quark-antiquark pairs in the ﬁnal state. The key aspect here is that the splitting does not introduce any additional color ﬂow in the large-Nc limit: in the double-line notation introduced for the large-Nc limit the only effect of such a vertex is to separate the two lines and allow for different transverse coordinates for the quark and the antiquark. Such a separation has no consequences in the trace structure of the leading part of the diagram when written entirely in terms of fundamental Wilson lines. C. Dijet production in DIS UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . PHYSICAL REVIEW D 87, 034007 (2013) B. Single-hadron production in pA As a ﬁrst attempt, it was shown in Refs. [19,20] that one can easily calculate the soft-gluon limit where the longitudinal momentum of the gluon is much smaller than that of the original quark and, therefore, the parent quark does not feel any recoil effect after the emission. This no-recoil condition is present in the transverse coordinate-space formalism in the form of the parent quark having the same transverse coordinate before and after the emission of the gluon, providing an easy way to rewrite the interference terms between scattering before and after the splitting in terms of only gluon dipoles 034007-6 D. Dijet production in pA Up until now, the large-Nc limit has been mainly invoked to be able to regard color traces as separate entities—even after one takes the average over the back- ground color ﬁeld—arguing that correlations between ﬁelds entering Wilson lines in different color traces are suppressed by factors of 1=N2c. The multiple-scattering factors of the processes considered so far, when expressed in terms of fundamental Wilson lines only, can all be expressed in terms of products of traces of either two or four Wilson lines in the fundamental representation. This will not be the case from now on when we start to consider more complicated processes, starting with the case of an initial gluon splitting into two gluons which fragment independently. This process will have contributions from diagrams with three and four gluons present at the moment of the multiple scattering, which will lead to traces of six and eight fundamental Wilson lines. Tr½U1taUy 2 U3taUy 4 ¼ 1 2 Tr½U1Uy 4 Tr½Uy 2 U3 1 2Nc Tr½U1Uy 2 U3Uy 4 : (3) (3) This relation is represented graphically in Fig. 8. Now consider the case where the interaction occurs after the splitting in the amplitude and before the splitting in the conjugate amplitude. Then, the partons involved in the scattering are a quark-antiquark pair and a gluon, and the multiple-scattering factor—in terms of the correspond- ing Wilson lines—is Let us consider ﬁrst the case where the multiple scatter- ing occurs after the splitting in the amplitude and before the splitting in the conjugate amplitude. It is easy to see that there are three gluons involved in the multiple scattering—each one contributing one adjoint Wilson line to the multiple-scattering factor—and that they are con- nected before and after the scattering by three-gluon ver- tices. Following Ref. [5], we can evaluate the relevant scattering matrices, which yield Tr½U1taUy 2 tbWba 3 ¼ 1 2 Tr½U1Uy 3 Tr½Uy 2 U3 1 2Nc Tr½U1Uy 2 : (4) (4) It is easy to see that the graphical representation of the color structure of this process is topologically equivalent to that shown in Fig. 6(b). As previously anticipated, the message to take from here is that the color ﬂow structure present in this process is the same for both cases presented above for the leading part in the large-Nc limit. D. Dijet production in pA Processes with two particles in the ﬁnal state of a proton- nucleus collision have become increasingly important in the last few years given the availability of new data and the unique character of the physics that can be probed through these particular sorts of measurements. By constraining the kinematics of the two outgoing particles, one can, at lead- ing order, separately ﬁx the longitudinal momentum frac- tions carried by the incoming partons (up to the additional fragmentation integrals involved in the case of di-hadron production), something that cannot be achieved by one- particle measurements. This advantage makes this kind of process a very attractive method to try to measure high-density effects characteristic of the small-x part of the target wave function, and in fact the Relativistic y The observation above can be easily illustrated when one compares the different contributions to the g ! q q FIG. 7. Diagrams for the quadrupole term in the dijet production in DIS. The photon is omitted in (b) since we are interested only in the color structure. (a) Amplitude squared. (b) Color structure. FIG. 7. Diagrams for the quadrupole term in the dijet production in DIS. The photon is omitted in (b) since we are interested only in the color structure. (a) Amplitude squared. (b) Color structure. 034007-7 034007-7 FIG. 8. Color structure of the g ! q q process with interaction after the splitting. DOMINGUEZ et al. PHYSICAL REVIEW D 87, 034007 (2013) DOMINGUEZ et al. FIG. 8. Color structure of the g ! q q process with interaction after the splitting. process arising from having the multiple scattering before or after the splitting. For deﬁniteness, consider ﬁrst the case where the scattering occurs after the splitting in both the amplitude and in the conjugate amplitude. The partons involved in the scattering are therefore two quark- antiquark pairs, as indicated on the left-hand side of Fig. 8. This multiple scattering term is written in terms of Wilson lines (after averaging over the initial color of the gluon) as Tr½U1taUy 2 U3taUy 4 . Using the Fierz identity to get rid of the color matrices one obtains emitting a gluon or a gluon splitting into two gluons. The four-gluon vertex can also be ignored since its color struc- ture is equivalent to a combination of three-gluon vertices. UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) FIG. 9. The graphs on the right-hand side of the equation should also include their Hermitian conjugates, which can be obtained by simply reversing all of the arrows. Here all the correlators are assumed to be real, and therefore the 1 2 factor is cancelled. FIG. 9. The graphs on the right-hand side of the equation should also include their Hermitian conjugates, which can be obtained by simply reversing all of the arrows. Here all the correlators are assumed to be real, and therefore the 1 2 factor is cancelled. FIG. 10. Double-line representation of the gluon vertex. Here we should include the Hermitian conjugates of the graphs on the right-hand side of the equation. Its graphical representation is given in Fig. 11, where once again we made use of the identity illustrated in Fig. 10. Similarly to the previous case, it is easy to see that the term with three color traces is the one that dominates in the large-Nc limit, and the term with a trace of eight Wilson lines (octupole) is subleading. We can then safely state that, for all the cases considered in this section, the leading contribution to the multiple-scattering term in the large-Nc limit can always be written in terms of dipole and quadru- pole correlators only. FIG. 10. Double-line representation of the gluon vertex. Here we should include the Hermitian conjugates of the graphs on the right-hand side of the equation. Both Eq. (5) and Fig. 9 show that the term with a trace of six Wilson lines (sextupole) is suppressed in the large-Nc limit. On the one hand, the terms with a product of three color traces are proportional to N3c, while the terms with only one trace are of order Nc. On the other hand, the graphical representation clearly shows that the term with the sextupole comes from a nonplanar diagram and there- fore is suppressed with respect to planar diagrams by at least a factor of 1=N2c. D. Dijet production in pA Moreover, this is the same structure we see when only two gluons are present in the multiple scattering, which are always the product of two fundamental dipoles. As opposed to pro- cesses where gluons are emitted, no extra color charge is created at the vertex and therefore the color structure remains unchanged; the same is also true for processes with a quark-antiquark pair merging into a gluon, which might be important for higher-order contributions. fadeWdb 1 Wec 2 ffbcWaf 3 ¼ 1 2 Tr½U1Uy 3 Tr½U3Uy 2 Tr½U2Uy 1 þ 1 2 Tr½U2Uy 3 Tr½U1Uy 2 Tr½U3Uy 1 1 2 Tr½U3Uy 2 U1Uy 3 U2Uy 1 1 2 Tr½U1Uy 2 U3Uy 1 U2Uy 3 : (5) fadeWdb 1 Wec 2 ffbcWaf 3 ¼ 1 2 Tr½U1Uy 3 Tr½U3Uy 2 Tr½U2Uy 1 þ 1 2 Tr½U2Uy 3 Tr½U1Uy 2 Tr½U3Uy 1 1 2 Tr½U3Uy 2 U1Uy 3 U2Uy 1 1 2 Tr½U1Uy 2 U3Uy 1 U2Uy 3 : (5) (5) The corresponding graphical representation is shown in Fig. 9. Finding the correct graphical representation—in terms of fermion lines only—without explicitly performing the algebra shown in Eq. (5) is possible as long as one knows how to represent two consecutive three-gluon ver- tices in the double-line representation. The identity to be used is illustrated in Fig. 10. The observations above allow us to neglect—from the point of view of the color structure of the process in the large-Nc limit—all of the vertices involving a gluon splitting into a quark-antiquark pair, and to focus our atten- tion on processes where all of the vertices involve a quark 034007-8 FIG. 9. The graphs on the right-hand side of the equation should also include their Hermitian conjugates, which can be obtained by simply reversing all of the arrows. Here all the correlators are assumed to be real, and therefore the 1 2 factor is cancelled. UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . PHYSICAL REVIEW D 87, 034007 (2013) UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . IV. THREE PARTICLES IN THE FINAL STATE The aim of this section is to show with explicit examples how the color structure of a given process is affected by the inclusion of additional gluons in the ﬁnal state. At the end of the previous section it was seen already that when the complexity of the problem increases—as well as the number of colored particles participating in the multiple scattering—it is natural that new higher-point correlations have to be included in the full description of the process. It was also seen that these higher-point correlations always appear suppressed by inverse powers of Nc when the multiple-scattering factor is expressed only in terms of fundamental Wilson lines. The same will be observed in the examples shown in this section: higher-point correla- tions keep appearing, but the leading term in the large-Nc limit can always be written in terms of dipole and quadru- pole amplitudes. The case where the multiple scattering occurs after the splitting, both in the amplitude and the conjugate ampli- tude, is treated similarly. Now, there is an extra gluon present in the multiple scattering, contributing an extra adjoint Wilson line, and the two three-gluon vertices are placed before the scattering. The color connections after the scattering are given by identifying the corresponding gluons from the amplitude and the conjugate amplitude. In summary, the relevant multiple-scattering term, in terms of adjoint Wilson lines as well as fundamental Wilson lines, is fadeðW1Wy 2 ÞdbfabcðW3Wy 4 Þec ¼ 1 2 Tr½U2Uy 1 Tr½U3Uy 4 Tr½U1Uy 2 U4Uy 3 þ 1 2 Tr½U4Uy 3 Tr½U1Uy 2 Tr½U3Uy 4 U2Uy 1 1 2 Tr½U2Uy 1 U3Uy 4 U1Uy 2 U4Uy 3 1 2 Tr½U1Uy 2 U3Uy 4 U2Uy 1 U4Uy 3 : (6) limit can always be written in terms of dipole and quadru- pole amplitudes. Let us start with one of the simplest cases at this level, which at the same time will show us a general feature of the further inclusion of gluons. Consider the process of production of a quark-antiquark pair and a gluon in DIS. As was observed in previous cases, it is sufﬁcient to con- sider the case where the maximum number of particles participate in the multiple scattering since any other com- bination would yield simpler correlators. The process is then described by a photon splitting into a quark-antiquark FIG. 11. IV. THREE PARTICLES IN THE FINAL STATE The relevant diagrams can all be obtained by considering all possible ways of attaching one extra gluon to the diagrams contributing to the q ! qg process. As an illustration, consider the diagrams corresponding to the square of the processes where the additional gluon is emitted either by the ﬁnal quark or by the ﬁrst gluon and all three particles participate in the interaction with the background ﬁeld. The color structure is given by the diagrams of Fig. 13.1 It is easy to see that for the case where the second gluon is emitted from the quark the color structure can be resolved by a straightforward application of the Fierz identity, leading to the conclu- sion that the leading term in the large-Nc limit is given by one dipole and two quadrupoles. Instead of getting into the algebraic details of this computation, we choose the graphical approach here to show how the additional gluon modiﬁes the structure of the correlator. Consider the diagram in the ﬁrst term of the right-hand side of Fig. 3, representing the leading piece of the q ! qg process, and include the additional gluon as shown in Fig. 14. Since the gluon is only attached to the quadru- pole part one can momentarily forget about the dipole part: it is clear that when one uses the Fierz identity to remove the gluon from the diagram one observes that the dominant piece of the diagram is the one in which the quadrupole is split into two quadrupoles. Tr½Uy 1 taU2Uy 4 tbU3ðW5Wy 6 Þab ¼ 1 2 Tr½U2Uy 4 U6Uy 5 Tr½U3Uy 1 U5Uy 6 1 2Nc Tr½U3Uy 1 U2Uy 4 : (7) Tr½Uy 1 taU2Uy 4 tbU3ðW5Wy 6 Þab ¼ 1 2 Tr½U2Uy 4 U6Uy 5 Tr½U3Uy 1 U5Uy 6 1 2Nc Tr½U3Uy 1 U2Uy 4 : (7) (7) Both graphically and algebraically it is easy to see that the effect of adding a new gluon to the process studied in Sec. III C can be understood in terms of the Fierz identity. Here we choose to interpret the result in terms of the graphical representation. The extra gluon can be removed from the diagram by the rule depicted in Fig. 4, regardless of it being involved in the multiple interaction. For this particular case, the ﬁrst term on the right-hand side of Fig. IV. THREE PARTICLES IN THE FINAL STATE The graphs on the right-hand side of the equation should also include their Hermitian conjugates, which can be obtained by simply reversing all of the arrows. Here all the correlators are assumed to be real, and therefore the 1 2 factor is cancelled. fadeðW1Wy 2 ÞdbfabcðW3Wy 4 Þec ¼ 1 2 Tr½U2Uy 1 Tr½U3Uy 4 Tr½U1Uy 2 U4Uy 3 þ 1 2 Tr½U4Uy 3 Tr½U1Uy 2 Tr½U3Uy 4 U2Uy 1 1 2 Tr½U2Uy 1 U3Uy 4 U1Uy 2 U4Uy 3 1 2 Tr½U1Uy 2 U3Uy 4 U2Uy 1 U4Uy 3 : Let us start with one of the simplest cases at this level, which at the same time will show us a general feature of the further inclusion of gluons. Consider the process of production of a quark-antiquark pair and a gluon in DIS. As was observed in previous cases, it is sufﬁcient to con- sider the case where the maximum number of particles participate in the multiple scattering since any other com- bination would yield simpler correlators. The process is then described by a photon splitting into a quark-antiquark (6) FIG. 11. The graphs on the right-hand side of the equation should also include their Hermitian conjugates, which can be obtained by simply reversing all of the arrows. Here all the correlators are assumed to be real, and therefore the 1 2 factor is cancelled. FIG. 11. The graphs on the right-hand side of the equation should also include their Hermitian conjugates, which can be obtained by simply reversing all of the arrows. Here all the correlators are assumed to be real, and therefore the 1 2 factor is cancelled. 034007-9 FIG. 12. q qg production in DIS. DOMINGUEZ et al. PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) DOMINGUEZ et al. FIG. 12. q qg production in DIS. pair, which emits a gluon before undergoing multiple scatterings with the target ﬁeld in both the amplitude and conjugate amplitude. The multiple-scattering term, there- fore, includes two quark-antiquark pairs and two gluons with the color connections as shown in Fig. 12, and can be written as Next, we switch our attention to processes present in pA collisions. First consider the quark-initiated process with one quark and two gluons in the ﬁnal state. 1Here we are only interested in the planar diagrams, in which gluon lines do not cross one another. The nonplanar diagrams are large-Nc suppressed, and thus are always discarded. A. Leading order (4) From the point of view of the color structure only, a four-gluon vertex can always be represented by the sum of different ways of combining two three-gluon vertices. Because of this, processes with four-gluon vertices are not considered since its color structure is already accounted for by a proper treatment of the three-gluon vertices. A. Leading order Before getting into the details of the inductive step of the proof, let us summarize some of the observations that have been made throughout the paper, which will be useful for the argument presented in this section. (8) (1) In the large-Nc limit, the Nc power counting is most easily done when scattering factors are expressed fully in terms of fundamental Wilson lines, where each trace contributes with a factor of Nc. One can clearly see that the ﬁrst two terms come from attaching the two legs of the new gluon to the same preexisting fermion loop, while the last two terms come from attaching the two legs of the new gluon to different fermion loops. Higher-point correlators appear in this expression but are always suppressed by a power of 1=N2c, as compared to the terms with only dipoles and quadrupoles. (2) Changing the moment of the multiple interaction does not change the Nc power counting. The case where all the ﬁnal-state particles participate in the multiple interaction is where the most complicated correlators can possibly be found, and therefore these will be the only cases under study in this section. For the gluon-initiated processes the situation is very similar. Even though the explicit calculations are more intricate and tedious, one can easily recognize that the leading terms for large-Nc for the process g ! ggg consist of a combination of two dipoles and two quadrupoles. One can perform a similar analysis to the one performed above for the q ! qgg process, starting with the leading piece of the g ! gg process and adding an extra gluon. Again, the terms that survive in the large-Nc limit will be the ones coming from attaching both legs of the new gluon to only one of the preexisting fermion loops—in this case two dipoles and one quadrupole—therefore creating one extra quadrupole (in the case where all produced particles par- ticipate in the multiple scattering in both the amplitude and conjugate amplitude). (3) A gluon splitting into a quark-antiquark pair does not add any color charge to the process and therefore leaves its color structure unchanged. For the argu- ment presented here one can neglect any new con- tributions from processes with extra quark-antiquark pairs in the ﬁnal state and always assume that the additional particles are gluons. UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . ect of the extra gluon in the leading piece for one diagram contributing to the q ! qgg process. FIG. 14. Illustration of the effect of the extra gluon in the leading piece for one diagram contributing to the q ! qgg process. leading order for a given number of particles, and then show how the argument can be further generalized to also include all-order contributions. Tr½U5 t t U6fadeðW1W2 Þ fa0bcðW3W4 Þ ¼ 1 4 Tr½U1Uy 2 U4Uy 3 Tr½U6Uy 5 U3Uy 4 Tr½U2Uy 1 þ 1 4 Tr½U3Uy 4 U2Uy 1 Tr½U6Uy 5 U1Uy 2 Tr½U4Uy 3 1 4 Tr½U6Uy 5 U1Uy 2 U4Uy 3 U2Uy 1 U3Uy 4 1 4 Tr½U6Uy 5 U3Uy 4 U2Uy 1 U4Uy 3 U1Uy 2 : (8) Tr½U5 t t U6fadeðW1W2 Þ fa0bcðW3W4 Þ ¼ 1 4 Tr½U1Uy 2 U4Uy 3 Tr½U6Uy 5 U3Uy 4 Tr½U2Uy 1 þ 1 4 Tr½U3Uy 4 U2Uy 1 Tr½U6Uy 5 U1Uy 2 Tr½U4Uy 3 1 4 Tr½U6Uy 5 U1Uy 2 U4Uy 3 U2Uy 1 U3Uy 4 1 4 Tr½U6Uy 5 U3Uy 4 U2Uy 1 U4Uy 3 U1Uy 2 : (8) IV. THREE PARTICLES IN THE FINAL STATE 4 cuts the already existing quadrupole in two, giving as a result a term with two color traces—which will clearly dominate in the large-Nc limit—as compared to the second term, which does not introduce any new traces and has an extra factor of 1=Nc in front. Here the original quadrupole is split into two quadrupoles, but it is easy to see that for slightly different cases—as for example if one considers the term with scattering before the emission of the gluon in the conjugate amplitude—one can end up with a quadru- pole and a dipole. These small variations of the same process can be seen as attaching the gluon to different places of the original quadrupole. At the end of the day the result is always the same: the leading term in the large-Nc limit will be given by the term that splits the original color trace into two different color traces. For the case just described, there was no other choice but to attach the additional gluon to the quadrupole from the leading piece of the q ! qg process. When one considers other diagrams—such as the one already described where the second gluon is emitted from the ﬁrst gluon—there are other ways of attaching this new gluon to the dominant part of the diagram. In general one can consider all possible ways of attaching the two legs of the new gluon and quickly realize that the pieces which will survive in the large-Nc limit are the ones coming from attaching both legs of the new gluon to the same fermion loop. As an illus- tration, let us consider the algebraic expression of the scattering term for the diagram in Fig. 13(b): FIG. 13. q ! qgg. (a) Additional gluon emitted from quark. (b) Additional gluon emitted from gluon. FIG. 13. q ! qgg. (a) Additional gluon emitted from quark. (b) Additional gluon emitted from gluon. 034007-10 FIG. 14. Illustration of the effect of the extra gluon in the leading piece for one diagram contributing to the q ! qgg process. UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . DOMINGUEZ et al. FIG. 15. Higher-order s contribution to the q q dijet. k particles in the ﬁnal state, its representation in terms of only fundamental Wilson lines is expressed in terms of dipoles and quadrupoles only. Now reattach the gluon removed in the previous step. There are two possibilities: either both of its legs are attached to the same fermion loop, or each leg is attached to a different fermion loop. In the ﬁrst case, one can see from the examples in Sec. IV that the insertion of the new gluon splits the corresponding fermion loop—either dipole or quadrupole—creating a new quadrupole (when the new dipole interacts in both the amplitude and the conjugate amplitude—as was pointed out—any other case would lead to simpler corre- lators). The second case can be easily seen to be suppressed by a factor of 1=N2c with respect to the ﬁrst case. The two terms one obtains after applying the Fierz identity to a gluon joining two separate fermion loops have one power of Nc less compared to the diagram without the extra gluon; one of them has one loop less while the other has an explicit factor of 1=Nc. This is to be compared to the ﬁrst case where one additional fermion loop is created, and therefore one extra power of Nc is present. FIG. 15. Higher-order s contribution to the q q dijet. dipole or a quadrupole, and thus higher-order virtual graphs can only generate more dipoles. Let us take the DIS dijet as an example, where we add a one-gluon loop in both the amplitude and complex conjugate amplitude, as shown in Fig. 15. Using the Fierz identity, it is obvious that the addition of the virtual gluons in the dijet processes does not introduce higher-point functions in the large-Nc limit. We have shown that, to all orders in s, all multiparticle production processes in dilute-dense collisions are expres- sible in terms of dipoles and quadrupoles only in the large-Nc limit. In particular, this includes the case where particles are emitted with a large rapidity difference, along with un-tagged emissions or gaps in between. We would like to point out that, in the case of strongly ordered gluon emissions, this result was ﬁrst obtained in Ref. [37]. Our derivation extends the result to the general case of noneikonal gluon, quark, or antiquark emissions. VI. CONCLUSION In conclusion, we ﬁnd that in the large-Nc limit, all multiparticle production processes in p þ A-type colli- sions up to all orders in s can be described in terms of only dipoles and quadrupoles under the color glass con- densate framework, excluding cases with large rapidity intervals or gaps between the measured particles. This conclusion can very possibly lead us to an effective kt factorization at small x for multiple-jet production pro- cesses in high-energy scatterings within a dilute-dense system. This effective kt factorization involves two funda- mental objects, namely, the dipole and the quadrupole, and it can only work in the large-Nc limit. Only in the large-Nc limit can one get rid of all of the higher-point functions, which are presumably new objects. Provided we under- stand both dipoles and quadrupoles well, we will be able to predict any multiple-jet production processes up to correc- tions of order 1 N2c by using this effective kt factorization. DOMINGUEZ et al. dipole or a quadrupole, and thus higher-order virtual graphs can only generate more dipoles. Let us take the DIS dijet as an example, where we add a one-gluon loop in both the amplitude and complex conjugate amplitude, as shown in Fig. 15. Using the Fierz identity, it is obvious that the addition of the virtual gluons in the dijet processes does not introduce higher-point functions in the large-Nc limit. We can therefore conclude that for any multiple-jet graphs, under the framework of saturation physics, the dipole and quadrupole are the only objects that appear in a physical multiple-jet production process in the large-Nc limit. One should note that so far our proof does not apply to multiparticle production processes with large rapidity intervals or gaps between the measured particles (all jets are produced in the same rapidity region). For such situ- ations, one needs to consider higher-order diagrams (and at least re-sum those that contain a logarithmic enhance- ment). In the following section, we explain that our proof holds to all orders in s, which encompasses those situ- ations where particles are emitted with large rapidity differences. We have shown that, to all orders in s, all multiparticle production processes in dilute-dense collisions are expres- sible in terms of dipoles and quadrupoles only in the large-Nc limit. In particular, this includes the case where particles are emitted with a large rapidity difference, along with un-tagged emissions or gaps in between. We would like to point out that, in the case of strongly ordered gluon emissions, this result was ﬁrst obtained in Ref. [37]. Our derivation extends the result to the general case of noneikonal gluon, quark, or antiquark emissions. B. Higher-order s corrections As brieﬂy mentioned earlier, within the framework of the dilute-dense factorization, the next-to-leading order s corrections of the single inclusive production cross sec- tions [25] in the large-Nc limit do not involve higher-point functions. Here we would like to generalize this conclusion up to all order. There are two classes of graphs at higher order, namely, the virtual graphs and real graphs. In terms of color structure in the coordinate space, these two classes of graphs are actually similar. They both introduce an additional gluon at a new coordinate. The only difference is that the additional gluon in the real graphs goes through a cut and is produced in the ﬁnal state. For the real graphs, we can follow the above discussion on the multiple-jet productions, and integrate over an arbitrary number of ﬁnal-state jets and arrive at the conclusion that all real graphs in the large-Nc limit can only involve dipoles and quadrupoles. For the virtual graphs, we can easily see that the large-Nc limit requires the additional gluon within a V. PROOF OF THE GENERAL CASE These observations reduce signiﬁcantly the number of cases we have to consider to show that the inductive step in our proof indeed works. Suppose that all the correlators needed to describe all processes with k particles in the ﬁnal state in the large-Nc limit are dipoles and quadrupoles. Now consider a planar diagram for a process with k þ 1 particles in the ﬁnal state: remove one gluon and consider the representation of the leading Nc piece of the remaining diagram in terms of only fundamental Wilson lines. Since it is a planar diagram corresponding to a process with The argument used in the previous section to go from processes with two particles in the ﬁnal state to processes with three particles in the ﬁnal state can be generalized in a straightforward manner to an arbitrary number of particles in the ﬁnal state. In order to do so in a consistent matter we will show by induction that adding a new particle to the ﬁnal state does not change the fact that the leading terms in the large-Nc limit are given in terms of only dipoles and quadrupoles. We ﬁnd it convenient to focus ﬁrst on the 034007-11 PHYSICAL REVIEW D 87, 034007 (2013) DOMINGUEZ et al. APPENDIX: LARGE-Nc EVALUATION OF CORRELATORS IN THE MCLERRAN-VENUGOPALAN MODEL In this appendix we show how one can compute general n-point correlators under the framework of the McLerran- Venugopalan model [38] in the large-Nc limit. The formal- ism we will employ is the one introduced in Refs. [39,40], which has also been successfully used to compute the full finite-Nc expressions for several correlators in Refs. [5,41–43]. This was done explicitly for correlators involving four fundamental Wilson lines (two quark-antiquark pairs) in Refs. [5,40,41], where only two overall singlet states are available and the corresponding transition matrix takes the following form: 1 2 Fðx1; x0 1; x0 2; x2Þ CFðLx1x0 1 þ Lx0 2x2Þ þ 1 2Nc Fðx1; x0 1; x0 2; x2Þ 1 A; (A2) M ¼ CFðLx1x2 þ Lx0 2x0 1Þ þ 1 2Nc Fðx1; x2; x0 2; x0 1Þ 1 2 Fðx1; x2; x0 2; x0 1Þ 0 @ M ¼ CFðLx1x2 þ Lx0 2x0 1Þ þ 1 2Nc Fðx1; x2; x0 2; x0 1Þ 1 2 Fðx1; x2; x0 2; x0 1Þ 0 @ M ¼ CFðLx1x2 þ Lx0 2x0 1Þ þ 1 2Nc Fðx1; x2; x0 2; x0 1Þ 1 2 Fðx1; x2; x0 2; x0 1Þ 0 @ (A2) with Fðx1; x2; x0 2; x0 1Þ ¼ Lx1x0 2 Lx1x0 1 þ Lx2x0 1 Lx2x0 2. One must perform an ordered integral of the longitu- dinal coordinates and then sum over n. Since in a McLerran-Venugopalan–like Gaussian model the longi- tudinal dependence of the correlations factors out, the ordered integral is equal to 1 n! times the full integral. From there it is easy to see that the nth powers appearing above become exponentials and the large-Nc formula for the quadrupole is recovered. 2 1 1 2 1 1 2 1 2 2 If one naively takes the large-Nc limit the matrix becomes diagonal, which is consistent with the fact that each color transition is suppressed by a factor of 1=N2c. For correlators where the singlet structure is not the same at both ends of the longitudinal extent, it is not appropri- ate to take the large-Nc limit at the level of the transition matrix since different singlet states receive different weights in the ﬁnal calculation. For example, for the quadrupole calculation in Ref. UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . This work was supported in part by the U.S. Department of Energy under the contracts DOE OJI Grant No. DE- SC0002145 and Polish NCN Grant No. DEC-2011/01/B/ ST2/03915. F. D.’s research is supported by the European Research Council under the Advanced Investigator Grant ERC-AD-267258. C. M.’s research is supported by the European Research Council Grant HotLHC ERC- 2011-StG-279579. A. M. S. is supported by the Sloan Foundation. The general strategy consists in expanding the Wilson lines, and then taking advantage of the fact that the only nontrivial correlation is the average of two gauge ﬁelds by using Wick’s theorem. The elementary correlator of two ﬁelds takes the form g2 ShA c ðzþ; xÞA d ðz0þ; yÞixg ¼ cdðzþ z0þÞ2xgðzþÞLxy; (A1) g2 ShA c ðzþ; xÞA d ðz0þ; yÞixg ¼ cdðzþ z0þÞ2xgðzþÞLxy; (A1) where Lxy can be written in terms of a two-dimensional massless propagator (see Ref. [5]). In order to be able to re-sum these two-point contractions it is necessary to pay close attention to the color algebra. Only overall singlet states need to be considered: one can therefore calculate the matrix indicating the possible transitions between such states, and then diagonalize it. ACKNOWLEDGMENTS We thank F. Gelis, A. Kovner, A. H. Mueller, R. Venugopalan, and F. Yuan for discussions and comments. 034007-12 PHYSICAL REVIEW D 87, 034007 (2013) APPENDIX: LARGE-Nc EVALUATION OF CORRELATORS IN THE MCLERRAN-VENUGOPALAN MODEL [5] one can see that the relevant combination of matrix elements for the nth-order term of the expansion takes the form ðMnÞ11 þ NcðMnÞ21, where the nondiagonal component appears multiplied by a factor of Nc. This way of taking the large-Nc limit at the matrix level can be generalized to more complicated correlators, with similar results. One can ﬁnd an ordering of the singlet states such that all the information necessary to ﬁnd the large-Nc version of the correlator is in a lower diagonal matrix organized in blocks, in which going away from the diagonal lowers the power of Nc of the corre- sponding matrix element. Here we describe this procedure for the case of the six-point correlator of the form 1 Nc hTrðU1Uy 2 U3Uy 4 U5Uy 6 Þi. One can see from the explicit expression for the matrix M that M11 and M22 have one additional factor of Nc when compared to M12 and M21. When taking the leading Nc term of the elements of Mn it is easy to see that ðMnÞ11 ¼ Mn 11 and ðMnÞ22 ¼ Mn 22, while ðMnÞ21 includes only terms with one factor of M21 and n 1 factors of M11 or M22, which is the same as taking M12 ¼ 0 right from the begin- ning (M11 and M22 can also be replaced by their large-Nc versions). The fact that the nondiagonal element enters only once signals that there was only one color transition. For such a system of three quarks and three antiquarks there are six singlet states available, corresponding to the six ways of pairing quarks with antiquarks. The proper way to organize the states in the matrix representation is accord- ing to the power of Nc of the overlap with the ﬁnal singlet state. In terms of the graphical representation introduced in Ref. [40], the six singlet states correspond, in that particu- lar order, to the topologies shown in Fig. 16, where the number of fermion loops gives the power of Nc associated with the overlap. One can easily see that ðMnÞ21 ¼ M21 X n1 k¼0 Mk 11Mnk1 22 ; ¼ M21 Mn 11 Mn 22 M11 M22 : (A3) (A3) 034007-13 FIG. 16. Graphical representation of the six topologies involved in the calculation. The power of Nc associated with each conﬁguration is equal to the number of fermion loops. DOMINGUEZ et al. APPENDIX: LARGE-Nc EVALUATION OF CORRELATORS IN THE MCLERRAN-VENUGOPALAN MODEL PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) DOMINGUEZ et al. FIG. 16. Graphical representation of the six topologies involved in the calculation. The power of Nc associated with each conﬁguration is equal to the number of fermion loops. In the large-Nc limit only transitions to states corresponding to a higher power of Nc are allowed, which—thanks to the speciﬁc order chosen for the singlet states—means that one can divide the corresponding transition matrix into blocks, and all of the blocks above the diagonal can be neglected. The corresponding transition matrix M then takes the following form: M ¼ M1 0 0 M4 M2 0 0 M5 M3 0 BB@ 1 CCA; (A4) (A4) where the matrices along the diagonal are diagonal, and the off-diagonal matrices have one power of Nc less than the ones along the diagonal. It is not difﬁcult to calculate the nth power of this matrix: where the matrices along the diagonal are diagonal, and the off-diagonal matrices have one power of Nc less than the ones along the diagonal. It is not difﬁcult to calculate the nth power of this matrix: Mn ¼ Mn 1 0 0 Pn1 i¼0 Mi 2M4Mni1 1 Mn 2 0 Pn2 i¼0 Pni2 j¼0 Mi 3M5Mj 2M4Mnij2 1 Pn1 i¼0 Mi 3M5Mni1 2 Mn 3 0 BB@ 1 CCA: (A5) (A5) One can easily see that the relevant matrix elements for the evaluation of the desired correlator are of the form One can easily see that the relevant matrix elements for the evaluation of the desired correlator are of the form the elements of Mn in the ﬁrst column. Following this observation, we drop completely all contributions from the second column (and second row), which correspond to the second topology proportional to Nc. the elements of Mn in the ﬁrst column. Following this observation, we drop completely all contributions from the second column (and second row), which correspond to the second topology proportional to Nc. 1 1 Nc Nc Nc N2c Mn 1 0 0 0 0 0 0 BBBBBBBBBBB@ 1 CCCCCCCCCCCA : (A6) 1 1 Nc Nc Nc N2c Mn 1 0 0 0 0 0 0 BBBBBBBBBBB@ 1 CCCCCCCCCCCA : (A6) This singles out the ﬁrst column of Mn. UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . By plugging these matrix elements back into the above equations, transforming the nth powers into exponentials, and including the tadpole contributions, one gets where Fijkl ¼ Lik Ljk þ Ljl Lil. By plugging these matrix elements back into the above equations, transforming the nth powers into exponentials, and including the tadpole contributions, one gets 1 Nc hTr½U1Uy 2 U3Uy 4 U5Uy 6 i ¼ e123456 F1234 F1324 ½e1234 e1432e56 F1256 F1526 ½e1256 e1652e34 F3456 F3546 ½e3456 e3654e12 þ F1234F1456 e123456 F1324G e143256 F1324F1546 þ e163254 F1546G þ F1256F2543 e123456 F1526G e163452 F1526F2453 þ e163254 F2453G þ F3456F1236 e123456 F3546G e123654 F3546F1326 þ e163254 F1326G ; (A11) (A11) where ij ¼ 2ðLii þ Ljj 2LijÞ and G ¼ L12 þ L34 þ L56 L16 L32 L54. where ij ¼ 2ðLii þ Ljj 2LijÞ and G ¼ L12 þ L34 þ L56 L16 L32 L54 j jj j In this expression one can easily recognize three different types of contributions, depending on the number of transitions between the singlet states. The ﬁrst term clearly comes from the ﬁrst row in Eq. (A9) and corresponds to the totally elastic part with no color transitions, the following three terms come from the second, third, and fourth rows of Eq. (A9) and correspond to terms with only one transition, and the rest of the terms come from the last row of Eq. (A9) and are associated with terms that have two transitions between singlet states. In this expression one can easily recognize three different types of contributions, depending on the number of transitions between the singlet states. The ﬁrst term clearly comes from the ﬁrst row in Eq. (A9) and corresponds to the totally elastic part with no color transitions, the following three terms come from the second, third, and fourth rows of Eq. (A9) and correspond to terms with only one transition, and the rest of the terms come from the last row of Eq. (A9) and are associated with terms that have two transitions between singlet states. 034007-15 UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . UNIVERSALITY OF MULTIPARTICLE PRODUCTION IN . . . The ﬁrst column of its nth power is mn 11 m21 Pn1 i¼0 mi 11mni1 22 m31 Pn1 i¼0 mi 11mni1 33 m41 Pn1 i¼0 mi 11mni1 44 P4 k¼2 m5kmk1 Pn2 i¼0 Pni2 j¼0 mi 11mj kkmnij2 55 0 BBBBBBBBBBB@ 1 CCCCCCCCCCCA ; (A8) which can be rewritten as mn 11 m21 m11m22 ½mn 11 mn 22 m31 m11m33 ½mn 11 mn 33 m41 m11m44 ½mn 11 mn 44 P4 k¼2 m5kmk1 mn 11 ðm11mkkÞðm11m55Þ þ mn kk ðmkkm11Þðmkkm55Þ þ mn 55 ðm55mkkÞðm55m11Þ 0 BBBBBBBBBBBB@ 1 CCCCCCCCCCCCA : (A9) p mn 11 m21 Pn1 i¼0 mi 11mni1 22 m31 Pn1 i¼0 mi 11mni1 33 m41 Pn1 i¼0 mi 11mni1 44 P4 k¼2 m5kmk1 Pn2 i¼0 Pni2 j¼0 mi 11mj kkmnij2 55 0 BBBBBBBBBBB@ 1 CCCCCCCCCCCA ; (A8) which can be rewritten as mn 11 m21 Pn1 i¼0 mi 11mni1 22 m31 Pn1 i¼0 mi 11mni1 33 m41 Pn1 i¼0 mi 11mni1 44 P4 k¼2 m5kmk1 Pn2 i¼0 Pni2 j¼0 mi 11mj kkmnij2 55 0 BBBBBBBBBBB@ 1 CCCCCCCCCCCA ; (A8) (A8) mn 11 m21 m11m22 ½mn 11 mn 22 m31 m11m33 ½mn 11 mn 33 m41 m11m44 ½mn 11 mn 44 P4 k¼2 m5kmk1 mn 11 ðm11mkkÞðm11m55Þ þ mn kk ðmkkm11Þðmkkm55Þ þ mn 55 ðm55mkkÞðm55m11Þ 0 BBBBBBBBBBBB@ 1 CCCCCCCCCCCCA : (A9) (A9) Now, as in the previous case, these expressions appear in the ﬁnal result summed over n with a factor of 1 n! due to the ordering in the longitudinal coordinate. Therefore the nth powers become exponentials. Now, as in the previous case, these expressions appear in the ﬁnal result summed over n with a factor of 1 n! due to the ordering in the longitudinal coordinate. Therefore the nth powers become exponentials. One can easily calculate explicitly the relevant leading-Nc piece of the transition matrix for the case of interest. This reduced version of the transition matrix then takes the form M ¼ 1 2 NcðL12 þ L34 þ L56Þ 0 0 0 0 F1243 NcðL14 þ L32 þ L56Þ 0 0 0 F1265 0 NcðL16 þ L34 þ L52Þ 0 0 F3465 0 0 NcðL12 þ L36 þ L54Þ 0 0 F1465 F2534 F1263 NcðL16 þ L32 þ L54Þ 0 BBBBBBBB@ 1 CCCCCCCCA ; (A10) (A10) where Fijkl ¼ Lik Ljk þ Ljl Lil. APPENDIX: LARGE-Nc EVALUATION OF CORRELATORS IN THE MCLERRAN-VENUGOPALAN MODEL Since M1 and M2 are diagonal matrices it is easy to see that the elements of M in the second column never enter the expressions for to the second topology proportional to Nc. As a 5 5 matrix, M has the form M ¼ m11 0 0 0 0 m21 m22 0 0 0 m31 0 m33 0 0 m41 0 0 m44 0 0 m52 m53 m54 m55 0 BBBBBBBB@ 1 CCCCCCCCA : (A7) As a 5 5 matrix, M has the form As a 5 5 matrix, M has the form 1 1 Nc Nc Nc N2c Mn 0 0 0 BBBBBB@ CCCCCCA : (A6) This singles out the ﬁrst column of Mn. Since M1 and M2 are diagonal matrices it is easy to see that the elements of M in the second column never enter the expressions for M ¼ m11 0 0 0 0 m21 m22 0 0 0 m31 0 m33 0 0 m41 0 0 m44 0 0 m52 m53 m54 m55 0 BBBBBBBB@ 1 CCCCCCCCA : (A7) (A7) This singles out the ﬁrst column of Mn. Since M1 and M2 are diagonal matrices it is easy to see that the elements of M in the second column never enter the expressions for M m31 0 m33 0 0 m41 0 0 m44 0 0 m52 m53 m54 m55 BBBBB@ CCCCCA : (A7) This singles out the ﬁrst column of Mn. Since M1 and M2 are diagonal matrices it is easy to see that the elements of M in the second column never enter the expressions for M m31 0 m33 0 0 m41 0 0 m44 0 0 m52 m53 m54 m55 BBBB@ CCCCA : (A7) This singles out the ﬁrst column of Mn. Since M1 and M2 are diagonal matrices it is easy to see that the elements of M in the second column never enter the expressions for M m31 0 m33 0 0 m41 0 0 m44 0 0 m52 m53 m54 m55 BBBB@ CCCCA : (A7) 034007-14 PHYSICAL REVIEW D 87, 034007 (2013) DOMINGUEZ et al. [1] A. Kovner and U. A. Wiedemann, Phys. Rev. D 64, 114002 (2001). [23] A. Dumitru, A. Hayashigaki, and J. Jalilian-Marian, Nucl. Phys. A765, 464 (2006). [24] T. Altinoluk and A. Kovner, Phys. Rev. D 83, 105004 (2011). [2] F. Gelis, E. Iancu, J. Jalilian-Marian, and R. Venugopalan, Annu. Rev. Nucl. Part. Sci. 60, 463 (2010). [3] J. Jalilian-Marian and Y. V. Kovchegov, Phys. Rev. D 70, 114017 (2004); 71, 079901E (2005). [25] G. A. Chirilli, B.-W. Xiao, and F. Yuan, Phys. Rev. Lett. 108, 122301 (2012); G. A. Chirilli, B.-W. Xiao, and F. Yuan, Phys. Rev. D 86, 054005 (2012). [4] C. Marquet, Nucl. Phys. A796, 41 (2007). [26] A. H. Mueller and S. Munier, Nucl. Phys. A893, 43 (2012). [5] F. Dominguez, C. Marquet, B.-W. Xiao, and F. Yuan, Phys. Rev. D 83, 105005 (2011). [6] F. Dominguez, A. H. Mueller, S. Munier, and B. W. Xiao, Phys. Lett. B 705, 106 (2011). [27] F. Dominguez, B.-W. Xiao, and F. Yuan, Phys. Rev. Lett. 106, 022301 (2011). [28] F. Gelis and J. Jalilian-Marian, Phys. Rev. D 67, 074019 (2003). [7] A. Dumitru and J. Jalilian-Marian, Phys. Rev. D 82, 074023 (2010). [29] E. Braidot for the STAR collaboration, arXiv:1005.2378. [8] E. Iancu and D. N. Triantafyllopoulos, J. High Energy Phys. 11 (2011) 105; 04 (2012) 025. [30] A. Adare et al. (PHENIX Collaboration), Phys. Rev. Lett. 107, 172301 (2011). [9] A. Dumitru, J. Jalilian-Marian, T. Lappi, B. Schenke, and R. Venugopalan, Phys. Lett. B 706, 219 (2011). [31] B.-W. Xiao and F. Yuan, Phys. Rev. Lett. 105, 062001 (2010); Phys. Rev. D 82, 114009 (2010). R. Venugopalan, Phys. Lett. B 706, 219 (2011) [10] A. H. Mueller, arXiv:hep-ph/0111244. [10] A. H. Mueller, arXiv:hep-ph/0111244. [32] N. N. Nikolaev, W. Schafer, B. G. Zakharov, and V. R. Zoller, Phys. Rev. D 72, 034033 (2005). [11] A. H. Mueller, Nucl. Phys. B335, 115 (1990); B415, 373 (1994). [12] L. D. McLerran and R. Venugopalan, Phys. Rev. D 59, 094002 (1999). [33] R. Baier, A. Kovner, M. Nardi, and U. A. Wiedemann, Phys. Rev. D 72, 094013 (2005). [34] J. L. Albacete and C. Marquet, Phys. Rev. Lett. 105, 162301 (2010). [13] Y. V. Kovchegov and L. D. McLerran, Phys. Rev. D 60, 054025 (1999); 62, 019901E (2000). [35] A. Stasto, B.-W. Xiao, and F. Yuan, Phys. Lett. B 716, 430 (2012). 14] A. H. Mueller, Nucl. Phys. B558, 285 (1999). 034007-15 PHYSICAL REVIEW D 87, 034007 (2013) PHYSICAL REVIEW D 87, 034007 (2013) DOMINGUEZ et al. DOMINGUEZ et al. [15] C. Marquet, B.-W. Xiao, and F. Yuan, Phys. Lett. B 682, 207 (2009). [36] T. Lappi and H. Ma¨ntysaari, arXiv:1209.2853. [16] I. Arsene et al. (BRAHMS Collaboration), Phys. Rev. Lett. 93, 242303 (2004). [37] A. Kovner and M. Lublinsky, J. High Energy Phys. 11 (2006) 083. [17] J. Adams et al. (STAR Collaboration), Phys. Rev. Lett. 97, 152302 (2006). [38] L. D. McLerran and R. Venugopalan, Phys. Rev. D 49, 2233 (1994); 49, 3352 (1994). [18] A. Dumitru and J. Jalilian-Marian, Phys. Rev. Lett. 89, 022301 (2002). [39] H. Fujii, Nucl. Phys. A709, 236 (2002). [40] J. P. Blaizot, F. Gelis, and R. Venugopalan, Nucl. Phys. A743, 57 (2004). [19] Y. V. Kovchegov and A. H. Mueller, Nucl. Phys. B529, 451 (1998). [41] F. Dominguez, C. Marquet, and B. Wu, Nucl. Phys. A823, 99 (2009). [20] A. Dumitru and L. D. McLerran, Nucl. Phys. A700, 492 (2002). [42] Y. V. Kovchegov, J. Kuokkanen, K. Rummukainen, and H. Weigert, Nucl. Phys. A823, 47 (2009). [21] Y. V. Kovchegov and K. Tuchin, Phys. Rev. D 65, 074026 (2002). [43] C. Marquet and H. Weigert, Nucl. Phys. A843, 68 (2010). [22] C. Marquet, Nucl. Phys. B705, 319 (2005). 034007-16
https://openalex.org/W4207044619	https://archpublichealth.biomedcentral.com/counter/pdf/10.1186/s13690-021-00776-0	English	null	Determinants of adverse birth outcomes among women delivered in public hospitals of Ethiopia, 2020	Archives of public health	2,022	cc-by	11,114	RESEARCH Open Access Determinants of adverse birth outcomes among women delivered in public hospitals of Ethiopia, 2020 Muktar Abadiga* , Getu Mosisa, Reta Tsegaye, Adugna Oluma, Eba Abdisa and Tilahun Bekele © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abadiga et al. Archives of Public Health (2022) 80:12 https://doi.org/10.1186/s13690-021-00776-0 Abadiga et al. Archives of Public Health (2022) 80:12 https://doi.org/10.1186/s13690-021-00776-0 Abstract Background: Adverse birth outcome is a common health problem consisting of several health effects involving pregnancy and the newborn infant. Infants with one or more adverse birth outcomes are at greater risk for mortality and a variety of health and developmental problems. Factors such as the age of the mother, antepartum hemorrhage, history of abortion, gestational age, anemia, and maternal undernutrition have predisposed the mother to adverse birth outcome. For appropriate prevention of the adverse birth outcomes, data pertaining to determinants of adverse birth outcomes are important. Therefore, this study was aimed to assess the determinants of adverse birth outcomes among women who give birth in public hospitals of western Ethiopia. Methods: An institutional-based unmatched prospective case-control study was conducted from February 15 to April 15, 2020, in selected public hospitals of western Ethiopia. From mothers who gave birth in public hospitals of Wollega zones, 165 cases and 330 controls were selected. Mothers with adverse birth outcomes were cases and mothers without adverse birth were controls. Data was collected by structured interviewer-administered questionnaires. In addition to the interview, the data collectors abstracted clinical data by reviewing the mother and the babies’ medical records. The collected data were entered into Epi info version 7 and exported to SPSS version 21 for analysis. Finally, multivariable logistic regression was used to identify determinants of adverse birth outcomes at P-value < 0.05. Results: A total of 495 mothers (165 cases and 330 controls) were included in the study with a mean age of 28.48 + 5.908. Low ANC visit (AOR = 3.92: 95% CI; 1.86, 8.2), premature rupture of membrane (AOR = 2.83: 95% CI; 1.72,4.64), being Anemic (AOR = 2: 95% CI; 1.16,3.44), pregnancy induced-hypertension (AOR = 2.3:95% CI; 1.4,3.85), not getting dietary supplementation (AOR = 2.47:95% CI; 1.6,3.82), and physical abuse (AOR = 2.13: 95% CI; 1.05,4.32) were significantly associated with the development of the adverse birth outcome. Conclusion: Low antenatal care visit, being anemic, premature rupture of membrane, pregnancy-induced hypertension, not getting dietary supplementation, and physical abuse were determinants of adverse birth outcomes. The clinicians should play a pivotal role to improve antenatal care follow up, counsel, and supplement recommended diets and minimize violence and abuse during pregnancy. Background Although most pregnancy and childbirth are a joyful ex- perience for most women, it sometimes ends up in ad- verse birth outcome [1]. The adverse birth outcome is a multifactorial outcome that mainly includes preterm birth, low birth weight, stillbirth, macrosomia, congenital anomaly, and infant/neonatal death. It is a common health problem consisting of a several health effects in- volving pregnancy and the newborn infant [2]. Preterm birth is a live birth before 37 completed weeks of gesta- tion and is the leading cause of neonatal mortality [3]. Low birthweight is infants who weigh less than 2500 g (about 5 ½ pounds) at birth [4]. On the other hand, still- birth is when the infant died in the womb after 28 weeks of gestation [5]. The etiologies of adverse birth outcomes are multifac- torial and not completely understood yet. Studies have shown that sociodemographic factors such as the age of the mother [12, 13, 16, 17, 23, 24], educational level [23], level of income [25, 26], and place of residence [16, 17, 26] showed association with adverse birth outcome. Obstetric factors such as the previous history of adverse birth outcome [13, 14], antepartum hemorrhage [14], history of abortion [16], gestational age [11], pregnancy interval [27], obstetric complications [11, 15–17] and the number of parities [12, 17] were associated with adverse birth outcome. Other factors such as the presence of chronic diseases [13], hypertensive disorder during preg- nancy [14], anemia [15, 27], no dietary counseling [17, 27], low antenatal care visit [12–15, 17, 27], substance use [25] and malaria [28] were also associated with ad- verse birth outcome. Factors associated with adverse birth outcomes are not the same across different cul- tures and socioeconomic statuses within a society. Birth outcomes are measures of health at birth and their magnitude is dramatically decreased in the past 40 years. However; there is still a large gap between devel- oping and developed countries [2]. Globally, about 15 million babies are born prematurely each year, from which Sub-Saharan Africa and South Asia account for more than 60% [6, 7]. Of all births occurred worldwide, about 20 million are low birth weight and 15% of this occurs in sub-Saharan Africa [4]. Worldwide, nearly 3 million stillbirths occur annually, 98% of which occur in developing countries [8]. On the other hand, more than 45% of under-fives death occur within the first 28 days of life [9]. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 17 Abadiga et al. Archives of Public Health (2022) 80:12 Plain English Summary The adverse birth outcome is a common health problem consisting of several health effects involving pregnancy and the newborn infant. Birth outcomes are measures of health at birth and their magnitude is dramatically decreased in the past 40 years. However; there is still a large gap between developing and developed countries. Infants with one or more adverse birth outcomes are at greater risk for mortality and a variety of health and developmental problems. For appropriate prevention of adverse birth outcomes, data pertaining to determinants of adverse birth outcomes are important. An institutional-based unmatched prospective case-control study was conducted from February 15 to April 15, 2020, in selected public hospitals of western Ethiopia. In this study, low Antenatal care (ANC) visits, being anemic, premature rupture of membrane, pregnancy-induced hypertension, not getting dietary supplementation, and physical abuse were determinants of adverse birth outcomes. The clinicians should play a pivotal role to improve ANC follow up, counsel, and supplement recommended diets and minimize violence and abuse during pregnancy. Keywords: Determinant, Adverse birth outcome, Mothers, Western Ethiopia outcomes are more likely to experience complications like respiratory, immunologic, nervous system, and be- havioral problems [7, 18]. Preterm birth and low birth weight are critical determinants of child survival, disabil- ities, stunting, and long-term adverse consequences [19]. Low birth weight infants may suffer the risk of develop- ing many complications such as heart problems, anemia, chronic lung disorders, growth retardation, and inhibited cognitive developments [20]. Similarly, preterm birth im- poses infants to physical and neurological problems which may lead them to lifelong disabilities [21]. Pre- term complications are also the leading cause of ap- proximately 27% of neonatal deaths which accounts for about four million every year [22]. Study design, setting and population Study design, setting and population An institutional-based unmatched prospective case- control study was conducted from February 15 to April 15, 2020, in selected public hospitals of Wollega zones, western Ethiopia. According to the 2010 Central Statis- tical Agency (CSA) report, the total population of Wol- lega zone is 3,345,675 (52% females and 48% males) [29]. The Wollega zone is administratively divided into 4 zones, namely, the East Wollega zone, Horro Guduru Wollega zone, West Wollega zone, and Kellem Wollega zone. In the Wollega zone, there are 2 comprehensive specialized hospitals, 9 general hospitals, 13 primary hospitals, 102 health centers, and 401 health posts. This study was conducted in six randomly selected public hospitals found in Wollega zones, namely; Nekemte spe- cialized hospital, Arjo hospital, Wollega university spe- cialized hospital, Shambu general hospital, Gimbi general hospital, and Nedjo general hospital. An esti- mated total number of 85,345 births have been regis- tered annually in the zones. The source population was all mothers who gave birth in public hospitals of the four Wollega zones. The study population was all mothers who give birth at the six selected public hospitals of Wollega zones during the study period. Eligible cases were selected consecutively and the con- secutive two controls were selected until the required sample size was achieved. The number of cases and con- trols were proportionally allocated to each hospital based on the number of mothers who gave birth at each of the selected hospitals in 4 months before the data collection time. Then, the average number of mothers expected to give birth per 2 months in each of the respective hospi- tals was estimated. Accordingly, we included 42 cases and 84 controls in Nekemte specialized Hospital, 38 cases and 76 controls in Wollega university specialized hospital, 27 cases and 54 controls in Gimbi general Hos- pital, 22 cases and 44 controls in Shambu general hos- pital, 29 cases and 58 controls in Nedjo hospital and 23 cases and 46 controls in Arjo Hospital. All mothers who gave birth at the selected hospitals of Wollega zones were included in the study. Women with induced termination of pregnancy for medical reasons, women with a serious general medical condition, un- known last menstrual period (LMP) or not reliable ultra- sonography and unable to communicate were excluded. Sample size determination and sampling techniques Sample size determination and sampling techniques The Sample size was calculated using the double popula- tion proportion using EPI-Info 3.5.1 statistical software. The proportion of low educational status, advanced ma- ternal age, rural residence, age at first sex ≤14, and the age difference between the partner at first intercourse ≥5 years was used to determine the sample size from a study done in Debre Tabor town [23]. Maternal age 45– 54 years was chosen as an independent variable since it brought a higher sample size among other computed ex- planatory variables. The assumptions for the sample size calculation were as follows: minimum detectable odds ratio of 2, a confidence level of 95% (Zα/2 = 1.96), power of 80% (Zβ = 0.80), a case to control ratio of 1:2, and proportion of case among an exposed group of 22.0% [23]. After adding a 10% non-response rate, the total cal- culated sample size was 543 (181 cases and 362 controls). Background The overall prevalence of adverse birth out- comes is 25.7% in rural India [10], 15.61% in Zimbabwe [11], and 10.8% in rural Uganda [12]. In Ethiopia, the prevalence of adverse birth outcome is 18.3% in Hawassa town [13], 23% in Gondor university hospital [14], 32.5% in Dessie referral hospital [15], 18.2% in Butajira general hospital [16], and 31.8% in North Wollo zone [17]. For appropriate planning and implementing proper in- terventions to prevent the adverse birth outcome, data pertaining to determinants of adverse birth outcome are very important. Although there are studies on the Infants with one or more adverse birth outcomes are at greater risk for mortality and a variety of health and developmental problems [7]. Babies with adverse birth Abadiga et al. Archives of Public Health (2022) 80:12 Page 3 of 17 various forms of adverse birth outcomes, there is limited data on the overall adverse birth outcomes in Ethiopia. To the best of our knowledge, this is the first study to assess adverse birth outcomes and their associated fac- tors in the western part of Ethiopia. Therefore, this study was aimed to assess the determinants of adverse birth outcomes among women who gave birth in public hospi- tals of western Ethiopia. The finding of this study will help the health care providers in planning health inter- ventions to improve the wellbeing of children and women. were mothers without adverse birth outcomes (≥37 ges- tational weeks at birth, live birth, weight ≥2.5 kg at birth and not greater than 4 kg, no congenital anomaly, the appropriate size for gestational age, and no neonatal death). Study design, setting and population Cases were mothers with at least one adverse birth out- come (preterm birth; < 37 gestational weeks at birth, low birth weight; the weight of < 2.5 kg at birth, stillbirth; in- fant died in the womb or during the intrapartum period after 28 weeks of gestation, macrosomia; birth-weight over 4000 g irrespective of gestational age, birth defect/ congenital anomaly; structural changes in one or more parts of the infant’s body that are present at birth, neo- natal death; the death of infant or neonates within 28 days of life, small for gestational age; birth weight below the 10th percentile for the gestational age). Controls Obstetrics related characteristics of the respondents Obstetrics related characteristics of the respondents Regarding the number of births, 106 (64.24%) of the cases and 217 (65.76%) of the controls have less than 3 births. The birth space for 54 (32.73%) cases and 121 (36.67%) of controls were more than 23 months. The majority of the study participants, 118 (71.52%) of the cases 247 (74.85%) of the controls were used family planning before the current pregnancy. The majority of participants, 125 (75.76%) of the cases and 260 (78.79%) of the controls were planned their pregnancy. About 60 Results Socio-demographic characteristics of the respondents From the total of 543 eligible participants (181 cases and 362 controls), 495 mothers (165 cases and 330 controls) were participated in the study making a response rate of 91.2%. The mother’s age was lying between 15 and 44 years with a mean of 28.48 + 5.908 SD. The majority of the study participants, 141 (85.45%) of the cases and 286 (86.67%) of the controls were Oromo. Regarding the reli- gion of the respondents, 86 (52.12%) of the cases, and 160 (48.48%) of the controls were protestant. About 153 (92.73%) of the cases and 311 (94.24%) of the controls were married. Regarding educational status, 47 (28.48%) of the cases and 108 (32.7%) of controls had a diploma and above. The majority of the study participants, 104 (63.03%) of the cases and 212 (64.24%) of controls were from rural. More than half of the study participants, 105 (63.64%) of the cases and 189 (57.27%) of the controls were married at an age between 18 and 23 years. Regard- ing monthly income, 37(22.42%) of the cases and 75 (22.73%) of the controls get a monthly income of 2001– 3000 Ethiopian birr. Concerning family size, 107 (64.85%) of the cases, and 197(59.7%) of the controls have 4–6 family size (Table 1). Data collection tool and procedure Six Master of Science qualified mid- wives supervised the overall data collection process. Data collection tool and procedure Data collection tool and procedure Data was collected by structured interviewer- administered questionnaire adapted from the Ethiopian Demographic and Health Survey (EDHS) and other simi- lar studies [11–14, 16, 17, 23, 27, 30]. The outcome vari- able was the adverse birth outcome and the independent variables were socio-demographic variables, obstetric re- lated factors, pre-conception related factors, nutritional and dietary related factors, behavior-related factors, mothers’ history of pre-existing medical illness, and health facility-related factors. The interview was held in a separate room after a woman is stabilized and ready to be discharged. In addition to the interview, the data col- lectors abstracted clinical data by reviewing the mother Abadiga et al. Archives of Public Health (2022) 80:12 Abadiga et al. Archives of Public Health (2022) 80:12 Page 4 of 17 Page 4 of 17 Low birth weight: is when a baby’s birth weight is below 2500 g or below 2.5 kg. Macrosomia: is defined as birth- weight over 4 kg irrespective of gestational age. Stillbirth: is when the infant died in the womb or during the intra- partum period after 28 weeks of gestation. Birth defect/ congenital anomaly: is when there are structural changes in one or more parts of the infant’s body that are present at birth. Adverse birth outcome: is when a woman met at least one of the above conditions. No adverse birth outcome: Those women who gave normal live birth without the above-mentioned abnormal birth outcome [5]. Perceived stress: A mother is considered as stressed if perceived stress score is above mean and not stressed if below calculated mean value of scores. Danger sign of pregnancy: A mother is considered as having danger sign if she developed at least one from nine danger signs stated in the world health organization (WHO) guide list and no danger sign if no any sign developed. and the babies’ medical records. Anthropometric mea- surements were also made for both the mothers and the newborns. The baby’s weight was measured by the data collectors within 15 min after delivery using a calibrated digital scale and rounded to the nearest 0.1 g. Mothers’ mid-upper arm circumference was measured using flex- ible non-stretchable tape measure. Maternal hemoglobin level was reviewed from mothers’ cards to determine anemia. The data was collected by 12 trained BSc mid- wives recruited from other hospitals not included in this study for 2 months. Data quality control The questionnaire was translated to the local language Afan Oromo and then back to English by two different language experts to check for consistency. Five percent of the questionnaire (27 participants) was pre-tested at the same study area 5 days before data collection and modification was made based on the pre-test result. The ratio of a case to control used in the pretest was 1:2 (9 cases and 18 controls). Five days of training on the ob- jectives of the study, sampling technique, ethical consid- eration, and data collection techniques was given for data collectors and supervisor. Continuous follow-up and supervision of data collection were made by the su- pervisors. The collected data were checked by the super- visor daily for completeness. Data processing and analysis The collected data were coded, cleaned, and entered into Epi info version 7 and exported to SPSS version 21 for analysis. Descriptive statistics like frequencies and per- centages were performed as univariate analysis. In this study, cases were coded as 1, and controls were coded as 0 for analysis. Bivariable logistic regression analysis was used to assess the unadjusted effect of each independent variable on the dependent variable. Variables that have a P-value less than 0.25 were entered into a multivariable logistic regression model. A multivariable logistic regres- sion model was fitted to identify independent determi- nants of adverse birth outcomes. Model fitness was tested with Hosmer-Lemeshow goodness of fit test and omnibus tests of model coefficients. Variance inflation factor (VIF) and tolerance test were also used to check multicollinearity. Adjusted odds ratio (AOR) with a 95% confidence interval (CI) was calculated to determine the strength of an association. P-value < 0.05 was considered statistically significant in multivariable logistic regression. Operational definitions More than half, 88 (53.33%) of the cases and 196 (59.39%) of the controls were delivered through SVD. Concerning the previous history of ad- verse birth outcomes, 131 (79.4%) of the cases, and 271 (82.12%) of the controls had no history of adverse birth outcomes. About 106 (64.24%) of the cases and 280 (84.85%) of the controls didn’t experience PROM, and most of the labor started spontaneously for 125 (75.76%) of cases and 269 (81.52%) of controls (Table 2). Operational definitions Preterm birth: is when a baby is born before 37 com- pleted weeks of gestation but after 28 weeks of gestation. Abadiga et al. Archives of Public Health (2022) 80:12 Page 5 of 17 Page 5 of 17 Table 1 Socio-demographic characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) Variables Control n (%) Cases n (%) Total n (%) Ethnicity of mothers Oromo 286 (86.67) 141 (85.45) 427 (86.3) Amhara 35 (10.6) 21 (12.73) 56 (11.3) Gurage 9 (2.73) 3 (1.82) 12 (2.4) Religion of mothers Protestant 160 (48.48) 86 (52.12) 246 (49.7) Orthodox 86 (26.06) 39 (23.64) 125 (25.3) Catholic 13 (3.94) 8 (4.85) 21 (4.2) Muslim 62 (18.78) 29 (17.58) 91 (18.4) Others 9 (2.73) 3 (1.82) 12 (2.4) Marital status Married 311 (94.24) 153 (92.73) 464 (93.7) Single 6 (1.82) 3 (1.82) 9 (1.8) Divorced 9 (2.73) 6 (3.64) 15 (3) Widowed 4 (1.21) 3 (1.82) 7 (1.4) Educational status Unable to read and write 72 (21.8) 40 (24.24) 112 (22.6) Completed grade 1–8 84 (25.5) 42 (25.45) 126 (25.5) Completed grade 9–12 66 (20) 36 (21.82) 102 (20.6) Diploma and above 108 (32.7) 47 (28.48) 155 (31.3) Residence Rural 212 (64.24) 104 (63.03) 316 (63.8) Urban 118 (35.76) 61 (36.97) 179 (36.2) Age category 15–24 89 (26.97) 43 (26.06) 132 (26.7) 25–34 176 (53.33) 93 (56.36) 269 (54.3) 35–44 65 (19.7) 29 (17.58) 94 (19) Age at 1st marriage < 18 years 110 (33.33) 51 (30.91) 161 (32.5) 18–23 years 189 (57.27) 105 (63.64) 294 (59.4) > 23 years 31 (9.39) 9 (5.45) 40 (8.1) Monthly income < 1000 birr 67 (20.3) 40 (24.24) 107 (21.6) 1000–2000 birr 54 (16.36) 31 (18.79) 85 (17.2) 2001–3000 birr 75 (22.73) 37 (22.42) 112 (22.6) 3001–4000 birr 59 (17.88) 28 (16.97) 87 (17.6) > 4000 75 (22.73) 29 (17.58) 104 (21) Family size 1–3 98 (29.7) 37 (22.42) 135 (27.3) 4–6 197 (59.7) 107 (64.85) 304 (61.4) > 6 35 (10.7) 21 (12.73) 56 (11.3) Table 1 Socio-demographic characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) (36.4%) of the cases and 182 (55.15%) of the controls were attended ANC more than four times. Concerning danger signs, 123 (74.55%) of the cases and 267 (80.9%) of the controls were didn’t experienced danger signs during pregnancy. Mothers medical history and health facility related characteristics of respondents The majority of the participants, 288 (87.27%) of the controls and 124 (75.15%) of the cases and nearly all, 322 (97.58%) of the controls and 160 (96.97%) of the cases had no history of hypertension and cardiac dis- eases respectively. Three hundred eleven (94.24%) of the controls and 154 (93.33%) of the cases were HIV nega- tive. The majority of participants, 291 (88.18%) of the controls and 120 (72.73%) of the cases, 317 (96.06%) of the controls and 154 (93.33%) of the cases, and 289 (87.58%) of the controls and 154 (93.3%) of the cases had no history of Anemia, malaria, and STI respectively. y p y Concerning pregestational BMI, 258 (78.18%) of the controls, and 122 (73.94%) of the cases had 18–24 kg/ m2. The majority of the participants, 281 (85.15%) of the controls and 108 (64.45%) of the cases had no pregnancy-induced hypertension. More than half, 192 (58.18%) of the controls and 111 (67.27%) of the cases had a height of < 150 cm. Regarding the time of ANC visit, 90 (54.55%) of the cases and 167 (50.6%) of the controls visited in the 2nd trimester. Almost all,321 (97.27%) of the controls and 163 (98.79%) of the cases delivered a single child. Labor duration has lasted for 8– 15 h in 143 (43.33%) of the controls and 65 (39.4%) of the cases. The majority of participants, 217 (65.75%) of the controls and 102 (61.82%) of the cases travel less than 1 h to reach the health facility (Table 3). Social and behavioral related characteristics of respondents The majority of the participants, 281 (85.15%) of the controls and 128 (77.58%) of the cases had no history of substance use. Three hundred seven (93.03%) of the controls and 144 (87.27%) of the cases had no history of physical abuse and harassment. Almost all, 313 (94.85%) of the controls and 159 (96.36%) of the cases were not used traditional medicine, and about 254 (76.97%) of the controls and 113 (68.48%) of the cases didn’t experience stress during pregnancy (Table 4). Abadiga et al. Bivariable logistic regression analysis of respondents In the bivariable analysis, age at first marriage, income, family size, birth space, ANC attendance, danger sign of pregnancy, mode of delivery, premature rupture of the Social and behavioral related characteristics of respondents Archives of Public Health (2022) 80:12 Page 6 of 17 Table 2 Obstetrics related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) Variables Control n (%) Cases n (%) Total n (%) Number of births < 3 217 (65.76) 106 (64.24) 323 (65.3) 3–5 81 (24.55) 47 (28.48) 128 (25.9) > 5 32 (9.70) 12 (7.27) 44 (8.9) Duration between births 1st pregnancy 79 (23.94) 49 (29.70) 128 (25.9) < 12 months 16 (4.85) 10 (6.06) 26 (5.3) 12–17 months 26 (7.88) 10 (6.06) 36 (7.3) 18–23 months 88 (26.67) 42 (25.45) 130 (26.3) > 23 months 121 (36.67) 54 (32.73) 175 (35.4) Family planning utilization Ye 247 (74.85) 118 (71.52) 365 (73.7) No 83 (25.15) 47 (28.48) 130 (26.3) Plan of pregnancy Yes 260 (78.79) 125 (75.76) 385 (77.8) No 70 (21.21) 40 (24.24) 110 (22.2) Antenatal care attendance One time 16 (4.85) 33 (20) 49 (9.9) Two times 46 (13.94) 33 (20) 79 (16) Three times 86 (26.06) 39 (23.6) 125 (25.3) > =4 times 182 (55.15) 60 (36.4) 242 (48.9) Danger sign of pregnancy Yes 63 (19.1) 42 (25.45) 105 (21.2) No 267 (80.9) 123 (74.55) 390 (78.8) Mode of delivery Spontaneous vaginal delivery 196 (59.39) 88 (53.33) 284 (57.4) Forceps 18 (5.45) 10 (6.06) 28 (5.7) Cesarean section 66 (20) 43 (26.06) 109 (22) Vacuum delivery 45 (13.64) 20 (12.12) 65 (13.1) Destructive delivery 5 (1.52) 4 (2.42) 9 (1.8) History of adverse birth outcome Yes 59 (17.88) 34 (20.6) 93 (18.8) No 271 (82.12) 131 (79.4) 402 (81.2) Premature rupture of membrane Yes 50 (15.15) 59 (35.76) 109 (22) No 280 (84.85) 106 (64.24) 386 (78) Labor started S t l 269 (81 52) 125 (75 76) 394 (79 6) Table 2 Obstetrics related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) Table 2 Obstetrics related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 d t l 330) membrane, labor duration, history of hypertension, anemia, malaria, STI, pregnancy-induced hypertension, the height of mother, dietary counseling, dietary supple- mentation, time of ANC attendance, time to reach a Bivariable logistic regression analysis of respondents In the bivariable analysis, age at first marriage, income, family size, birth space, ANC attendance, danger sign of pregnancy, mode of delivery, premature rupture of the Bivariable logistic regression analysis of respondents Bivariable logistic regression analysis of respondents Abadiga et al. Bivariable logistic regression analysis of respondents Archives of Public Health (2022) 80:12 Page 8 of 17 Table 3 Mothers medical history and health facility related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) Variables Control n (%) Cases n (%) Total n (%) 2nd trimester 167 (50.6) 90 (54.55) 257 (51.9) 3rd Trimester 38 (11.51) 29 (17.57) 67 (13.5) Number of children born Multiple 9 (2.73) 2 (1.21) 11 (2.2) Singleton 321 (97.27) 163 (98.79) 484 (97.8) Labour duration category < 8 h 111 (33.64) 59 (35.76) 170 (34.3) 8-15 h 143 (43.33) 65 (39.4) 208 (42) 16-22 h 61 (18.48) 34 (20.6) 95 (19.2) > 22 h 15 (4.55) 7 (4.24) 22 (4.4) Time to reach health facility < 1 h 217 (65.75) 102 (61.82) 319 (64.4) 1-2 h 70 (21.21) 35 (21.21) 105 (21.2) > 2 h 43 (13.03) 28 (16.97) 71 (14.3) Table 3 Mothers medical history and health facility related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) health facility, history of substance abuse, physical abuse and stress were significantly associated with the adverse birth outcome at a p-value 0.25 (Table 5). developing adverse birth outcomes were 2.83 times higher among mothers who developed PROM than mothers who do not develop (AOR = 2.83: 95% CI; 1.72,4.64). The odds of developing adverse birth out- comes were 2 times higher among anemic mothers than their counterparts (AOR = 2.0: 95% CI; 1.16,3.44). The study revealed that mothers who developed pregnancy- induced hypertension were 2.3 times higher odds of de- veloping adverse birth outcomes than their counterparts (AOR = 2.3:95% CI;1.4,3.85). The odds of developing ad- verse birth outcomes were 2.47 times higher among mothers who didn’t get dietary supplementation than mothers who got dietary supplementation (AOR = 2.47: 95% CI;1.6,3.82). Mothers who experienced physical abuse were 2.13 times more likely to develop adverse birth outcomes than mothers who do not exposed to physical abuse/harassment (AOR = 2.13: 95% CI; 1.05,4.32) (Table 6). Multivariable logistic regression analysis In a multivariable logistic regression analysis, ANC at- tendance, PROM, being Anemic, PIHTN, dietary supple- mentation, and physical abuse/harassment were significantly associated with the adverse birth outcome. The odds of developing adverse birth outcomes were 3.92 times higher in mothers who had one-time ANC at- tendance than mothers who attended four and above times (AOR = 3.92: 95% CI; 1.86, 8.2). The odds of Table 4 Social and behavioral related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) 495; Cases: 165 and controls: 330) Variables Control n (%) Cases n (%) Total n (%) History of substance abuse Yes 49 (14.85) 37 (22.42) 86 (17.4) No 281 (85.15) 128 (77.58) 409 (82.6) Physical abuse Yes 23 (6.97) 21 (12.73) 44 (8.9) No 307 (93.03) 144 (87.27) 451 (91.1) Use of traditional Medicine Yes 17 (5.15) 6 (3.64) 23 (4.6) No 313 (94.85) 159 (96.36) 472 (95.4) Experienced stress Yes 76 (23.03) 52 (31.52) 128 (25.9) No 254 (76.97) 113 (68.48) 367 (74.1) Variables Control n (%) Cases n (%) Total n (%) Bivariable logistic regression analysis of respondents Archives of Public Health (2022) 80:12 Page 7 of 17 Table 3 Mothers medical history and health facility related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) Variables Control n (%) Cases n (%) Total n (%) History of diabetes mellitus Yes 21 (6.36) 10 (6.06) 31 (6.3) No 309 (93.64) 155 (93.94) 464 (93.7) History of hypertension Yes 42 (12.73) 41 (24.85) 83 (16.8) No 288 (87.27) 124 (75.15) 412 (83.2) History of Cardiac disease Yes 8 (2.42) 5 (3.03) 13 (2.6) No 322 (97.58) 160 (96.97) 482 (97.4) Status of Human immunodeficiency virus status Positive 9 (2.73) 4 (2.42) 13 (2.6) Negative 311 (94.24) 154 (93.33) 465 (93.9) Unknown 10 (3.03) 7 (4.24) 17 (3.4) Anemia Yes 39 (11.82) 45 (27.27) 84 (17) No 291 (88.18) 120 (72.73) 411 (83) Malaria Yes 13 (3.94) 11 (6.67) 24 (4.8) No 317 (96.06) 154 (93.33) 471 (95.2) Sexually transmitted infection Yes 41 (12.42) 12 (7.27) 53 (10.7) No 289 (87.58) 154 (93.3) 442 (89.3) Pregestational body mass index < 18 kg/m2 35 (10.6) 17 (10.3) 52 (10.5) 18–24 kg/m2 258 (78.18) 122 (73.94) 380 (76.8) > 24 kg/m2 37 (11.21) 26 (15.75) 63 (12.7) Pregnancy induced hypertension Yes 49 (14.85) 57 (34.55) 106 (21.4) No 281 (85.15) 108 (65.45) 389 (78.6) Maternal Height > =150 cm 138 (41.82) 54 (32.73) 192 (38.8) < 150 cm 192 (58.18) 111 (67.27) 303 (61.2) Dietary counselling Yes 216 (65.45) 92 (55.76) 308 (62.2) No 114 (34.55) 73 (44.24) 177 (37.8) Mid-upper arm circumference < 23 cm 97 (29.4) 49 (29.7) 146 (29.5) = > 23 cm 233 (70.6) 116 (70.3) 349 (70.5) Dietary supplementation Yes 236 (71.52) 78 (47.27) 314 (63.4) N ( ) ( ) ( ) Table 3 Mothers medical history and health facility re zones, 2020 (n = 495; Cases: 165 and controls: 330) Variables Control n (% History of diabetes mellitus Yes 21 (6.36) No 309 (93.64) History of hypertension Yes 42 (12.73) No 288 (87.27) History of Cardiac disease Yes 8 (2.42) No 322 (97.58) Status of Human immunodeficiency virus status Positive 9 (2.73) Negative 311 (94.24) Unknown 10 (3.03) Anemia Yes 39 (11.82) No 291 (88.18) Malaria Yes 13 (3.94) No 317 (96.06) Sexually transmitted infection Yes 41 (12.42) No 289 (87.58) Pregestational body mass index < 18 kg/m2 35 (10.6) 18–24 kg/m2 258 (78.18) > 24 kg/m2 37 (11.21) Pregnancy induced hypertension Yes 49 (14.85) No 281 (85.15) Maternal Height > =150 cm 138 (41.82) < 150 cm 192 (58.18) Dietary counselling Yes 216 (65.45) No 114 (34.55) Mid-upper arm circumference < 23 cm 97 (29.4) = > 23 cm 233 (70.6) Dietary supplementation Yes 236 (71.52) No 94 (28.48) Time of 1st antenatal care visit 1st Trimester 125 (37.88) Table 3 Mothers medical history and health facility related characteristics of mothers attending birth at public hospitals of Wollega zones, 2020 (n = 495; Cases: 165 and controls: 330) thers attending birth at public hospitals of Wollega Cases n (%) Total n (%) 10 (6.06) 31 (6.3) 155 (93.94) 464 (93.7) 41 (24.85) 83 (16.8) 124 (75.15) 412 (83.2) 5 (3.03) 13 (2.6) 160 (96.97) 482 (97.4) 4 (2.42) 13 (2.6) 154 (93.33) 465 (93.9) 7 (4.24) 17 (3.4) 45 (27.27) 84 (17) 120 (72.73) 411 (83) 11 (6.67) 24 (4.8) 154 (93.33) 471 (95.2) 12 (7.27) 53 (10.7) 154 (93.3) 442 (89.3) 17 (10.3) 52 (10.5) 122 (73.94) 380 (76.8) 26 (15.75) 63 (12.7) 57 (34.55) 106 (21.4) 108 (65.45) 389 (78.6) 54 (32.73) 192 (38.8) 111 (67.27) 303 (61.2) 92 (55.76) 308 (62.2) 73 (44.24) 177 (37.8) 49 (29.7) 146 (29.5) 116 (70.3) 349 (70.5) 78 (47.27) 314 (63.4) 87 (52.73) 181 (36.6) 46 (27.88) 171 (34.5) others attending birth at public hospitals of Wollega Cases n (%) Total n (%) 10 (6.06) 31 (6.3) 155 (93.94) 464 (93.7) 41 (24.85) 83 (16.8) 124 (75.15) 412 (83.2) 5 (3.03) 13 (2.6) 160 (96.97) 482 (97.4) 4 (2.42) 13 (2.6) 154 (93.33) 465 (93.9) 7 (4.24) 17 (3.4) 45 (27.27) 84 (17) 120 (72.73) 411 (83) 11 (6.67) 24 (4.8) 154 (93.33) 471 (95.2) 12 (7.27) 53 (10.7) 154 (93.3) 442 (89.3) 17 (10.3) 52 (10.5) 122 (73.94) 380 (76.8) 26 (15.75) 63 (12.7) 57 (34.55) 106 (21.4) 108 (65.45) 389 (78.6) 54 (32.73) 192 (38.8) 111 (67.27) 303 (61.2) 92 (55.76) 308 (62.2) 73 (44.24) 177 (37.8) 49 (29.7) 146 (29.5) 116 (70.3) 349 (70.5) 78 (47.27) 314 (63.4) 87 (52.73) 181 (36.6) 46 (27.88) 171 (34.5) Abadiga et al. Discussion Globally, adverse birth outcomes remained the major public health problems in the world, particularly in de- veloping countries where the accessibility and utilization of health care facilities are limited. This study was con- ducted to identify determinants of adverse birth out- comes among mothers who gave birth at public hospitals of Wollega zones. In this study, Antenatal care attendance, premature rupture of the membrane, being anemic, pregnancy-induced hypertension, dietary sup- plementation, and physical abuse were significantly asso- ciated with the development of the adverse birth outcome. Abadiga et al. Discussion Archives of Public Health (2022) 80:12 Page 9 of 17 Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) Characteristics Adverse birth outcome COR (95%) CI P- value Control n (%) Cases n (%) Ethnicity Oromo 286 (86.67) 141 (85.45) 1 Amhara 35 (10.6) 21 (12.73) 1.21 (0.68,2.17) 0.5 Gurage 9 (2.73) 3 (1.82) 0.67 (0.18,2.54) 0.56 Religion of the mother Protestant 160 (48.48) 86 (52.12) 1 Orthodox 86 (26.06) 39 (23.64) 0.84 (0.53,1.34) 0.47 Catholic 13 (3.94) 8 (4.85) 1.14 (0.457,2.87) 0.77 Muslim 62 (18.78) 29 (17.58) 0.87 (0.52,1.45) 0.6 Others 9 (2.73) 3 (1.82) 0.62 (0.16,2.35) 0.48 Marital status Married 311 (94.24) 153 (92.73) 1 Single 6 (1.82) 3 (1.82) 1.02 (0.25,4.12) 0.98 Divorced 9 (2.73) 6 (3.64) 1.35 (0.47,3.87) 0.57 Widowed 4 (1.21) 3 (1.82) 1.52 (0.34,6.89) 0.58 Educational status Unable to read and write 72 (21.8) 40 (24.24) 1.28 (0.76,2.14) 0.35 Completed grade 1–8 84 (25.5) 42 (25.45) 1.15 (0.69,1.9) 0.59 Completed grade 9–12 66 (20) 36 (21.82) 1.25 (0.74,2.13) 0.4 Diploma and above 108 (32.7) 47 (28.48) 1 Residence Rural 212 (64.24) 104 (63.03) 1 Urban 118 (35.76) 61 (36.97) 1.05 (0.7,1.55) 0.79 Age category 15–24 years 89 (26.97) 43 (26.06) 1.08 (0.6,1.9) 0.78 25-34 years 176 (53.33) 93 (56.36) 1.18 (0.71,1.96) 0.5 35–44 years 65 (19.7) 29 (17.58) 1 Age at 1st marriage < 18 years 110 (33.33) 51 (30.91) 1.6 (0.71,3.6) 0.26 18–23 years 189 (57.27) 105 (63.64) 1.9 (0.88,4.17) 0.1* > 23 years 31 (9.39) 9 (5.45) 1 Monthly income < 1000 birr 67 (20.3) 40 (24.24) 1.54 (0.86,2.76) 0.14* 1000–2000 birr 54 (16.36) 31 (18.79) 1.48 (0.8,2.75) 0.21* 2001–3000 birr 75 (22.73) 37 (22.42) 1.27 (0.7,2.28) 0.41 3001–4000 birr 59 (17.88) 28 (16.97) 1.23 (0.66,2.28) 0.52 > 4000 birr 75 (22.73) 29 (17.58) 1 Family size 1–3 98 (29.7) 37 (22.42) 1 4–6 197 (59.7) 107 (64.85) 1.44 (0.92,2.25) 0.11* > 6 35 (10.7) 21 (12.73) 1.59 (0.82,3.07) 0.17* Number of live births Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) Page 10 of 17 Abadiga et al. Discussion Archives of Public Health (2022) 80:12 Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) Characteristics Adverse birth outcome COR (95%) CI P- value Control n (%) Cases n (%) < 3 217 (65.76) 106 (64.24) 1 3–5 81 (24.55) 47 (28.48) 1.18 (0.77,1.82) 0.43 > 5 32 (9.70) 12 (7.27) 0.77 (0.38,1.55) 0.46 Duration between pregnancy 1st pregnancy 79 (23.94) 49 (29.70) 1.39 (0.86,2.24) 0.18* < 12 months 16 (4.85) 10 (6.06) 1.4 (0.6,3.28) 0.44 12–17 months 26 (7.88) 10 (6.06) 0.86 (0.39,1.9) 0.71 18–23 months 88 (26.67) 42 (25.45) 1.07 (0.657,1.74) 0.78 > 23 months 121 (36.67) 54 (32.73) 1 Family planning utilization Yes 247 (74.85) 118 (71.52) 1 No 83 (25.15) 47 (28.48) 1.18 (0.78,1.8) 0.427 Plan of pregnancy Yes 260 (78.79) 125 (75.76) 1 No 70 (21.21) 40 (24.24) 1.19 (0.76,1.85) 0.44 Antenatal care attendance One time 16 (4.85) 33 (20) 6.25 (3.22,12.16) < 0.001* Two times 46 (13.94) 33 (20) 2.17 (1.27,3.71) 0.004* Three times 86 (26.06) 39 (23.6) 1.37 (0.85,2.22) 0.19* > =4 times 182 (55.15) 60 (36.4) 1 Danger sign of pregnancy Yes 63 (19.1) 42 (25.45) 1.45 (0.93,2.26) 0.1* No 267 (80.9) 123 (74.55) 1 Mode of delivery Spontaneous vaginal delivery 196 (59.39) 88 (53.33) 1 Forceps 18 (5.45) 10 (6.06) 1.24 (0.55,2.79) 0.61 Cesarean section 66 (20) 43 (26.06) 1.45 (0.92,2.29) 0.11* Vacuum delivery 45 (13.64) 20 (12.12) 0.99 (0.55,1.77) 0.97 Destructive delivery 5 (1.52) 4 (2.42) 1.78 (0.47,6.79) 0.39 History of adverse birth outcome Yes 59 (17.88) 34 (20.6) 1.19 (0.745,1.91) 0.46 No 271 (82.12) 131 (79.4) 1 Premature rupture of membrane Yes 50 (15.15) 59 (35.76) 3.12 (2.01,4.83) < 0.001* No 280 (84.85) 106 (64.24) 1 Labour started Spontaneously 269 (81.52) 125 (75.76) 1 Induced 61 (18.48) 40 (24.24) 1.41 (0.9,2.21) 0.135* History of diabetes mellitus Yes 21 (6.36) 10 (6.06) 0.95 (0.44,2.07) 0.9 No 309 (93.64) 155 (93.94) 1 History of hypertension Page 11 of 17 Abadiga et al. Discussion Archives of Public Health (2022) 80:12 Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) Characteristics Adverse birth outcome COR (95%) CI P- value Control n (%) Cases n (%) Multiple 9 (2.73) 2 (1.21) 1 Singleton 321 (97.27) 163 (98.79) 2.28 (0.48,10.7) 0.29 Labor duration category < 8 h 111 (33.64) 59 (35.76) 1 8-15 h 143 (43.33) 65 (39.4) 0.85 (0.56,1.32) 0.47 16-22 h 61 (18.48) 34 (20.6) 1.05 (0.62,1.77) 0.86 > 22 h 15 (4.55) 7 (4.24) 0.87 (0.34,2.27) 0.79 Time to reach health facility < 1 h 217 (65.75) 102 (61.82) 0.72 (0.42,1.23) 0.23* 1-2 h 70 (21.21) 35 (21.21) 0.77 (0.41,1.43) 0.41 > 2 h 43 (13.03) 28 (16.97) 1 History of substance abuse Yes 49 (14.85) 37 (22.42) 1.66 (1.03,2.67) 0.037* No 281 (85.15) 128 (77.58) 1 Physical abuse Yes 23 (6.97) 21 (12.73) 1.95 (1.04,3.63) 0.036* No 307 (93.03) 144 (87.27) 1 Use of traditional medicine Yes 17 (5.15) 6 (3.64) 0.69 (0.27,1.79) 0.45 No 313 (94.85) 159 (96.36) 1 Experienced stress Yes 76 (23.03) 52 (31.52) 1.54 (1.01,2.33) 0.043* No 254 (76.97) 113 (68.48) 1 COR Crude Odd Ratio, CI Confidence Interval shows significant at P-value < 0.25 Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) In this study, the odds of developing adverse birth out- comes were 3.92 times higher in mothers who have one- time ANC attendance than mothers who attended four and above times (AOR = 3.92: 95% CI; 1.86, 8.2). This finding is congruent with the results from Gonder [14], Hawassa [13], Dessie [15], North Wollo [17], and Tigray [27], in which adverse birth outcome is higher among mothers with limited ANC visit. This might be due to the fact that counseling given during the ANC follow up helps the mothers to get information on diet, danger signs of pregnancy, and pregnancy-related complication. It is known that ANC visits during pregnancy help to monitor the wellbeing of the fetus. The lack of adequate ANC visits during pregnancy decreases the chance of identifying risks of adverse birth outcomes and providing appropriate interventions for its prevention. Discussion Archives of Public Health (2022) 80:12 Page 11 of 17 Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) Characteristics Adverse birth outcome COR (95%) CI P- value Control n (%) Cases n (%) Yes 42 (12.73) 41 (24.85) 2.27 (1.4,3.66) 0.001 No 288 (87.27) 124 (75.15) 1 History of cardiac disease Yes 8 (2.42) 5 (3.03) 1.26 (0.405,3.91) 0.69 No 322 (97.58) 160 (96.97) 1 Human immunodeficiency virus status Positive 9 (2.73) 4 (2.42) 1 Negative 311 (94.24) 154 (93.33) 1.1 (0.34,3.67) 0.86 Unknown 10 (3.03) 7 (4.24) 1.57 (0.34,7.22) 0.56 Anemia Yes 39 (11.82) 45 (27.27) 2.8 (1.73,4.5) < 0.001* No 291 (88.18) 120 (72.73) 1 Malaria Yes 13 (3.94) 11 (6.67) 1.74 (0.76,3.97) 0.18* No 317 (96.06) 154 (93.33) 1 Sexually transmitted infection Yes 41 (12.42) 12 (7.27) 0.55 (0.28,1.08) 0.08* No 289 (87.58) 154 (93.3) 1 Pregestational body mass index < 18 kg/m2 35 (10.6) 17 (10.3) 0.97 (0.52,1.81) 0.93 18–24 kg/m2 258 (78.18) 122 (73.94) 1.45 (0.67,3.1) 0.34 > 24 kg/m2 37 (11.21) 26 (15.75) 1 Pregnancy induced hypertension Yes 49 (14.85) 57 (34.55) 3.03 (1.95,4.7) < 0.001* No 281 (85.15) 108 (65.45) 1 Maternal height > =150 cm 138 (41.82) 54 (32.73) 1 < 150 cm 192 (58.18) 111 (67.27) 1.47 (0.99,2.18) 0.051* Dietary counselling Yes 216 (65.45) 92 (55.76) 1 No 114 (34.55) 73 (44.24) 1.5 (1.03,2.2) 0.036* Mid upper arm circumference < 23 cm 97 (29.4) 49 (29.7) 1.01 (0.67,1.53) 0.94 = > 23 cm 233 (70.6) 116 (70.3) 1 Dietary supplementation Yes 236 (71.52) 78 (47.27) 1 No 94 (28.48) 87 (52.73) 2.8 (1.9,4.13) < 0.001* Time of 1st antenatal care visit 1st Trimester 125 (37.88) 46 (27.88) 1 2nd trimester 167 (50.6) 90 (54.55) 1.46 (0.96,2.24) 0.078* 3rd Trimester 38 (11.51) 29 (17.57) 2.07 (1.15,3.74) 0.015* Number of born child Table 5 Bivariable logistic regression analysis of adverse birth outcome among women gave birth at public hospitals of Wollega Zones, west Ethiopia, 2020 (n = 495; Cases: 165 and controls: 330) (Continued) Page 12 of 17 Abadiga et al. Discussion Women should access to information related to nutrition and danger signs of pregnancy and ANC follow up will also help a woman seek early treatment for pregnancy related problems. Therefore, the regional health bureau should give more attention on expanding antenatal care espe- cially in the rural areas, and create awareness about fam- ily planning methods for all women of reproductive age group both in rural and urban setting. g g The odds of developing adverse birth outcomes were 2 times higher among anemic mothers than their counter- parts (AOR = 2.0: 95% CI; 1.16,3.44). This finding is con- sistent with the study done in Dessie [15], Tigray [27], India [31]. Anemia, which is common during pregnancy, particularly in developing countries, highly contributed to the adverse birth outcome. Anemia may lead to de- creased blood flow to the placenta and results in pre- term labor which in turn results in preterm birth and other adverse birth outcomes. Also, anemia may cause hypoxia which can induce fetal stress, which stimulates the production of the corticotrophin-releasing hormone (CRH) leading to preterm labor. Iron deficiency may also increase the risk of maternal infections which can again stimulate the production of CRH predisposing to pre- term birth. Therefore, evaluation of hemoglobin level Abadiga et al. Discussion Archives of Public Health (2022) 80:12 Page 13 of 17 Table 6 Multivariate logistic regression analysis of adverse birth outcome among mothers gave birth at public hospitals of Wollega Zones, west Ethiopia,2020 (n = 495; Cases: 165 and controls: 330) Characteristics Adverse birth outcome COR (95%) CI AOR (95%) CI P-value Control n (%) Cases n (%) Age at 1st marriage 110 (33.33) 51 (30.91) < 18 years 189 (57.27) 105 (63.64) 1.6 (0.71,3.6) 1.44 (0.57,3.68) 0.44 18–23 years 31 (9.39) 9 (5.45) 1.9 (0.88,4.17) 1.98 (0.83,4.7) 0.12 > 23 years 110 (33.33) 51 (30.91) 1 1 Monthly income < 1000 birr 67 (20.3) 40 (24.24) 1.54 (0.86,2.76) 1.03 (0.5,2.09) 0.93 1000–2000 birr 54 (16.36) 31 (18.79) 1.48 (0.8,2.75) 1.48 (0.7,3.14) 0.31 2001–3000 birr 75 (22.73) 37 (22.42) 1.27 (0.7,2.28) 1.29 (0.64,2.59) 0.48 3001–4000 birr 59 (17.88) 28 (16.97) 1.23 (0.66,2.28) 1.24 (0.59,2.6) 0.56 > 4000 birr 75 (22.73) 29 (17.58) 1 1 Family size 1–3 98 (29.7) 37 (22.42) 1 1 4–6 197 (59.7) 107 (64.85) 1.44 (0.92,2.25) 1.56 (0.87,2.82) 0.13 > 6 35 (10.7) 21 (12.73) 1.59 (0.82,3.07) 1.84 (0.79,4.3) 0.15 Birth space 1st pregnancy 79 (23.94) 49 (29.70) 1.39 (0.86,2.24) 1.22 (0.68,2.17) 0.49 < 12 months 16 (4.85) 10 (6.06) 1.4 (0.6,3.28) 1.35 (0.47,3.85) 0.57 12–17 months 26 (7.88) 10 (6.06) 0.86 (0.39,1.9) 0.4 (0.16,1.0) 0.07 18–23 months 88 (26.67) 42 (25.45) 1.07 (0.657,1.74) 1.1 (0.64,1.96) 0.7 > 23 months 121 (36.67) 54 (32.73) 1 Antenatal care attendance One time 16 (4.85) 33 (20) 6.25 (3.22,12.16) 3.92 (1.86,8.2) < 0.001 Two times 46 (13.94) 33 (20) 2.17 (1.27,3.71) 1.6 (0.9,2.9) 0.1 Three times 86 (26.06) 39 (23.6) 1.37 (0.85,2.22) 1.33 (0.78,2.26) 0.29 > =4 times 182 (55.15) 60 (36.4) 1 1 Danger sign of pregnancy Yes 63 (19.1) 42 (25.45) 1.45 (0.93,2.26) 1.58 (0.94,2.65) 0.08 No 267 (80.9) 123 (74.55) 1 1 Mode of delivery Spontaneous vaginal delivery 196 (59.39) 88 (53.33) 1 1 Forceps 18 (5.45) 10 (6.06) 1.24 (0.55,2.79) 1.05 (0.35,3.1) 0.93 Cesarean section 66 (20) 43 (26.06) 1.45 (0.92,2.29) 1.21 (0.65,2.27 0.54 Vacuum delivery 45 (13.64) 20 (12.12) 0.99 (0.55,1.77) 1.1 (0.52,2.23) 0.83 Destructive delivery 5 (1.52) 4 (2.42) 1.78 (0.47,6.79) 1.27 (0.25,6.5) 0.77 Premature rupture of membrane Yes 50 (15.15) 59 (35.76) 3.12 (2.01,4.83) 2.83 (1.72,4.64) < 0.001 No 280 (84.85) 106 (64.24) 1 1 Labour started Spontaneously 269 (81.52) 125 (75.76) 1 1 Induced 61 (18.48) 40 (24.24) 1.41 (0.9,2.21) 0.94 (0.53,1.65) 0.82 History of hypertension Yes 42 (12.73) 41 (24.85) 2.27 (1.4,3.66) 1.16 (0.55,2.46) 0.69 Table 6 Multivariate logistic regression analysis of adverse birth outcome among mothers gave birth at public hospitals of Wollega Zones, west Ethiopia,2020 (n = 495; Cases: 165 and controls: 330) Abadiga et al. Discussion Archives of Public Health (2022) 80:12 Page 15 of 17 and dietary supplementation is highly recommended throughout pregnancy time. birth complications. The reason for this might be due to the fact that supplementation of diet during pregnancy helps the mothers and fetuses to get adequate nutrient which contributes to normal growth and prevent com- plications. When the mother’s nutritional status is poor; they will be prone to chronic infection which may lead to the activation of the maternal-fetal immune system causing preterm labor. So, health care personnel working on antenatal care should appropriately counsel the preg- nant mother on the importance of dietary supplementa- tion for the health of the mother and fetus. Premature rupture of the membrane (PROM) is one of the significant factors associated with adverse birth out- comes [32]. The finding of the study showed that the odds of developing adverse birth outcomes were 2.83 times higher among mothers who developed PROM than mothers who do not develop (AOR = 2.83: 95% CI; 1.72,4.64). The reason for this might be due to the fact that premature rupture of the membrane leads to umbil- ical cord prolapse, placental abruption, and fetal distress. PROM can lead to uterine contraction as amniotic fluid contains prostaglandin which in turn may result in ad- verse birth outcomes. Besides, PROM elevates fetal plasma interleukin-6 which may trigger preterm labor and leads to preterm delivery. PROM is also associated with lower latency from membrane rupture until deliv- ery and causes around 25–30% of all preterm deliveries. The study findings also indicated that mothers who experienced physical abuse were 2.13 times more likely to develop adverse birth outcomes than mothers who do not exposed to physical abuse (AOR = 2.13: 95% CI; 1.05,4.32). This finding is supported by the study done in Tigray [37] and Hosanna Town [38], in which phys- ical abuse and violence were associated with adverse birth outcomes. The reason for this might be physical abuse during pregnancy may lead to physical injury to the fetus, abortion, and premature rupture of the mem- brane and finally may cause adverse birth outcomes. The hypertensive disorder is one of the most common problems encountered during pregnancy and contribute to the adverse birth outcome. This study revealed that mothers who developed pregnancy-induced hyperten- sion were 2.3 times higher odds of developing adverse birth outcomes than their counterparts (AOR = 2.3:95% CI;1.4,3.85). Discussion Archives of Public Health (2022) 80:12 Page 14 of 17 Table 6 Multivariate logistic regression analysis of adverse birth outcome among mothers gave birth at public hospitals of Wollega Zones, west Ethiopia,2020 (n = 495; Cases: 165 and controls: 330) (Continued) Characteristics Adverse birth outcome COR (95%) CI AOR (95%) CI P-value Control n (%) Cases n (%) No 288 (87.27) 124 (75.15) 1 1 Anemia Yes 39 (11.82) 45 (27.27) 2.8 (1.73,4.5) 2.0 (1.16,3.44) 0.01 No 291 (88.18) 120 (72.73) 1 1 Malaria Yes 13 (3.94) 11 (6.67) 1.74 (0.76,3.97) 2.11 (0.76,5.83) 0.14 No 317 (96.06) 154 (93.33) 1 1 Sexually transmitted infection Yes 41 (12.42) 12 (7.27) 0.55 (0.28,1.08) 0.55 (0.25,1.2) 0.13 No 289 (87.58) 154 (93.3) 1 1 Pregnancy induced hypertension Yes 49 (14.85) 57 (34.55) 3.03 (1.95,4.7) 2.3 (1.4,3.85) 0.001 No 281 (85.15) 108 (65.45) 1 1 Maternal height > =150 cm 138 (41.82) 54 (32.73) 1 1 < 150 cm 192 (58.18) 111 (67.27) 1.47 (0.99,2.18) 0.99 (0.587,1.67) 0.97 Dietary counselling Yes 216 (65.45) 92 (55.76) 1 1 No 114 (34.55) 73 (44.24) 1.5 (1.03,2.2) 1.46 (0.94,2.27) 0.09 Dietary supplementation No 236 (71.52) 78 (47.27) 2.8 (1.9,4.13) 2.47 (1.6,3.82) < 0.001 Yes 94 (28.48) 87 (52.73) 1 1 Time of 1st antenatal care visit 1st Trimester 125 (37.88) 46 (27.88) 1 1 2nd trimester 167 (50.6) 90 (54.55) 1.46 (0.96,2.24) 1.45 (0.89,2.37) 0.13 3rd Trimester 38 (11.51) 29 (17.57) 2.07 (1.15,3.74) 1.76 (0.88,3.53) 0.11 Time to reach health facility < 1 h 217 (65.75) 102 (61.82) 0.72 (0.42,1.23) 0.59 (0.317,1.11) 0.1 1-2 h 70 (21.21) 35 (21.21) 0.77 (0.41,1.43) 0.8 (0.39,1.64) 0.54 > 2 h 43 (13.03) 28 (16.97) 1 1 History of substance abuse Yes 49 (14.85) 37 (22.42) 1.66 (1.03,2.67) 1.37 (0.78,2.42) 0.27 No 281 (85.15) 128 (77.58) 1 Physical abuse Yes 23 (6.97) 21 (12.73) 1.95 (1.04,3.63) 2.13 (1.05,4.32) 0.036 No 307 (93.03) 144 (87.27) 1 1 Experienced stress Yes 76 (23.03) 52 (31.52) 1.54 (1.01,2.33) 0.76 (0.42,1.36) 0.35 No 254 (76.97) 113 (68.48) 1 1 COR Crude Odd Ratio, AOR Adjusted Odd Ratio, CI Confidence Interval *shows significant at P value < 0 05 Table 6 Multivariate logistic regression analysis of adverse birth outcome among mothers gave birth at public hospitals of Wollega Zones, west Ethiopia,2020 (n = 495; Cases: 165 and controls: 330) (Continued) Abadiga et al. Abbreviations ANC: Antenatal care; AOR: Adjusted odds ratio; BMI: Body mass index; CI: Confidence interval; COR: Crude odd ratio; CSA: Central statistical agency; HIV: Human immunodeficiency virus; KG: Kilogram; LMP: Last menstrual period; PIHTN: Pregnancy-induced hypertension; PROM: Premature rupture of membrane; SPSS: Statistical package for social sciences; SVD: Spontaneous vaginal delivery; VIF: Variance inflation factor Discussion This finding is supported by a study done in Suhul hospital Shire [33] and Gonder [14] in which pregnancy-induced hypertension is a high-risk factor for adverse birth outcomes. Another prospective cohort study done in Ethiopia indicated that a higher number of adverse prenatal outcomes occurred among pregnancy-induced hypertensive women [34]. This might be due to the fact that hypertension which is oc- curred during pregnancy increase the risk of fetus intra- uterine growth restriction which further contributed to adverse birth outcomes. Hypertensive disorder can cause vascular damage to the placenta causing abruption pla- centa. When the blood pressure becomes uncontrollable, the quickest means of emptying the uterus become the choice accounting for the preterm delivery. In addition, elevated blood pressure in pregnancy compromises per- fusion to the fetus and has a medical risk of cardiovascu- lar complications for the mother. So, early screening of hypertensive disorders of pregnancy at the time of ante- natal follow up is very important, and the health care professionals especially midwives should emphasize on this issue. Acknowledgments g We acknowledge Wollega University for funding this research project. We also would like to acknowledge the Wollega zone health bureau and each hospital administrative office for their cooperation. We are also grateful to the study participants who voluntarily agreed to be interviewed and participated in the study. The study results also revealed that the odds of devel- oping adverse birth outcomes were 2.47 times higher among mothers who didn’t get dietary supplementation than their counterparts (AOR = 2.47:95% CI;1.6,3.82). A systematic review study indicated that nutrients have a profitable effect on reducing adverse birth outcomes [35]. Another study conducted in Iran [36], also con- cluded as dietary supplementation reduced maternal and Conclusions In this study, low ANC visits, being anemic, premature rupture of membrane, pregnancy-induced hypertension, not getting dietary supplementation, and physical abuse were determinants of adverse birth outcomes. The clini- cians should play a pivotal role to improve ANC follow up, counsel, and supplement recommended diets and minimize violence and abuse during pregnancy. Supplementary Information y The online version contains supplementary material available at https://doi. org/10.1186/s13690-021-00776-0. y The online version contains supplementary material available at https://doi. org/10.1186/s13690-021-00776-0. Additional file 1. Additional file 1. 71/journal.pone.0217770. 26. Kent ST, McClure LA, Zaitchik BF, Gohlke GM. Area-level risk factors for adverse birth outcomes: trends in urban and rural settings. BMC Pregnancy Childbirth. 2013;13:129 http://www.biomedcentral.com/1471-2393/13/129. 4. Department of Health and Human Services. Health resources and services administration, Maternal and Child Health Bureau. USA: Child Health; 2011. 4. Department of Health and Human Services. Health resources and services administration, Maternal and Child Health Bureau. USA: Child Health; 2011. 5. World Health Organization (WHO). International statistical classification of diseases and related health problems. Geneva: World Health Organization; 2004. 5. World Health Organization (WHO). International statistical classification of diseases and related health problems. Geneva: World Health Organization; 2004. 27. Hailemichael HT, Debelew GT, Alema HB, Weldu MG, Misgina KH. Determinants of adverse birth outcome in Tigrai region, North Ethiopia: hospital based case-control study. BMC Pediatr. 2020;20:10 https://doi.org/1 0.1186/s12887-019-1835-6. 6. Howson CP, Kinney MV, McDougall L, Lawn JE. Born too soon: preterm birth matters. Reprod Health. 2013;10(1):S1. 6. Howson CP, Kinney MV, McDougall L, Lawn JE. Born too soon: preterm birth matters. Reprod Health. 2013;10(1):S1. 28. Mikomangwa WP, OMS M, Aklillu E, Kamuhabwa AA. Adverse birth outcomes among mothers who received intermittent preventive treatment with Sulphadoxine- Pyrimethamine in the low malaria transmission region. BMC Pregnancy Childbirth. 2019;19:236 https://doi.org/10.1186/s12884-01 9-2397-1. 7. Lee AC, et al. National and regional estimates of term and preterm babies born small for gestational age in 138 low-income and middle-income countries in 2010. Lancet Glob Health. 2013;1(1):e26–36. 8. Lawn JE, Blencowe H, Waiswa P, Amouzou A, Mathers C, Hogan D, et al. Stillbirths: rates, risk factors, and acceleration towards 2030. Lancet. 2016; 387(10018):587–603. 29. Authority CS. Population and housing census of Ethiopia. Addis Ababa: Authority CS; 2012. 9. You D, Hug L, Ejdemyr S, Idele P, Hogan D, Mathers C, et al. Global, regional, and national levels and trends in under-5 mortality between 1990 and 2015, with scenario-based projections to 2030: a systematic analysis by the UN inter-agency Group for Child Mortality Estimation. Lancet. 2015;386: 2275–86. 30. Central Statistical Agency (CSA) [Ethiopia] and ICF. Ethiopia Demographic and Health Survey 2016. Addis Ababa and Rockville: CSA and ICF; 2016. 31. Kumari S, et al. Maternal and severe anemia in delivering women is associated with risk of preterm and low birth weight: a cross sectional study from Jharkhand, India. One Health. 2019;8:100098 Available from: https://doi. org/10.1016/j.onehlt.2019.100098. 10. Padhi BK, Baker KK, Dutta A, Cumming O, Freeman MC, Satpathy R, et al. Funding Pregnancy Childbirth. 2015;15:279. https://doi.org/10.1186/s12884-015-0708- g This study was funded by Wollega university. The funding organization has no role in designing and organization of the study, data collection and analysis, and result writing. 13. Tsegaye, Kassa. Prevalence of adverse birth outcome and associated factors among women who delivered in Hawassa town governmental health institutions, South Ethiopia, in 2017. Reprod Health. 2018;15:193 https://doi. org/10.1186/s12978-018-0631-3. The authors declare that no competing interests concerning the research, authorship, and/or publication of this article. The authors declare that no competing interests concerning the research, authorship, and/or publication of this article. 22. Lawn JE, Gravett MG, Nunes TM, Rubens CE, Santon C. Global report on preterm birth and still birth (1 of 7): definitions, description of the burden and opportunities to improve data. Maryland: The Johns Hopkins Bloomberg school of public health; 2010. Received: 6 August 2020 Accepted: 21 December 2021 Availability of data and materials 14. Adane, et al. Adverse birth outcomes among deliveries in Gonder university hospital, Northwest Ethiopia. BMC Pregnancy Childbirth. 2014;14:90 http:// www.biomedcentral.com/1471-2393/14/90. All data used in this study are available from the corresponding author on reasonable request. 15. Cherie N, Mebratu A. Adverse birth out comes and associated factors among delivered mothers in Dessie Referral Hospital, North East Ethiopia. J Womens Health Reprod Med. 2017;1(1):4. Received: 6 August 2020 Accepted: 21 December 2021 23. Kebede AS, Muche AA, Alene AG. Factors associated with adverse pregnancy outcome in Debre Tabor town, Northwest Ethiopia: a case control study. BMC Res Notes. 2018;11:820 https://doi.org/10.1186/s13104-01 8-3932-2. Consent for publication Not applicable. 20. Negrato CA, Gomes MB. Low birth weight: causes and consequences. Diabetol Metab Syndr. 2013;5(1):49. 20. Negrato CA, Gomes MB. Low birth weight: causes and consequences. Diabetol Metab Syndr. 2013;5(1):49. 21. World Health Organization (WHO). Born too soon: the global action report on preterm birth. Geneva: World Health Organization; 2012. Authors’ contributions MA and GM involved in the conceptualization of the study, designing, result writing, and analyzing the finding. RT and AO are involved in designing of the study, writing, and revising a manuscript. TB and EA were involved in the organization of the research project, writing and revising the manuscript. All authors read and approved the final manuscript. Page 16 of 17 Page 16 of 17 Abadiga et al. Archives of Public Health (2022) 80:12 Abadiga et al. Archives of Public Health Ethics approval and consent to participate 16. Abdo RA, Halil HM, Kebede BA. Prevalence and predictors of adverse birth outcome among deliveries at Butajira general hospital, Gurage zone, southern nations, nationalities, and people’s region, Ethiopia. J Womens Health Care. 2019;8:474. https://doi.org/10.35248/2167-0420.19.8.474. The study was approved by the institutional review boards of Wollega University ethical review board. A permission letter was also obtained from each hospital administrative office. All participants of the study were provided written consent, clearly stating the objectives of the study and their right to refuse. Then, written informed consent was obtained from the study participants. To ensure confidentiality, names or identifying information was not indicated on the questionnaires. Mothers were interviewed in private rooms to ensure their privacy. The filled questionnaires were carefully handled ensuring confidentiality and were kept under the secured custody of the corresponding author. 17. Kassahun, et al. Adverse birth outcomes and its associated factors among women who delivered in north Wollo zone, northeast Ethiopia: a facility based cross-sectional study. BMC Res Notes. 2019;12:357 https://doi.org/1 0.1186/s13104-019-4387-9. 18. Physician for Social Responsibility (PSR). Adverse birth outcomes and environmental health threats. 2009. 19. Lawn JE, Wilczynska-Ketende K, Cousens SN. Estimating the causes of 4 million neonatal deaths in the year 2000. Int J Epidemiol. 2006;35:706–18 PMID:16556647. References 1. Lawn JE, Lee AC, Kinney M, Sibley L, Carlo WA, Paul VK, et al. Two million intrapartum-related stillbirths and neonatal deaths: where, why, and what can be done? Int J Gynecol Obstet. 2009;107:S5–19. 1. Lawn JE, Lee AC, Kinney M, Sibley L, Carlo WA, Paul VK, et al. Two million intrapartum-related stillbirths and neonatal deaths: where, why, and what can be done? Int J Gynecol Obstet. 2009;107:S5–19. 24. Weng Y-H, Yang C-Y, Chiu YW. Risk assessment of adverse birth outcomes in relation to maternal age. PLoS One. 2014;9(12):e114843. https://doi.org/1 0.1371/journal.pone.0114843. 2. Hornstra G, Uauy R, Yang X. The impact of maternal nutrition on the offspring. Basel: Karger Medical and Scientific Publishers; 2005. 2. Hornstra G, Uauy R, Yang X. The impact of maternal nutrition on the offspring. Basel: Karger Medical and Scientific Publishers; 2005. 25. Ferguson KK, Rosario Z, McElrath TF, Vélez Vega C, Cordero JF, Alshawabkeh A, et al. Demographic risk factors for adverse birth outcomes in Puerto Rico in the PROTECT cohort. PLoS One. 2019;14(6):e0217770. https://doi.org/10.13 71/journal.pone.0217770. 3. Gladstone M, Oliver C, Van den Broek N. Survival, morbidity, growth and developmental delay for babies born preterm in low- and middle-income countries—a systematic review of outcomes measured. PLoS One. 2015; 10(3):e0120566. 3. Gladstone M, Oliver C, Van den Broek N. Survival, morbidity, growth and developmental delay for babies born preterm in low- and middle-income countries—a systematic review of outcomes measured. PLoS One. 2015; 10(3):e0120566. Res. 2017;1(7):BJSTR. MS.ID.000619. https://doi.org/10.26717/BJSTR.2017.01. 000619. 34. Berhe, et al. Effect of pregnancy induced hypertension on adverse perinatal outcomes in Tigray regional state, Ethiopia: a prospective cohort study. BMC Pregnancy Childbirth. 2020;20:7 Available from: https://doi.org/10.1186/s12 884-019-2708-6. 35. Zerfu TA, Ayele HT. Micronutrients and pregnancy; effect of supplementation on pregnancy and pregnancy outcomes: a systematic review. Nutr J. 2013;12:20 Available from: https://doi.org/10.1186/1475-2 891-12-20. 35. Zerfu TA, Ayele HT. Micronutrients and pregnancy; effect of supplementation on pregnancy and pregnancy outcomes: a systematic review. Nutr J. 2013;12:20 Available from: https://doi.org/10.1186/1475-2 891-12-20. 36. Tabrizi, et al. Effects of food supplementation during pregnancy on maternal weight gain, hemoglobin levels and pregnancy outcomes in Iran. Matern Child Health J. 2018; Available from: https://doi.org/10.1007/s10995- 018-2648-1. 36. Tabrizi, et al. Effects of food supplementation during pregnancy on maternal weight gain, hemoglobin levels and pregnancy outcomes in Iran. Matern Child Health J. 2018; Available from: https://doi.org/10.1007/s10995- 018-2648-1. 37. Berhanie E, Gebregziabher D, Berihu H, Gerezgiher A, Kidane G. Intimate partner violence during pregnancy and adverse birth outcomes: a case- control study. Reprod Health. 2019;16:22 https://doi.org/10.1186/s12978-019- 0670-4. 37. Berhanie E, Gebregziabher D, Berihu H, Gerezgiher A, Kidane G. Intimate partner violence during pregnancy and adverse birth outcomes: a case- control study. Reprod Health. 2019;16:22 https://doi.org/10.1186/s12978-019- 0670-4. 38. Laelago T, Belachew T, Tamrat M. Effect of intimate partner violence on birth outcomes. Afr Health Sci. 2017;17(3):681–9. 38. Laelago T, Belachew T, Tamrat M. Effect of intimate partner violence on birth outcomes. Afr Health Sci. 2017;17(3):681–9. 38. Laelago T, Belachew T, Tamrat M. Effect of intimate partner violence on birth outcomes. Afr Health Sci. 2017;17(3):681–9. 71/journal.pone.0217770. Risk of adverse pregnancy outcomes among women practicing poor sanitation in rural India: a population-based prospective cohort study. PLoS Med. 2015;12(7):e1001851. https://doi.org/10.1371/journal.pmed.1001851. 32. American College of Obstetricians and Gynecologists. Practice bulletin no. 160: premature rupture of membranes. Obstet Gynecol. 2016;127(1):e39–51. (ISSN: 1873-233X). 11. Chaibva, et al. Adverse pregnancy outcomes, ‘stillbirths and early neonatal deaths’ in Mutare district, Zimbabwe. BMC Pregnancy Childbirth. 2019;19:86 https://doi.org/10.1186/s12884-019-2229-3. 33. Tesfay A, Abera H, Brhane G. Assessment of magnitude and associated factors of adverse birth outcomes among deliveries at Suhul Hospital Shire, Tigray, Ethiopia from September, 2015 to February, 2016. Biomed J Sci Tech 12. Asiki, et al. Adverse pregnancy outcomes in rural Uganda (1996–2013): trends and associated factors from serial cross-sectional surveys. BMC Page 17 of 17 Abadiga et al. Archives of Public Health (2022) 80:12 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.
https://openalex.org/W2889901485	https://www.matec-conferences.org/articles/matecconf/pdf/2018/65/matecconf_iccems2018_01011.pdf	English	null	Monitoring of Daily Temperature Effect on Deck Deformation of Concrete Arch Bridge	MATEC web of conferences	2,018	cc-by	3,494	1 Introduction continuous beam bridge and concrete bridge. Tian et al. 3 utilized the numerical method to get the quasi-static deformation of railway bridge resulted from temperature. Peiretti et al. 4 conducted experiment and found that temperature was uniform, linear variation and nonlinear distribution in concrete bridge deck based on a four-year temperature monitoring data. It is obvious that temperature makes great differences on stress, strain, bending moment, deformation and other behaviour of bridge structure. Solar heat transfer Heat is divided into convection, conduction and radiation, whose effect can be replaced by temperature in some degree in practice. Temperature change makes greatly differences on concrete in many aspects, including stress, strain and deformation. Some software can compute deformation, stress and bending moment of bridge structure due to temperature load, such as Midas Civil. Many scholars have studied influences on the concrete box girder, tower and other components of bridges. Researches not only concentrated on the temperature distribution of bridge components, but also investigated the behaviour of the bridge structures due to temperature change. Recently, in order to further investigate behaviour of bridges, the healthy monitoring system is more and more popular. Generally, the system was composed by several subsystems to monitor global performance, local performance and surrounding environment conditions. On the other hand, temperature is an important and continuous input variable not only for bridge components but also for the entire structure. Shashi Moorty and Charles W. Roeder 5 analysed temperature-dependent bridge movement, suggesting that concrete bridges sometimes are designed for smaller temperature ranges and corresponding thermal movements than may be expected in practice. In Chinese, Zhou Guangdong and Yi Tinghua 6 proved that thermal load played a significant role in structural condition evaluation and bridge design procedure, especially for those statically indeterminate bridges and cable-supported bridges. Therefore, the temperature load has been taken into account in General Specifications for Design of Highway Bridges and Culverts 7. Other papers further studied the correlation between available thermal variables and deformation induced by thermal loading. Battista et al. 8 established a temperature-displacement model according to four type temperature data. It can be applied to On the one hand, many researches focus on the temperature distribution of critical bridge component. J. Suzuki et al. 1 focus on the pre-stressed concrete box- girder bridge. They found that air temperature was higher than surface temperature during the night and opposite during the daytime. Lu Zhifang et al. Monitoring of Daily Temperature Effect on Deck Deformation of Concrete Arch Bridge 1 School of Highway, Chang’an University, 710064 Xi’an, China 2 School of Science, Chang’an University, 710064 Xi’an, China Abstract. Temperature makes greatly differences on concrete in many aspects, including stress, strain and deformation, especially for arch concrete bridge structure. Some software can compute theoretical deformation, stress and bending moment of bridge structure due to temperature load, such as Midas Civil. In recent years, in order to learn about conditions of structure, many bridges have installed the healthy monitoring system. In this research, the monitoring data lasting approach a year was obtained from Haierwa Bridge, a concrete truss arch bridge, in Hebei province. The bridge belongs to Xuanda Highway, which undertook the main traffic flow of coal transportation. The proportion of heavy vehicles, exceeding 50t, ups to 30%, different with common highway. The objective of this paper is to monitor and analyse the deformation of concrete truss arch bridge due to air temperature change. Firstly, results show that the daily tendencies of temperature and bridge vertical deformation at mid-span, L/4 and arch foot in the winter and summer. The linear relationship was calculated between the temperature and deformation of critical sections based on the monitoring data. In addition, the finite element model was established to calculate the theoretical value, and further compared with practice values. © 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/). https://doi.org/10.1051/matecconf/201820601011 https://doi.org/10.1051/matecconf/201820601011 MATEC Web of Conferences 206, 01011 (2018) ICCEMS 2018 2.1 Bridge overview Xuanda Highway, namely xuanhua - datong highway, is one of the main channels for transporting coal from Datong to other places in Hebei Province. The Highway is started from xuanhua, via a Shaohua Ying, YangYuan to shanxi provincial boundary, whose total length is 127.8 km. Xuanda Highway is the first mountainous area heavy highway in Hebei province. It is a two-way four-lane highway, with a speed limit range of 90 km/h to 110 km/h. Figure 2. Sensors arrangement on Haierwa Bridge The deck deformation sensors and air temperature sensors placement of Haierwa Bridge were as follows: 771018 771012 771011 771004 769204 174411 ( Temperature) 771018 771012 771011 771004 769204 174411 ( Temperature) 174411 ( Temperature) Figure 2. Sensors arrangement on Haierwa Bridge The deck deformation sensors and air temperature sensors placement of Haierwa Bridge were as follows: Figure 2. Sensors arrangement on Haierwa Bridge Th d k d f i d i Figure 2. Sensors arrangement on Haierwa Bridge Figure 2. Sensors arrangement on Haierwa Bridge The deck deformation sensors and air temperature sensors placement of Haierwa Bridge were as follows: The Haierwa Bridge (as figure 1) is located in the Xuanda Highway in Hebei province, nearly by Xuanhua. The number of the starting mileage pile is K168+ 400, and the terminal mileage pile is K168+ 612. The total length of bridge is 191.6m, and the width is 24.5m. It was completed and opened to traffic in December 2000. Bridge design span is combination of 14m+138m+10m+2 ×8m, which design load is car-20, trailer-120. The main span is composite truss arch bridge with pre-stressed concrete structure. The bridge is two-way four-lane. Especially, lane 3 and lane 4, from Datong to Xuanhua direction, are heavy traffic lanes of the heavy highway, regarded as the research object of the paper. Table. 1 Sensors placement Type of sensor Number of sensor Placement Deformation sensors 771018 Arch foot on the side of Datong 771012 L/4 on the side of Datong 771011 Mid-span 771004 L/4 on the side of Xuanhua 799204 Arch foot on the side of Xuanhua Air temperature sensors 174411 Bridge deck of L/4 on the side of Datong Figure 1. Haierwa Bridge 1 Introduction 2 analysed environment temperature and relative humidity. The research provides a new reasonable and reliable method for creep effect analysis of steel-concrete composite MATEC Web of Conferences 206, 01011 (2018) ICCEMS 2018 https://doi.org/10.1051/matecconf/201820601011 2.2 Monitoring systems estimate the bridge girder displacement. Xia et al. 9 analysed the temperature and structure data of a long- span suspension bridge. There was a linear correlation between temperature, displacement and stresses. GD zhou 10 proposed a general approach for modeling closed-form thermal correlation of deformation applied in a long-span arch bridge according to long-term monitoring data. estimate the bridge girder displacement. Xia et al. 9 analysed the temperature and structure data of a long- span suspension bridge. There was a linear correlation between temperature, displacement and stresses. GD zhou 10 proposed a general approach for modeling closed-form thermal correlation of deformation applied in a long-span arch bridge according to long-term monitoring data. The healthy monitoring system was conducted to monitor the behaviours of the Haierwa Bridge. The bridge monitoring system consists of monitoring center and five monitoring subsystems to monitor dynamic characteristics, environment, traffic, deformation and stress-strain. It is total of 101 monitoring points installed on Haierwa Bridge. There were 24 stress-strain sensors installed along the main arch ring, which can also monitor the structure temperature. The research main used data collected from 5 displacement sensors and a corresponding air temperature sensors. Five displacement sensors were installed at five places on bridge deck, respectively above arch foot, 1/4 span and mid-span. Sensors numbers are 771018, 771012, 771011, 771004, 769204 as figure 2. While the air temperature sensors were placed on corresponding positions at 174411 (as figure 2) to monitor the air temperature, replaced the effect of solar heat transfer. The monitoring data to analyse the relationship between deformation and solar heat transfer is from March to November 2015, lasting for three seasons. The writer selected one day in the summer and winter respectively to show the daily distribution of temperature and deformation. It is certain that temperature changing plays a role in deformation of concrete bridges. However, the attention pay on the whole bridge structure was not enough for all kinds of bridge structure, such as concrete truss arch bridge. The objective of this paper is to analyse the effect of temperature on bridge deck deformation, based on the temperature and deformation monitoring data of Haierwa Bridge. Moreover, the finite element model was established to calculate the theoretical value, and further compared with practice values. 3.1 Thermal actions in the winter Five critical positions were selected to analysis, including arch feet, 1/4 span and mid-span. It is symmetrical for the concrete truss arch bridge, so the following figures were presented by three critical sections. The deformation and air temperature conditions at mid-span was shown figure 4. The downward deflection is positive for all monitoring data. The curve of deflection is more stable than temperature. Figure 5. Deformation of arch bridge on 2015/8/5 Figure 5. Deformation of arch bridge on 2015/8/5 3 Thermal actions and deck deformation The air temperature sensor monitoring data was shown in figure 3, which presented the temperature tendency from March to October in 2015. The lowest temperature was - 2 ℃ in March, and the highest temperature was 26.5 ℃ between June and August. Therefore, the temperature daily effect analysis used March 15th and August 5th monitoring data. The biggest different temperature value was 28.5, which can be regarded as temperature load of finite element model. Figure 1. Haierwa Bridge 2 https://doi.org/10.1051/matecconf/201820601011 MATEC Web of Conferences 206, 01011 (2018) MATEC Web of Conferences 206, 01011 (2018) ICCEMS 2018 -5 0 5 10 15 20 25 30 2015-03-14 2015-04-02 2015-06-10 2015-06-14 2015-06-19 2015-06-22 2015-08-02 2015-08-03 2015-08-04 2015-08-05 2015-09-01 2015-09-02 2015-09-03 2015-09-04 2015-09-06 2015-09-07 2015-09-27 2015-09-30 2015-10-02 2015-10-05 2015-10-07 2015-10-15 Temperature (℃) Date Figure 3. Temperature tendency in 2015 increasing. The different deformation values were respectively 13mm, 8mm, 8mm for mid-span, L/4, arch foot, when temperature dropped about 10℃. In a word, the amplitude of temperature variation was smaller than that of winter, but the amplitude of deformation variation was larger in the summer. Figure 5. Deformation of arch bridge on 2015/8/5 3.3 Linear relationships between temperature and deformation The monitoring data on March 15th was selected to present the daily distribution in the winter. The picture shows that the deck deformation change of L/4 and arch foot was similar to the air temperature tendency. While the downward deflection of bridge mid-span decreased with temperature increasing in the winter, and increased with temperature declining. But there was a little delay between deformation and temperature. As for arch foot and 1/4 span, the deflection upward increased with temperature decreasing. The different deformation values were respectively 11mm, 6mm, 4mm for mid-span, L/4, arch foot, when temperature dropped about 12℃. According to figure 4 and figure 5, there were strong relationship between temperature and deformation, which need to further investigation. All monitoring data in 2015 was spotted to regress the linear relationship. As shown in figure 6, the temperature effect on deck deformation was biggest at mid-span. Although the correlation coefficient was not enough, only was 0.75. As for arch foot and 1/4 span (figure 7 and 8), the deflection upward increased with temperature decreasing. The relationship between deflection and temperature change is linear, and the correlation coefficient can exceed 0.9 when the temperature higher than 10℃. In other words, the linear relationship was weak under 10 ℃. The slopes of temperature-deflection are 1.1, 1.3 and -2.3 at arch foot, 1/4 span and mid span, respectively. It proved that temperature effect on mid-span deformation was much more than L/4 and arch foot. Figure 4. Deformation of arch bridge on 2015/3/15 Figure 6. Relationship between deformation and temperature of mid-span Figure 4. Deformation of arch bridge on 2015/3/15 Figure 4. Deformation of arch bridge on 2015/3/15 3.2 Thermal actions in the summer Relationship between deformation and temperature of arch foot g p p L/4 Figure 8. Relationship between deformation and temperature of arch foot 3.4 Verification of Finite Element Haierwa Bridge is a concrete truss arch bridge. The main span is up to 138m. There are double columns on the main arch at L/4 span, and expansion joint is between the two columns, which is different from common truss arch bridges. In order to verify the rule got from monitoring data above, the write establish the finite element (FE) model of the bridge. The FE model has 408 nodes and 414 beam elements. The concrete selected C50 to simulate. At the same time, the corresponding temperature load was applied to Midas Civil to obtain theoretical deflection value as figure 9. Because the traffic load and self-weight of bridge are stable g g q g , deck of mid-span declined 6.598cm, while the theoretical was 8.623cm. The deflection is stable from arch foot to 1/4 span with temperature changing, which is not exceed 1 cm in the finite element model. However, the monitoring data were about 2~3cm, and the direction is different. In other words, when temperature dropping, the practical downward deformation will much over than the nominal data at L/4 and arch foot. However the mid-span different deformation is opposite. In conclusion, there were about 2~3.5cm different deformation value between theoretical and monitoring data. The different may be led by shrinkage, creep, material property, traffic conditions and others, which need to be further studied. Table. 2 Comparison of monitoring data and theoretical value Correlation of temperature and deformation Mid-span L/4 span Arch foot Monitori ng data t≧10℃ d=-2.3151∆t d=1.3069∆t d=1.1478∆t t<10℃ d=0.5069∆t d=0.3913∆t FE model d=-3.028∆t d=-0.2517∆t d=-0.2567∆t ∆T(℃) -2~26.5 -2~26.5 -2~26.5 Monitoring value (cm) 6.598 -2.751 -2.300 FE theoretical value (cm) 8.623 0.717 0.732 Different value (cm) 2.027 3.468 2.080 Figure 9. Deformation of structure when temperature dropped Table. 2 Comparison of monitoring data and theoretical value Correlation of temperature and deformation Mid-span L/4 span Arch foot Monitori ng data t≧10℃ d=-2.3151∆t d=1.3069∆t d=1.1478∆t t<10℃ d=0.5069∆t d=0.3913∆t FE model d=-3.028∆t d=-0.2517∆t d=-0.2567∆t ∆T(℃) -2~26.5 -2~26.5 -2~26.5 Monitoring value (cm) 6.598 -2.751 -2.300 FE theoretical value (cm) 8.623 0.717 0.732 Different value (cm) 2.027 3.468 2.080 Table. 2 Comparison of monitoring data and theoretical value Figure 8. Relationship between deformation and temperature of arch foot 3.2 Thermal actions in the summer The monitoring data on August 5th was selected to present the daily distribution in the summer. The picture shows that the deck deformation change of L/4 and arch foot was similar to the air temperature tendency. However, the downward deflection of bridge mid-span decreased with temperature increasing in the summer. It needs to note that, the downward deformation is later than temperature decreased. As for arch foot and 1/4 span, the deflection upward increased with temperature Figure 6. Relationship between deformation and temperature of mid-span 3 3 MATEC Web of Conferences 206, 01011 (2018) ICCEMS 2018 https://doi.org/10.1051/matecconf/201820601011 Figure 7. Relationship between deformation and temperature of L/4 Figure 7. Relationship between deformation and temperature of L/4 Figure 8. Relationship between deformation and temperature of arch foot respectively and always existence, the write only take temperature load into the model. Compared with the monitoring data, most of the deformation of mid-span bridge is led by the change of temperature. As table 2 shown, the deformation growth rate was obtained due to temperature changing based on regression equation. It is obvious that the deformation of mid-span is far more than L/4 span and arch foot. The conclusion is similar to the FE model. But the deformation growth rate is different. In all monitoring data, the maximum temperature is 26.5 ℃ and the minimum temperature is -2 ℃. Therefore, the biggest different value of air temperature can up to 28.5 ℃. According to regression equation of monitoring data, the deck of mid-span declined 6.598cm, while the theoretical was 8.623cm. The deflection is stable from arch foot to 1/4 span with temperature changing, which is not exceed 1 cm in the finite element model. However, the monitoring data were about 2~3cm, and the direction is different. In other words, when temperature dropping, the practical downward deformation will much over than the nominal data at L/4 and arch foot. However the mid-span different deformation is opposite. In conclusion, there were about 2~3.5cm different deformation value between theoretical and monitoring data. The different may be led by shrinkage, creep, material property, traffic conditions and others, which need to be further studied. Figure 7. Relationship between deformation and temperature of L/4 Figure 7. Relationship between deformation and temperature of L/4 Figure 8. Relationship between deformation and temperature of arch foot Figure 7. Relationship between deformation and temperature of L/4 Figure 8. References 1. J. Suzuki, Y. Ohba, Y. Uchikawa, K. Hoshikawa and K. Kimura, Monitoring temperatures on a real box- girder bridge and energy budget analysis for basic information on bridge cooling and surface freezing, J. Bridge Eng., 12, 7 (2007) In conclusion, air temperature change has influence on structure deformation in concrete arch bridge, especially for mid-span. There is a strong linear relationship between the structure deformation and temperature. Combined with the finite element model, conclusions were as follows: 2. Z Lu, M Liu and Q Li, Creep effect analysis of steel- concrete composite bridge considering mutative temperature and relative humidity, J. Central South University, 46, 2650-2657 (2015) Deformation curves were more stable than the temperature curve. For instance, some small fluctuations of temperature were not reflected at the same time. 3. Y Tian, N Zhang and H Xia, Temperature effect on service performance of high-speed railway concrete bridges, Adv. Struct. Eng., 20, (6) 865-883 (2017). The deck deformation varied with temperature. The deck deformation change of L/4 and arch foot was similar to the air temperature tendency, while there was a little delay between deformation and temperature. The downward deflection of bridge mid-span decreased with temperature increasing, and increased with temperature declining. In addition, the amplitude of temperature variation was smaller than that of winter, but the amplitude of deformation variation was larger in the summer. 4. H. C. Peiretti, J. I. E. Parrotta, A. B. Oregui, A. P. Caldentey and F. A. Fernandez, Experimental study of thermal actions on a solid slab concrete deck bridge and comparison with Eurocode 1 J. Bridge Eng., 19, 10 (2014) 5. S. Moorty and C. W. Roeder, Temperature-dependent bridge moment, J. Struct. Eng., 118, 1090-1105 (1992) There were strong linear relationships between air temperature and deck deformation at L/4 and arch foot of concrete truss arch bridge. As for mid-span, the linear relationship is weaker. However, the temperature effect on mid-span deformation was much more than L/4 and arch foot. 6. GD Zhou and TH Yi, Thermal load in large-scale Bridges: a state-of-the-art review, Int. J. Distrib. Sens. N., 17 (2013) 7. CCCC Highway consultants CO. Ltd. General Code for Design of Highway Bridges and Culverts (China Communications Press, Beijing, 2004) In addition, the finite element model was established to calculate the theoretical value, and further compared with practice values. There were about 2cm different deformation value between theoretical and monitoring data. References It is essential to control deformation due to temperature change for concrete arch bridge, especial its middle span in long term operation. 8. N. D. Battista, J. D. Brown, H. P. Tan and K. Y. Koo, Measuring and modelling the thermal performance of the Tamar Suspension Bridge using a wireless sensor network, Struct. Infrastruct. E., 11, 176-193 (2015) 9. Q Xia, J Zhang, YD Tian and YF Zhang, Experimental study of thermal effects on a long-span suspension bridge, J. Bridge Eng., 22, 7(2017) 10. GD Zhou, TH Yi, B Chen and X Chen, Modeling deformation induced by thermal loading using long- term bridge monitoring data, J. Perform. Constr. Facil., 32, 3 (2018) 3.4 Verification of Finite Element Haierwa Bridge is a concrete truss arch bridge. The main span is up to 138m. There are double columns on the main arch at L/4 span, and expansion joint is between the two columns, which is different from common truss arch bridges. In order to verify the rule got from monitoring data above, the write establish the finite element (FE) model of the bridge. The FE model has 408 nodes and 414 beam elements. The concrete selected C50 to simulate. At the same time, the corresponding temperature load was applied to Midas Civil to obtain theoretical deflection value as figure 9. Because the traffic load and self-weight of bridge are stable Figure 9. Deformation of structure when temperature dropped Figure 9. Deformation of structure when temperature dropped Figure 9. Deformation of structure when temperature dropped 4 https://doi.org/10.1051/matecconf/201820601011 MATEC Web of Conferences 206, 01011 (2018) MATEC Web of Conferences 206, 01011 (2018) ICCEMS 2018 Acknowledgments This study is supported by the National Science Foundation of PR China (51208056), and the Fundamental Research Funds for the Central Universities (310821161013, 300102218213). 5 5
https://openalex.org/W2212487443	https://europepmc.org/articles/pmc4700156?pdf=render	English	null	Two Cases of Sarcoma Arising in Giant Cell Tumor of Bone Treated with Denosumab	Case reports in medicine	2,015	cc-by	4,255	Correspondence should be addressed to Cory Julian Broehm; cjbroehm@gmail.com Received 29 August 2015; Revised 2 December 2015; Accepted 3 December 2015 Received 29 August 2015; Revised 2 December 2015; Accepted 3 December 2015 Academic Editor: Gottfried J. Locker Academic Editor: Gottfried J. Locker Copyright © 2015 Cory Julian Broehm et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Giant cell tumor (GCT) of bone is a generally benign, but often locally aggressive, neoplasm of bone, with a propensity for recurrence. Sarcomatous transformation is rare and typically occurs with a history of recurrences and radiation treatment. Denosumab, an inhibitor of the RANK ligand involved in bone resorption in GCT, is increasingly used in treatment of recurrent or unresectable giant cell tumor of bone. We report two cases of sarcomatous transformation of GCT to osteosarcoma in patients receiving denosumab. One was a 59-year-old male with a 12-year history of GCT and multiple recurrences taking denosumab for 2.5 years. The second case was in a 56-year-old male with a seven-year history of GCT taking denosumab for six months. Review of the literature shows one case report of malignant transformation of GCT in a patient being treated with denosumab. As the use of denosumab for treatment of GCT will likely increase, larger, controlled studies are needed to ascertain whether denosumab may play a role in malignant transformation of giant cell tumor of bone. Hindawi Publishing Corporation Case Reports in Medicine Volume 2015, Article ID 767198, 6 pages http://dx.doi.org/10.1155/2015/767198 Hindawi Publishing Corporation Case Reports in Medicine Volume 2015, Article ID 767198, 6 pages http://dx.doi.org/10.1155/2015/767198 Hindawi Publishing Corporation Case Reports in Medicine Volume 2015, Article ID 767198, 6 pages http://dx.doi.org/10.1155/2015/767198 Cory Julian Broehm, Erika L. Garbrecht, Jeff Wood, and Therese Bocklage 1Department of Pathology, University of New Mexico School of Medicine, MSC08 4640, 1 University of New Mexico, Albuquerque, NM 87131, USA 1Department of Pathology, University of New Mexico School of Medicine, MSC08 4640, 1 University of New Mexico, Albuquerque, NM 87131, USA 1Department of Pathology, University of New Mexico School of Medicine, MSC08 4640, 1 University of New Mexi Albuquerque, NM 87131, USA q q 2Department of Orthopaedics, University of New Mexico School of Medicine, MSC10 5600, Albuquerque, NM 87131, USA 3Department of Radiology, University of New Mexico School of Medicine, MSC10 5530, 1 University of New Mexico, Albuquerque, NM 87131, USA 2Department of Orthopaedics, University of New Mexico School of Medicine, MSC10 5600, Albuquerque, NM 87131, USA 3Department of Radiology, University of New Mexico School of Medicine, MSC10 5530, 1 University of New Mexico, Albuquerque, NM 87131, USA Correspondence should be addressed to Cory Julian Broehm; cjbroehm@gmail.com Correspondence should be addressed to Cory Julian Broehm; cjbroehm@gmail.com 2. Case Reports 2.1. Case 1. A 46-year-old male presented in 2002 to an outside institution with five to six years of right hip pain radiating down the lateral thigh. Initial imaging is unavailable but by report radiographs showed a large mass involving the ischial tuberosity and portions of the adjacent inferior and superior rami of the right pelvis. He subsequently transferred care to our institution. Biopsies performed at the outside institution were reviewed and confirmed giant cell tumor of bone. He subsequently underwent partial internal hemipelvectomy in November 2002. Histopathologic analysis showed GCT (Figure 1). Figure 3: Axial CT of the pelvis (February 2015) showed extensive cortical irregularity and cystic spaces (arrows) of the right inferior pubic ramus (bone window algorithm). Postoperatively, the patient was started on alendronate (dose unknown). Follow-up MRI in February 2003 showed an enhancing, high T2 signal, 2 cm soft tissue mass within the right adductor musculature, adjacent to the former location of the ischial tuberosity, suspicious of recurrence. CT imaging in August 2003 showed a mass within the surgical bed, within the obturator externus and pectineus muscles, and abutting the root of the penis. CT guided biopsy confirmed recurrent benign GCT. Reexcision of the mass was performed in September 2003. The pathology specimen showed a 4 cm mass embedded within excised soft tissue. Giant cell tumor was present at multiple resection margins. 11.0 × 8.9 cm in January 2012, at which time there was possible invasion into the right corpus cavernosum.h The patient was started on denosumab (120 mg subcu- taneous monthly) in July 2012. Imaging over the next 2 years showed stable disease, with possible slight decrease in size of the tumor. In November 2014, the patient developed osteonecrosis of the left mandible after tooth extraction, and denosumab was held for two doses. Denosumab was restarted in late December 2014 after presenting to clinic with severe right groin pain radiating inferiorly. Follow-up CT scan in February 2015 showed enlargement of the pelvic mass to 13.6 × 13.0 cm with extension into the ischioanal fossa and subsequent mass effect on the rectum, prostate, bladder base, and base of penis (Figures 3 and 4). The imaging findings were highly suggestive of sarcomatous transformation of the GCT. Multiple nodules were also now noted throughout the lungs. 1. Introduction radiofrequency thermal ablation, or chemoembolization, may be used in cases where surgery is not possible. Wide resection may be reserved for cases in which surgery results in relatively minor functional impairment or for tumors with extensive local destruction [2, 3, 10]. However, consid- erable morbidity may be involved in resection. Giant cell tumor is composed of neoplastic mononuclear stromal cells and reactive nonneoplastic multinucleated giant cells that are responsible for bone resorption, which is mediated by interaction between receptor activator of nuclear factor-kB (RANK) expressed by giant cells and RANK ligand (RANKL) on stromal cells [11]. Denosumab, a monoclonal antibody inhibitor of RANKL, has proven effective in limited clinical trials in halting tumor progression in patients with recurrent or unresectable giant cell tumors [12, 13]. Giant cell tumor (GCT) of bone is a generally benign tumor that is often locally aggressive, causing significant destruction of bone [1]. Recurrence may be seen in 15–50% of cases after treatment, usually within 2 years, depending on the location of the tumor and treatment modality [2–5]. Pulmonary metastasis may occur in less than 5% of cases [2, 4, 6]. Malignant transformation of GCT is rare, occurring in less than one percent of cases [1]. Secondary transformation, which follows radiation therapy or less commonly surgical intervention, accounts for approximately 70% of malignant GCT [7, 8]. Primary malignant GCT, which arise de novo alongside typical GCT, make up the remainder of malignant cases [7–9].t Treatment often involves curettage, with or without bone filler or adjuvants such as polymethylmethacrylate (PMMA) or phenol. Less invasive procedures, such as radiotherapy, A recent report described a case of high grade sarcoma arising in a giant cell tumor of bone treated with denosumab [14]. We report an additional two cases of high grade sarcoma 2 Figure 1: Histology of hemipelvectomy specimen showed mononu- clear cells with interspersed multinucleated cells (hematoxylin and eosin stain, 20x). 2 Case Reports in Medicine Figure 2: Gadolinium enhanced axial T1 CT (December 2005) of the pelvis with fat saturation demonstrates postsurgical change and irregularity of the right inferior pubic ramus, with an adjacent associated enhancing soft tissue mass (arrows). Figure 1: Histology of hemipelvectomy specimen showed mononu- clear cells with interspersed multinucleated cells (hematoxylin and eosin stain, 20x). 1. Introduction Figure 2: Gadolinium enhanced axial T1 CT (December 2005) of the pelvis with fat saturation demonstrates postsurgical change and irregularity of the right inferior pubic ramus, with an adjacent associated enhancing soft tissue mass (arrows). arising in giant cell tumor of bone in patients receiving denosumab. Figure 3: Axial CT of the pelvis (February 2015) showed extensive cortical irregularity and cystic spaces (arrows) of the right inferior pubic ramus (bone window algorithm). 2. Case Reports No additional potential primary tumor that could account for the lung nodules was identified on imaging, and metastases from a malignant GCT were suspected. Denosumab was discontinued. Postoperatively, the patient did well, with intermittent complaints of pain. Follow-up MRI eight months after surgery showed an enhancing multilobulated mass at the margins of the original hemipelvectomy surgical bed, the largest mass measuring up to 2.7 cm. Edema and abnormal enhancement were noted within the obturator externus, obturator internus, and quadratus femoris muscles. Another 1.5 cm nodule was noted in the proximal adductor brevis muscle. He was subsequently followed up with approximately yearly MRI studies. The recurrent tumor showed progressive, interval growth, reaching a measurement of 3.2 × 3.6 cm in December 2005 (Figure 2), 9.2 × 3.4 cm in January 2008, and CT guided biopsy of a right lower lobe nodule showed a high grade spindle cell sarcoma with nuclear atypia and 3 Figure 6: Biopsy of enlarging pelvic mass showed a sarcoma comprised of high grade round to epithelioid cells (hematoxylin and eosin stain, 40x). 3 Case Reports in Medicine Figure 4: Axial CT of the pelvis (February 2015) showed severe interval enlargement of the soft tissue mass (arrows) surrounding the right inferior ramus (soft tissue algorithm). Figure 6: Biopsy of enlarging pelvic mass showed a sarcoma comprised of high grade round to epithelioid cells (hematoxylin and eosin stain, 40x). Figure 4: Axial CT of the pelvis (February 2015) showed severe interval enlargement of the soft tissue mass (arrows) surrounding the right inferior ramus (soft tissue algorithm). Figure 7: Histology of curettage specimen showed bland spindle cells and intermixed giant cells (hematoxylin and eosin stain, 20x). Figure 5: Biopsy of lung nodule revealed a sarcoma comprised of high grade spindle-shaped cells, consistent with metastatic sarcoma (hematoxylin and eosin stain, 20x). Figure 7: Histology of curettage specimen showed bland spindle cells and intermixed giant cells (hematoxylin and eosin stain, 20x). Figure 5: Biopsy of lung nodule revealed a sarcoma comprised of high grade spindle-shaped cells, consistent with metastatic sarcoma (hematoxylin and eosin stain, 20x). surrounding bone. GCT was strongly suspected and the patient underwent curettage with PMMA packing in August 2007. Histologic evaluation confirmed giant cell tumor of bone (Figure 7).h low mitotic rate (up to 4 mitoses per high power field) (Figure 5). By immunohistochemistry, the tumor stained for smooth muscle actin (SMA) and p63, with focal S-100 protein staining. 2. Case Reports Subsequent CT guided biopsy of the pelvic mass revealed a high grade sarcoma with discohesive round to epithelioid cells that expressed SATB2 and weak SMA and lacked p63 (Figure 6). The morphology and IHC staining results were consistent with transformation to osteosarcoma. The patient was followed postoperatively with radio- graphs of the knee. One month after surgery, a rim of lucency around the cement packing was noted (Figure 8). There was interval increase in the lucency until February 2009, when MRI showed an 8.15 × 3.9 × 1.8 cm area of marrow infiltration corresponding to the lucency, consistent with recurrence. There was minimal interval increase in size on MRI through November 2013, during which time the patient experienced some intermittent left knee pain, when an additional 2 × 1 cm mass was identified, along with mild periostitis along the lateral distal femur (Figure 9).hf Subsequent CT scan showed interval increase in size of the pelvic mass, an increase in the number and size of lung nodules, pulmonary lymphadenopathy, and a hepatic lesion suspicious of additional metastasis. The patient was started on doxorubicin and ifosfamide. CT scan showed slightly decreased primary tumor but an increased number of pul- monary nodules, two liver lesions, inguinal lymphadenopa- thy, and a probable 3.7 cm metastasis to the penis. He was then started on docetaxel and gemcitabine. He transferred care from our institution shortly thereafter. The patient was offered, and declined, both denosumab and surgery at that time. With continued knee pain and weakness, however, he opted for denosumab treatment in January 2014. He initially did well, denying pain except with running or other significant impact activities. But, by July 2014, he reported two months of significantly increasing knee pain and swelling. Radiographs showed continued increase in size of the lucency, measuring up to 5.0 cm, with periosteal reaction suggestive of impending pathologic fracture (Figure 10). Because of the intolerable pain, the patient opted for surgery. Due to the tumor’s proximity to 2.2. Case 2. A 49-year-old male presented in July 2007 with a six-month history of left knee pain and swelling. Radiographs from an outside institution showed a cystic lesion in the left femur condyle with sclerotic borders and expansion of 2.2. Case 2. A 49-year-old male presented in July 2007 with a six-month history of left knee pain and swelling. 2. Case Reports Radiographs from an outside institution showed a cystic lesion in the left femur condyle with sclerotic borders and expansion of 4 Case Reports in Medicine 4 Figure 11: Distal femoral amputation demonstrated an ill-defined mass (arrows) proximal to the PMMA packing. Figure 8: Frontal left knee radiograph (August 2007) demonstrates postsurgical curettage and packing with radiopaque polymethyl- methacrylate (PMMA) of a well-defined, solitary, mixed lytic, and sclerotic lesion with a narrow zone of transition (arrows), located within the distal femoral metaphysis. Figure 11: Distal femoral amputation demonstrated an ill-defined mass (arrows) proximal to the PMMA packing. Figure 8: Frontal left knee radiograph (August 2007) demonstrates postsurgical curettage and packing with radiopaque polymethyl- methacrylate (PMMA) of a well-defined, solitary, mixed lytic, and sclerotic lesion with a narrow zone of transition (arrows), located within the distal femoral metaphysis. Figure 12: Histologic examination of area shown in Figure 11 showed atypical, hyperchromatic spindle cells and osteoid forma- tion, consistent with osteosarcoma (hematoxylin and eosin stain, 20x). Figure 9: Coronal T1 postcontrast CT (November 2013) demon- strated a heterogeneously enhancing soft tissue mass (arrows) within the lateral aspect of the distal femoral condyle, proximal to the hypointense focus of PMMA. Figure 12: Histologic examination of area shown in Figure 11 showed atypical, hyperchromatic spindle cells and osteoid forma- tion, consistent with osteosarcoma (hematoxylin and eosin stain, 20x). the bone cortex and articular surface, curettage was not pos- sible and arthroplasty was scheduled. The patient underwent wide resection with endoprosthetic reconstruction in August 2014.h Figure 9: Coronal T1 postcontrast CT (November 2013) demon- strated a heterogeneously enhancing soft tissue mass (arrows) within the lateral aspect of the distal femoral condyle, proximal to the hypointense focus of PMMA. The pathology specimen consisted of a 15 cm length of distal femur with minimal attached soft tissue and muscle and with an up to 4.0 cm ill-defined heterogeneous mass proximal to the PMMA (Figure 11). Histologic examination showed that the mass was comprised of pleomorphic osteoblastic cells in a fibrosarcomatous arrangement with bone, osteoid, and focal cartilage formation (Figure 12). Mitoses averaged 18 per high powered field. By immunohistochemistry, the tumor cells stained with SATB2, p53, and p63. Multiple soft tissue margins were positive. The findings were consistent with a high grade osteosarcoma arising from giant cell tumor. 3. Discussion Recently, the first case report of sarcoma arising in the setting of denosumab treatment for giant cell tumor of bone was published [14]. The patient was a 20-year-old female with a five-year history of GCT of the proximal tibia, initially treated with intralesional resection. The tumor recurred twice over the next 3.5 years and was treated both times with intralesional resection. After the second resection, she was treated with denosumab and presented one year later with a rapidly growing mass in the proximal tibia. Open biopsy revealed a high grade, mitotically active sarcoma with extensive necrosis. The patient subsequently underwent amputation. The potential relationship between, and mechanism of, sarcomatous transformations of GCT during denosumab therapy is unclear due at least in part to the limited published data on this population. In vivo and in vitro studies of the effect of denosumab on GCT at the cellular level show loss of giant cells, usually a reduction in the neoplastic stromal cells with reduced RANKL expression and proliferation, and reactive and woven bone and/or osteoid formation [13, 15, 16]. Proliferation of spindle cells with reactive bone and osteoid formation has also been reported [17, 18]. Patients taking denosumab for GCT likely represent a subset that are at higher baseline risk for sarcomatous trans- formation, often having a long-standing history of disease with multiple recurrences and treatments. More extensive studies of the side effects of denosumab in patients with osteoporosis [19] and cancer [20–22] have not noted any increase in the risk of sarcoma, though denosumab dosing is lower in the former group and long-term follow-up is not consistent in the latter group. At our institution, 14 patients with giant cell tumor of bone have had recurrences (from a total of 40 patients with GCT with 4 patients lost to follow-up <6 months after treatment); of these, two patients have had malignant transformation: the two patients reported herein who also represent half of the patients treated with denosumab for at least six months (average follow-up: 83 months, range of 6–154). Additional controlled studies and long-term follow-up are needed before more definitive conclusions can be drawn regarding denosumab treatment and sarcomatous transformation of giant cell tumor of bone. Two Phase II trials have thus far evaluated the safety and efficacy of denosumab in the treatment of giant cell tumor of bone. 3. Discussion In a study of 37 patients with recurrent or unresectable GCT who received 120 mg of denosumab monthly (and loading doses on days 8 and 15 of the first month), 30 of 35 patients with available follow-up data had tumor response, defined as elimination of at least 90% of giant cells on biopsy or no radiologic progression. Denosumab was well tolerated, with pain and nausea as the primary complaints. One patient developed high grade sarcoma arising in a GCT, found after an abnormal elevated human chorionic gonadotropin level and subsequent tumor resection. Another patient with recurrent GCT metastatic to lung developed malignant GCT eight months after discontinuing treatment [13]. t A second Phase II trial utilizing the same doses and scheduling of denosumab as the first trial enrolled 282 patients with GCT into three cohorts: those with surgically unsalvageable disease (𝑛= 170); those with salvageable dis- ease with planned surgery (𝑛= 101); and a third cohort (𝑛= 11) that included patients from the original Phase II study of 37 patients. Ninety-six percent of patients in the surgically unsalvageable disease cohort showed no progression. Only 26 patients in the planned surgery cohort ultimately required surgery, 16 of these with a less morbid surgery than originally planned. Again, denosumab was well tolerated, with pain, nausea, and fatigue as the primary complaints. Hypophos- phatemia, hypocalcemia, infection, and osteonecrosis of the jaw were noted in a handful of patients. Two cases of sarcoma arising in GCT were recorded, though it is unclear in which cohort(s) these patients were. One case was suspected to have been present but unrecognized prior to treatment. The other case arose during the study. The investigators did not believe in a connection between denosumab and sarcomatous transformation of GCT. Interestingly, a case of thyroid carcinoma with high grade sarcoma was also reported, arising in an area of previous radiation [12]. At our institution, only six patients with giant cell tumor of bone have been treated with denosumab. Of these, four received more than six months of therapy. Two of these patients with a longer duration of denosumab therapy are the two patients presented in this paper. One of the remaining two patients, a 30-year-old woman, has recently developed a 1 cm soft tissue mass near the site of her original tumor following three recurrences in the radius over 60 months and following 36 2. Case Reports Cytogenetic analysis of tumor tissue revealed a complex karyotype that included loss of chromosome 17, a recurrent finding in osteosarcoma.h Figure 10: Frontal left knee radiograph (July 2014) demon- strated increased well-defined lytic focus (arrows) surrounding the radiopaque PMMA, suggestive of malignant transformation. i The patient was given four cycles of doxorubicin and cisplatin in September 2014 but developed skeletal metas- tases. He was then started on high dose methotrexate. He initially had stable disease but again progressed after several months, with development of pleural and additional skeletal metastases. He was then placed on gemcitabine and docetaxel but died soon after. Figure 10: Frontal left knee radiograph (July 2014) demon- strated increased well-defined lytic focus (arrows) surrounding the radiopaque PMMA, suggestive of malignant transformation. 5 Case Reports in Medicine months of denosumab therapy. That mass has not yet been biopsied.h Acknowledgment The authors thank Michael Grady for his creation of the figures. Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper. References [1] N. Athanasou, M. Bansal, R. Forsyth, R. Reid, and Z. Sapi, “Giant cell tumour of bone,” in WHO Classification of Tumours of Soft Tissue and Bone, C. D. M. Fletcher, J. Bridge, P. Hogendoorn, and F. Mertens, Eds., pp. 321–324, IARC Press, Lyon, France, 4th edition, 2013. [2] F. M. Klenke, D. E. Wenger, C. Y. Inwards, P. S. Rose, and F. H. Sim, “Giant cell tumor of bone: risk factors for recurrence,” Clinical Orthopaedics and Related Research, vol. 469, no. 2, pp. 591–599, 2011. [3] F. M. Klenke, D. E. Wenger, C. Y. Inwards, P. S. Rose, and F. H. Sim, “Recurrent giant cell tumor of long bones: analysis of surgical management,” Clinical Orthopaedics and Related Research, vol. 469, no. 4, pp. 1181–1187, 2011. Case Reports in Medicine 6 [4] F. Masui, S. Ushigome, and K. Fujii, “Giant cell tumor of bone: a clinicopathologic study of prognostic factors,” Pathology International, vol. 48, no. 9, pp. 723–729, 1998. [20] K. Fizazi, M. Carducci, M. Smith et al., “Denosumab versus zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer: a randomised, double- blind study,” The Lancet, vol. 377, no. 9768, pp. 813–822, 2011. [5] D. J. McDonald, F. H. Sim, R. A. McLeod, and D. C. Dahlin, “Giant-cell tumor of bone,” The Journal of Bone & Joint Surgery—American Volume, vol. 68, no. 2, pp. 235–242, 1986. [21] P. Peddi, M. A. Lopez-Olivo, G. F. Pratt, and M. E. Suarez- Almazor, “Denosumab in patients with cancer and skeletal metastases: a systematic review and meta-analysis,” Cancer Treatment Reviews, vol. 39, no. 1, pp. 97–104, 2013. [6] K. A. Siebenrock, K. K. Unni, and M. G. Rock, “Giant-cell tumour of bone metastasising to the lungs,” The Journal of Bone & Joint Surgery—British Volume, vol. 80, no. 1, pp. 43–47, 1998. [22] M. R. Smith, F. Saad, R. Coleman et al., “Denosumab and bone-metastasis-free survival in men with castration-resistant prostate cancer: results of a phase 3, randomised, placebo- controlled trial,” The Lancet, vol. 379, no. 9810, pp. 39–46, 2012. [7] F. Bertoni, P. Bacchini, and E. L. Staals, “Malignancy in giant cell tumor of bone,” Cancer, vol. 97, no. 10, pp. 2520–2529, 2003. [8] M. G. Rock, F. H. Sim, K. K. Unni et al., “Secondary malignant giant-cell tumor of bone,” The Journal of Bone and Joint Surgery—American Volume, vol. 68, no. 7, pp. 1073–1079, 1986. [9] A. G. References Nascimento, A. G. Huvos, and R. C. Marcove, “Primary malignant giant cell tumor of bone: a study of eight cases and review of the literature,” Cancer, vol. 44, no. 4, pp. 1393–1402, 1979. [10] D. M. Thomas, “RANKL, denosumab, and giant cell tumor of bone,” Current Opinion in Oncology, vol. 24, no. 4, pp. 397–403, 2012. [11] L. Huang, J. Xu, D. J. Wood, and M. H. Zheng, “Gene expression of osteoprotegerin ligand, osteoprotegerin, and receptor activa- tor of NF-kappaB in giant cell tumor of bone: possible involve- ment in tumor cell-induced osteoclast-like cell formation,” The American Journal of Pathology, vol. 156, no. 3, pp. 761–767, 2000.fi [12] S. Chawla, R. Henshaw, L. Seeger et al., “Safety and efficacy of denosumab for adults and skeletally mature adolescents with giant cell tumour of bone: interim analysis of an open-label, parallel-group, phase 2 study,” The Lancet Oncology, vol. 14, no. 9, pp. 901–908, 2013. [13] D. Thomas, R. Henshaw, K. Skubitz et al., “Denosumab in patients with giant-cell tumour of bone: an open-label, phase 2 study,” The Lancet Oncology, vol. 11, no. 3, pp. 275–280, 2010. [14] L. A. Aponte-Tinao, N. S. Piuzzi, P. Roitman, and G. L. Farfalli, “A high-grade sarcoma arising in a patient with recurrent benign giant cell tumor of the proximal tibia while receiving treatment with denosumab,” Clinical Orthopaedics and Related Research, vol. 473, no. 9, pp. 3050–3055, 2015. [15] D. G. Branstetter, S. D. Nelson, J. C. Manivel et al., “Denosumab induces tumor reduction and bone formation in patients with giant-cell tumor of bone,” Clinical Cancer Research, vol. 18, no. 16, pp. 4415–4424, 2012. [16] I. W. Y. Mak, N. Evaniew, S. Popovic, R. Tozer, and M. Ghert, “A translational study of the neoplastic cells of giant cell tumor of bone following neoadjuvant denosumab,” The Journal of Bone & Joint Surgery—American Volume, vol. 96, no. 15, article e127, 2014. [17] M. Hakozaki, T. Tajino, H. Yamada et al., “Radiological and pathological characteristics of giant cell tumor of bone treated with denosumab,” Diagnostic Pathology, vol. 9, no. 1, article 111, 2014. [18] N. Santosh, J. L. Mayerson, and O. H. Iwenofu, “Pseudosar- comatous spindle cell proliferation with osteoid matrix mim- icking osteosarcoma: a distinct histologic phenotype in giant cell tumor of bone following denosumab therapy,” Applied Immunohistochemistry & Molecular Morphology, 2015.hf [19] H. G. Bone, R. Chapurlat, M.-L. References Brandi et al., “The effect of three or six years of denosumab exposure in women with postmenopausal osteoporosis: results from the FREEDOM extension,” Journal of Clinical Endocrinology and Metabolism, vol. 98, no. 11, pp. 4483–4492, 2013.
https://openalex.org/W4361248624	https://figshare.com/articles/journal_contribution/Supplementary_Figure_Legends_1-4_from_Functional_Interplay_of_p53_and_Mus81_in_DNA_Damage_Responses_and_Cancer/22368524/1/files/39813593.pdf	English	null	Supplementary Figure Legends 1-4 from Functional Interplay of p53 and Mus81 in DNA Damage Responses and Cancer	null	2,023	cc-by	325	Figure S2: Embryonic Lethality and Neural Tube Defects Of Mus81-/-p53-/- Females. (A) Based on Mendelian ratio’s and litter sizes, 86 Mus81-/-p53-/- mice were expected. However, only 47 double knockout mice were obtained. Of the 47 Mus81-/-p53-/- mice that survived embryonic development, 44 were males and only 3 were females, indicating that females were dying in utero. (B) Representative pictures of day E9.5 Mus81-/-p53-/- female displaying neural tube defects (arrow; right panel) and E9.5 normal Mus81-/- p53-/- male embryo (left panel). Timed matings were performed from crossing Mus81+/-p53+/- male with Mus81+/-p53+/- female mice or Mus81-/-p53-/- male with Mus81-/-p53+/- female mice. Embryos were dissected at E8.5, E9.5, and E10.5 embryonic stages. Sex determination of embryos was performed as previously described (Nat. Genet, 10: 175-180, 1995). Pamidi et al. Pamidi et al. Supplemental Data Figure S1: Mus81-/- And Mus81+/- MEFs Do Not Have Proliferation Defects. Proliferation curves for WT, Mus81+/-, and Mus81-/- MEFs. For each genotype, 3 x 105 MEFs were initially plated. Cells were counted and passaged every 3 days. MEFs generated from two different embryos for each genotype were used. Counts are plotted as cumulative growth, and are representative of two independent experiments. Figure S3: Normal Immune Development Of Mus81-/-p53-/- Male Mice. Representative FACS dot blot analysis of immune cells from WT, Mus81-/-, p53-/-, Mus81-/-p53-/- mice. Similar distribution of all subpopulations was observed in thymus (A: CD4 and CD8 staining), bone marrow (B: B220/CD43 and C: B220/IgM staining), lymph nodes (D: B220 and Thy1 staining) and spleen (E: B220 and Thy1 staining). Pamidi et al. Pamidi et al. Figure S4: Lack of Loss of of Heterozygosity of Mus81 Locus in Sarcomas Isolated From Mus81+/-p53-/- Mice. DNA isolated from three sarcomas from Mus81+/-p53-/- mice was digested with Hind III. Southern blot analysis was performed using a Mus81 specific probe that recognizes a 9.6 kb Hind III genomic fragment for the wild-type Mus81 allele and a 2.6 kb Hind III genomic fragment for the mutant Mus81 allele. 2
https://openalex.org/W3069848044	https://europepmc.org/articles/pmc7821168?pdf=render	English	null	<scp>IPDmada</scp>: An R Shiny tool for analyzing and visualizing individual patient data meta‐analyses of diagnostic test accuracy	Research synthesis methods	2,020	cc-by	4,991	S P E C I A L I S S U E P A P E R S P E C I A L I S S U E P A P E R Junfeng Wang and Willem R. Keusters contributed equally to this study. 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. © 2020 The Authors. Research Synthesis Methods published by John Wiley & Sons Ltd. Junfeng Wang1 \| Willem R. Keusters1 \| Lingzi Wen2 \| Mariska M. G. Leeflang3 Junfeng Wang1 \| Willem R. Keusters1 \| Lingzi Wen2 \| Mariska M. G. Leeflang3 1Julius Center for Health Sciences and Primary Care, UMC Utrecht, Utrecht University, Utrecht, The Netherlands 2Centre for Evidence-Based Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China 3Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Amsterdam UMC, Amsterdam Public Health, University of Amsterdam, Amsterdam, The Netherlands 1Julius Center for Health Sciences and Primary Care, UMC Utrecht, Utrecht University, Utrecht, The Netherlands 2Centre for Evidence-Based Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China Background: Individual patient data meta-analyses (IPD-MA) are regarded as the gold standard for systematic reviews, which also applies to systematic reviews of diagnostic test accuracy (DTA) studies. An increasing number of DTA systematic reviews with IPD-MA have been published in recent years, but there is much variation in how these IPD-MA were performed. A number of existing methods were found, but there is no consensus as to which methods are preferred as the standard methods for statistical analysis in DTA IPD-MA. Objectives: To create a web-based tool which integrates recommended statisti- cal analyses for DTA IPD-MA, and allows researchers to analyse the data and visualize the results with interactive plots. 2Centre for Evidence-Based Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China 3Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Amsterdam UMC, Amsterdam Public Health, University of Amsterdam, Amsterdam, The Netherlands Objectives: To create a web-based tool which integrates recommended statisti- cal analyses for DTA IPD-MA, and allows researchers to analyse the data and visualize the results with interactive plots. Received: 29 February 2020 Revised: 18 June 2020 Accepted: 11 August 2020 Received: 29 February 2020 Revised: 18 June 2020 Accepted: 11 August 2020 Received: 29 February 2020 Revised: 18 June 2020 Accepted: 11 August 2020 DOI: 10.1002/jrsm.1444 DOI: 10.1002/jrsm.1444 Res Syn Meth. 2021;12:45–54. 1 \| BACKGROUND Embase and Medline databases were searched from 2000 to 2013 (and updated to 2014) by using a well validated search strategy from a research on DTA meta-analyses and adjusted for IPD. Twenty-nine DTA IPD-MA articles published between 2006 and 2014 were selected as the subjects for the final analysis. The detailed search strat- egy and a flow chart for study selection were provided in Data S2. Compared to conventional meta-analysis (ie, meta-analy- sis of interventions), meta-analysis of diagnostic test accu- racy (DTA) is more prone to heterogeneous studies due to both the patient characteristics and the threshold effect.1 Individual patient data meta-analyses (IPD-MA) may help to overcome such difficulties. The richness of individual patient data, in both patient-level variables and the quan- tity of data, can facilitate advanced analyses which could not be done with aggregated data, but it also leads to big variation in the methods of IPD-MA. Individual patient data meta-analyses (IPD-MA) may help to overcome such difficulties. The richness of individual patient data, in both patient-level variables and the quan- tity of data, can facilitate advanced analyses which could not be done with aggregated data, but it also leads to big variation in the methods of IPD-MA. Articles included for the analysis were carefully examined and data was extracted with a data extraction form (in Data S2) designed by two authors (Junfeng Wang and Mariska M. G. Leeflang). Information on sta- tistical methods used was extracted by one author with good knowledge in statistics in test evaluation (Junfeng Wang) and figures and plots for data visualization were extracted by another author (Lingzi Wen). However, not like other types of meta-analyses, the standard approach for DTA IPD-MA is underdeveloped. Many different ways of performing DTA IPD-MA were used in practice, but there was no clue which are the preferred methods. In this technical notes, we will introduce our method- ological review of statistical methods in DTA IPD-MA in practice, and give our recommendations on preferred analyses. Based on the findings of the review, we devel- oped a web-based tool IPDmada with R Shiny, which integrates all the preferred statistical analyses for DTA IPD-MA. IPDmada allows researchers to analyse the data, investigate the study-level and/or patient-level het- erogeneity as well as the threshold effect, and finally visualize the results with interactive plots. K E Y W O R D S covariate-adjusted ROC, diagnostic test accuracy, individual meta-analysis, Shiny, summary ROC 2 \| METHODS IN INDIVIDUAL PATIENT DATA META-ANALYSIS OF DIAGNOSTIC ACCURACY In IPD-MA, researchers not only collected test results of index test(s) and reference standard, but also pursued other patient-level information, which included but not limited to patient characteristics, for example, sex, age, and BMI, and values of other biomarkers or medical tests from the same individual. 2.2 \| Recommended analyses Summary of findings related to statistical analyses are briefly described here, and the full list of findings is pro- vided in Data S2, which was presented in 2015 Cochrane Colloquium.2 DTA IPD meta-analysis is most useful when original continuous test results were provided for each individual. If only binary test results (ie, test result is positive or neg- ative) were provided on individual level, this information could be obtained from the degenerated 2-by-2 tables as well, such IPD-MA will not have much added value com- pared with meta-analysis on aggregated data. Correspondence Correspondence Junfeng Wang, Julius Center for Health Sciences and Primary Care, UMC Utrecht, Utrecht University, Utrecht, The Netherlands. Email: j.wang-4@umcutrecht.nl Methods: A systematic methodological review was performed to identify statis- tical analyses and data visualization methods used in DTA IPD-MA. Methods were evaluated by the authors and recommended analyses were integrated into the IPDmada tool which is freely available online with the user interface developed with R Shiny package. Results: IPDmada allows users to upload their own data, perform the meta- analysis with both continuous and dichotomized tests, and incorporate individ- ual level covariate-adjusted analysis. All tables and figures can be exported as . csv or .pdf files. A hypothetical dataset was used to illustrate the application of IPDmada. Conclusions: IPDmada will be very helpful to researchers doing DTA IPD-MA, since it not only facilitates the statistical analysis but also provides a standard framework. The introduction of IPDmada will harmonize the methods used in DTA IPD-MA and ensure the quality of such analyses. Highlights • IPDmada is a newly developed web-based tool for performing statistical analysis of individual patient data meta-analysis of diagnostic accuracy and visualizing the results. • IPDmada is a newly developed web-based tool for performing statistical analysis of individual patient data meta-analysis of diagnostic accuracy and visualizing the results. wileyonlinelibrary.com/journal/jrsm Res Syn Meth. 2021;12:45–54. 46 WANG ET AL. 46 • The tool is freely available to all the researchers, and requiring no installa- tion of statistical software/packages. • The tool has an user-friendly interface, and allows meta-analysis on both dichotomized and continuous test results. Researchers can easily use this tool to investigate the threshold effect and covariate effect on the summary accuracy. • The introduction and implementation of IPDmada will serve as a useful tool for DTA IPD-MA and increase the quality of such studies. WANG ET AL. 47 IPD meta-analysis of continuous test results and patient-level covariates offers the possibility of per- forming advanced analyses, such as investigating the effect of threshold, for example, reconciling thresholds from primary studies to a predefined value, or using opti- mal threshold within each primary study; directly using continuous test results instead of dichotomizing them, which facilitated the calculation of area under ROC curve instead of summary ROC curve in the conventional meta-analysis; adjusting for baseline differences in study- level as well as patient-level characteristics if these char- acteristics have some confounding effects.3 available on the left side of each page, to display the options available for the analyses on the same page, which can be defined by users. Operation of the tool can be simply done by “point and click” on the web browser. All the analysis results will be presented in tables and figures, which can be exported as .csv or .pdf files. In “Analysis of dichotomized test results,” sensitivi- ties and specificities from primary studies can be redefined by tuning the thresholds, either with a predefined value, an optimal value or any value input by the “slider” bar, and real time forest plot and SROC curve will be plotted accordingly; in Analysis of continuous test results, IPD from primary studies will be integrated into one big dataset and ROC curves for each study and covar- iate-adjusted analysis will be plotted and summary AUC will be presented in a forest plot. In Section 4, we pro- vided an illustrative example of how to use this tool. We found that, like DTA meta-analysis of aggregated data, sensitivity and specificity and area under the ROC curve (AUC) are still the most commonly used measures of test accuracy in DTA IPD-MA. So we will provide all these performance measures for all primary studies in our tool. Given individual level data is available, regression models including fixed and random effects, multilevel and GEE logistic regression models are also used in DTA IPD meta-analysis. However, these models were used for multivariable prediction model development, which is out of the scope of DTA meta-analysis. IPD-MA of diag- nostic modeling studies was discussed elsewhere.4 4 \| AN ILLUSTRATIVE EXAMPLE WITH HYPOTHETICAL DATA In most of the published IPD-MA, patient-level data was not provided. So a hypothetical individual patient data (provided as Data S2) was used as an example for illustra- tive purpose. There were 15 primary studies evaluating one index test and all studies reported the test results as a continuous variable. Disease status and some patient-level covariates were also provided. We assumed no missing values in this example dataset. When missing values exist in one variable, they will be removed from the analyses including that variable and kept for other analyses. So in the IPDmada tool, we will focus on advanced statistical analyses facilitated by IPD which could not be done with aggregated data, in particular cut-off value analysis and covariate adjustment analysis.5 In the mean- while, analyses of aggregated data in the two-stage approach were also provided by using bivariate model6 and HSROC model,7 which are the standard approaches and best practices of DTA meta-analyses.8 2.1 \| Review of methods used in practice A systematic search was performed to identify published articles containing IPD meta-analyses of DTA studies. FIGURE 1 Screenshot of Import Data [Colour figure can be viewed at wileyonlinelibrary.com] Screenshot of Import Data [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 1 Screenshot of Import Data [Colour figure can be viewed at wileyonlinelibrary.com] threshold should be used across all primary studies to get the performance estimates. The properties of this thresh- old should be known to the clinicians. study are also calculated with R package table1 and shown in tab “Summary table.” Data entry of the example data is shown in Figure 1. After the threshold values had been defined as described above, users need to click “Calculate new results!,” then all the results will be presented in the five tabs on the right side. The tab “Test accuracy per study I” shows the threshold value (as define on the left sidebar), true positive number (TP), false negative number (FN), false positive number (FP), true negative number (TN), and sensitivity and specificity according to these numbers (Figure 2). The tab “Test accuracy per study II” shows further analysis results including positive predictive value (PPV), negative prediction value (NPV), likelihood ratio test positive (LR+), likelihood ratio test negative (LR−), diagnostic odds ratio (DOR), and the area under ROC curve (AUC). All these test accuracy measures were cal- culated with R packages epiR12 and pROC. 3 \| THE IPDMADA TOOL On the “Import Data” page, users can upload their pooled individual patient data with a comma delimited (. csv) file. The separator can be either comma, semicolon or tab, users are reminded to choose the appropriate one for their data. The dataset should be constructed as fol- low: three columns contain study name (can be author's name or numerical code, but the variable name must be “Study”), test results of index test (must be a numerical variable named “test.results”), and disease statues (must only contain 0 or 1 and named “disease”). Other columns are for covariates, there are no limitations on the num- bers and names of the covariates. We used R Shiny package9 to create a web-based user interface, which allows interactive data analysis and visualization with R. The advantage of this framework is, we can make use of the R environment and existing R packages, while users do not need any experience with using R. IPDmada was implemented on the webserver pro- vided by shinyapps.io, so installation of R or RStudio on user's device is not needed. The web-based tool can be accessed via the following link https://jwang7.shinyapps. io/ipdmada/. After data was imported, study ID was automatically generated as a sequential number. If display option is Head, then only the first six rows will be presented; option All should be selected to see the full dataset. Beside the raw data, summary statistics of each primary We aimed to design the interface in an user-friendly way, and also guide the users to conduct the IPD-MA step by step. Thus, three pages were created for “Import Data,” “Analysis of dichotomized test results,” and “Analysis of continuous test results.” A sidebar is WANG ET AL. WANG ET AL. 48 FIGURE 1 Screenshot of Import Data [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 2 Screenshot of Analysis of dichotomized test results: Test accuracy table [Colour figure can be viewed at wileyonlinelibrary com] FIGURE 2 Screenshot of Analysis of dichotomized test results: Test accuracy table [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 3 Screenshot of Analysis of dichotomized test results: Forest Plot [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 3 Screenshot of Analysis of dichotomized test results: Forest Plot [Colour figure can be viewed at wileyonlinelibrary.com] All tables and figures shown in the tabs can be down- loaded as .csv files or .pdf files. and one covariate can be chosen for the covariate- adjusted analyses. The first tab “Distribution of test results” shows the distributions of the index test in diseased and non- diseased groups in each study (Figure 5). Ridgelineplot can make multiple density plots of a numeric variable for several groups in a staggered fashion, which is a very informative way to visualize the different distributions of test results in primary studies. R packages ggplot214 and ggridges15 were used to generate the ridgelineplot. 4.2 \| Analysis of dichotomized test results The second page is “Analysis of dichotomized test results,” which provides three options to define the posi- tivity threshold of the index test. With the first option “User selected threshold per study,” N (N = number of primary studies included in MA) slider bars were created and user can tune the threshold value for each study. The default value is set to the median of all the test results. If the option “Optimal threshold” was selected, all slider bars will disappear and optimal threshold defined by optimizing Youden's index was calculated for each pri- mary study with R package pROC.10 The option “Pre- defined threshold” will ask the user to enter a number in the box below. The third tab “Forest Plot” presents the forest plots for sensitivity and specificity separately (Figure 3), and the fourth tab “SROC” presents the summary ROC curve derived from bivariate model6 of sensitivity and specific- ity implemented in R package mada13 (Figure 4). The model parameter estimates for bivariate model and HSROC model7 were provided in the last tab “Model parameter estimates.” Please note that, using optimal threshold in primary studies may lead to bias estimates of test performance11 in meta-analyses, and using different thresholds may not be clinically meaningful. These analyses were mainly for exploration and sensitivity analysis purposes, and same FIGURE 2 Screenshot of Analysis of dichotomized test results: Test accuracy table [Colour figure can be viewed at wileyonlinelibrary.com] WANG ET AL. 4 49 WANG ET AL. 49 4.3 \| Analysis of continuous test results 51 FIGURE 7 Screenshot of Analysis of continuous test results: Forest Plot [Colour figure can be viewed at wileyonlinelibrary.com] Screenshot of Analysis of continuous test results: Forest Plot [Colour figure can be viewed at wileyonlinelib FIGURE 7 Screenshot of Analysis of continuous test results: Forest Plot [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 8 Screenshot of Analysis of continuous test results: Distribution of covariate: Continuous covariate [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 8 Screenshot of Analysis of continuous test results: Distribution of covariate: Continuous covariate [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 9 Screenshot of Analysis of continuous test results: Distribution of covariate: Categorical covariate [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 9 Screenshot of Analysis of continuous test results: Distribution of covariate: Categorical covariate [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 10 Screenshot of Analysis of continuous test results: Correlation between test results and covariate [Colour figure can be viewed at wileyonlinelibrary.com] 52 WANG ET AL. WANG ET AL. WANG ET AL. WANG ET AL. 52 FIGURE 10 Screenshot of Analysis of continuous test results: Correlation between test results and covariate [Colour figure can be viewed at wileyonlinelibrary.com] analysis of diagnostic test of study-level data.22 To our knowledge, IPDmada is the first tool available for DTA IPD-MA. The second tab “ROC curve by study” shows all ROC curves from primary studies in one plot with R packages ggplot2 and plotROC16 (Figure 6). The third tab “Meta- analysis of AUC” shows both fixed effect and random effect meta-analyses of AUC17 in a forest plot, generated by R meta package18 (Figure 7). The development and implementation of IPDmada makes DTA IPD-MA easier to both researchers with or without strong statistical background. IPDmada will also serve as a useful tool for DTA IPD-MA and increase the quality of such studies. Analyses in table 4 to table 8 are based on the covari- ate selected from the left sidebar. The fourth tab “Distri- bution of covariate” is similar to “Distribution of test results” but focused on covariate instead of index test. For continuous covariate ridgelineplot is again used (Fig- ure 8), and for categorical covariate, stacked barchart is used to present the percentages in diseased and non- diseased groups (Figure 9). This figure is aimed to detect different distributions of covariates among primary stud- ies. 4.3 \| Analysis of continuous test results The second page is “Analysis of continuous test results,” which allows direct analysis of the index test on its con- tinuous scale. All the covariates read from the imported data are presented on the left sidebar with a radio button, WANG ET AL. FIGURE 4 Screenshot of Analysis of dichotomized test results: SROC curve [Colour figure can be viewed at wileyonlinelibrary.com] 50 WANG ET AL 50 FIGURE 4 Screenshot of Analysis of dichotomized test results: SROC curve [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 5 Screenshot of Analysis of continuous test results: Distribution of test results [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 5 Screenshot of Analysis of continuous test results: Distribution of test results [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 6 Screenshot of Analysis of continuous test results: ROC curve by study [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 6 Screenshot of Analysis of continuous test results: ROC curve by study [Colour figure can be viewed at wileyonlinelibrary com] FIGURE 6 Screenshot of Analysis of continuous test results: ROC curve by study [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 7 Screenshot of Analysis of continuous test results: Forest Plot [Colour figure can be viewed at wileyonlinelibrary.com] WANG ET AL. 51 51 WANG ET AL. 51 FIGURE 7 Screenshot of Analysis of continuous test results: Forest Plot [Colour figure can be viewed at wileyonlinelibrary.com] FIGURE 8 Screenshot of Analysis of continuous test results: Distribution of covariate: Continuous covariate [Colour figure can be viewed at wileyonlinelibrary.com] WANG ET AL. 4.3 \| Analysis of continuous test results The fifth tab “Correlation between test results and covariate” presents the relation between test and covari- ate, by visualizing the study specific liner regression models in a scatter plot (Figure 10). Besides the basic analysis provided in IPDmada, the most important functions of IPDmada are interactive anal- ysis of threshold effect and covariate-adjusted test accu- racy, which are the key advantages of using IPD-MA. The tool has been back to back tested by the authors with several extreme situations, for example, all the values are missing in one study, wrong input data. It can work properly in such situations. IPDmada also has some limitations. A comprehensive systematic review was performed to identify the rec- ommended analyses for DTA IPD-MA, however, the review only included publications till the end of 2014. Future work is needed to consider other new methods introduced recently, and to the best of our knowledge, such methods are still underdeveloped. Furthermore, the selection was based on most often used methods observed in the survey and considered meaningful and useful by the research team, which was a little subjective. It should be seen as a recommendation rather than compulsory requirement, and alternative methods can be still used by researchers in their own IPD-MAs. Different methods were only compared conceptually, and we did not com- pare the performance of these methods or check the robustness through simulations, given they are all exis- ting methods and were tested when they were firstly pro- posed. For data entry, variable names of important variables (study name, test results, disease status) are The last two tabs contain figures similar to Figures 5 and 6, however, the ROC curves and AUC were calcu- lated with covariate-adjusted ROC analysis5 following R package AROC.19 ORCID Junfeng Wang https://orcid.org/0000-0001-5157-5355 WANG ET AL. 53 fixed, users need to prepare their data in the correct for- mat before uploading to the webpage. For covariate ana- lyses, the current version only allows the users to analyze one covariate at one time. Researchers might be inter- ested in adjusting multiple covariates simultaneously in one model, and we are planning to add this function in future updates. In all analyses, complete cases analyses were performed when there were missing values in pri- mary studies. Advanced methods, for example, multiple imputation, would be a better approach to handle miss- ing data. However, multiple imputation may rely on strong assumptions on the mechanisms of missingness. The imputed datasets need to be carefully examined, and analyses of multiply imputed data are also complicated. This requires more statistical knowledge from the user and does not fit the purpose of this easy-to-use tool. fixed, users need to prepare their data in the correct for- mat before uploading to the webpage. For covariate ana- lyses, the current version only allows the users to analyze one covariate at one time. Researchers might be inter- ested in adjusting multiple covariates simultaneously in one model, and we are planning to add this function in future updates. In all analyses, complete cases analyses were performed when there were missing values in pri- mary studies. Advanced methods, for example, multiple imputation, would be a better approach to handle miss- ing data. However, multiple imputation may rely on strong assumptions on the mechanisms of missingness. The imputed datasets need to be carefully examined, and analyses of multiply imputed data are also complicated. This requires more statistical knowledge from the user and does not fit the purpose of this easy-to-use tool. REFERENCES 1. Macaskill P, Gatsonis C, Deeks J, Harbord R, Takwoingi Y. Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy. The Cochrane Collaboration; 2010. http://srdta. cochrane.org/. 2. Wang J, Bossuyt P, Geskus R, Zwinderman A, Leeflang M. Individual patient data meta-analysis for diagnostic test accu- racy studies: a review of methods used in practice. Filtering the Information Overload for Better Decisions. Abstracts of the 23rd Cochrane Colloquium. Vienna, Austria: John Wiley & Sons; 2015, 2015:3-7. 3. Wang J, Bossuyt P, Geskus R, et al. Using individual patient data to adjust for indirectness did not successfully remove the bias in this case of comparative test accuracy. J Clin Epidemiol. 2015;68(3):290-298. IPDmada only has an online version for now, since it is easier for maintenance and update. Users may want to run the analysis locally at their own device, considering IPD usually contains confidential patient-level data. We are working on the IPDmada package to fulfill this requirement. 4. Debray TP, Riley RD, Rovers MM, Reitsma JB, Moons KG. Individual participant data (IPD) meta-analyses of diagnostic and prognostic modeling studies: guidance on their use. PLoS Med. 2015;12(10):e1001886. 5. Janes H, Longton G, Pepe MS. Accommodating covariates in receiver operating characteristic analysis. Stata J. 2009;9(1): 17-39. It should be noted that, a easy-to-use tool is some- times a double-edge sword: it provides convenience to researches but also lowers the knowledge requirement for performing the analyses. Even a perfect tool cannot by itself ensure that the data will be analyzed properly and the results will be interpreted correctly. It is highly recommended and encouraged to have a methodologist and a statistician in a review team. 6. Reitsma JB, Glas AS, Rutjes AW, Scholten RJ, Bossuyt PM, Zwinderman AH. Bivariate analysis of sensitivity and specific- ity produces informative summary measures in diagnostic reviews. J Clin Epidemiol. 2005;58(10):982-990. 7. Rutter CM, Gatsonis CA. A hierarchical regression approach to meta-analysis of diagnostic test accuracy evaluations. Stat Med. 2001;20(19):2865-2884. 8. Macaskill P, Gatsonis C, Deeks J, Harbord R, Takwoingi Y. Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy. Version 0.9. 0. London, England: The Cochrane Col- laboration; 2010. ACKNOWLEDGMENT We would like to thank Bowen Li for his advice on the design of the Shiny tool. 9. Chang W, Cheng J, Allaire J, Xie Y, McPherson J. Shiny: web application framework for R. R package version 1.4.0. 2019. https://cran.r-project.org/package=shiny. CONFLICT OF INTEREST 10. Robin X, Turck N, Hainard A, et al. pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinform. 2011;12(1):77. The author reported no conflict of interest. The author reported no conflict of interest. 11. Ewald B. Post hoc choice of cut points introduced bias to diag- nostic research. J Clin Epidemiol. 2006;59(8):798-801. AUTHOR CONTRIBUTIONS Junfeng Wang and Mariska M. G. Leeflang designed the methodology review and recommended methods for DTA IPD-MA. Junfeng Wang and Lingzi Wen reviewed selected articles and extracted information. Junfeng Wang and Willem R. Keusters designed and developed the Shiny tool. Junfeng Wang, Willem R. Keusters, and Lingzi Wen generated data and tested the tool. All authors provided a substantial contribution to the design and implementation of the web tool, as well as writing or editing the manuscript, and approving the final version. 12. Stevenson M, Nunes T, Sanchez J, et al. epiR: Tools for the analysis of epidemiological data. R package version 1.0-11. 2020. https://CRAN.R-project.org/package=epiR. 13. Doebler P, Holling H. Meta-analysis of diagnostic accuracy with mada. R Package. 2015;1:15. 14. Wickham H, Chang W, Wickham MH. Package ‘ggplot2’. Cre- ate elegant data visualisations using the grammar of graphics. Version. 2016;2(1):1-89. 15. Wilke CO. ggridges: Ridgeline Plots in ‘ggplot2’. R package version 0.5.2. 2020. https://CRAN.R-project.org/package=ggridges. 16. Sachs MC. plotROC: a tool for plotting ROC curves. J Stat Softw. 2017;79:1-19. 5 \| DISCUSSION In this article, we introduced IPDmada, a newly devel- oped web-based tool for individual patient data meta- analysis of diagnostic accuracy. In this article, we introduced IPDmada, a newly devel- oped web-based tool for individual patient data meta- analysis of diagnostic accuracy. Web-based meta-analysis tools developed with R Shiny gained popularity in recent years. Several tools are available as packages or online tools: MAVIS for meta- analysis of effect size,20 MetaInsight for network meta- analysis of interventions,21 and MetaDTA for meta- DATA AVAILABILITY STATEMENT 17. Zhou XH. Empirical Bayes combination of estimated areas under ROC curves using estimating equations. Med Decis Mak- ing. 1996;16(1):24-28. The data that supports the findings of this study are avail- able in the supplementary material of this article. 54 WANG ET AL. 54 conduct and interrogate meta-analysis of diagnostic test accu- racy studies: MetaDTA. BMC Med Res Methodol. 2019;19(1):81. 18. Balduzzi S, Rücker G, Schwarzer G. How to perform a meta-anal- ysis with R: a practical tutorial. Evidence-based mental health. 2019;22(4):153-60. https://cran.r-project.org/web/packages/meta/ citation.html. conduct and interrogate meta-analysis of diagnostic test accu- racy studies: MetaDTA. BMC Med Res Methodol. 2019;19(1):81. SUPPORTING INFORMATION 19. Rodriguez-Alvarez MX, de Carvalho VI. AROC: Covariate- adjusted receiver operating characteristic curve inference. R package version 1.0-2. 2019. https://CRAN.R-project.org/ package=AROC. Additional supporting information may be found online in the Supporting Information section at the end of this article. 20. Hamilton WK, Aydin B, Mizumoto A. MAVIS: Meta Analysis Via Shiny. Merced, CA: Comprehensive R Archive Network (CRAN); 2016. How to cite this article: Wang J, Keusters WR, Wen L, Leeflang MMG. IPDmada: An R Shiny tool for analyzing and visualizing individual patient data meta-analyses of diagnostic test accuracy. Res Syn Meth. 2021;12:45–54. https://doi.org/10.1002/ jrsm.1444 How to cite this article: Wang J, Keusters WR, Wen L, Leeflang MMG. IPDmada: An R Shiny tool for analyzing and visualizing individual patient data meta-analyses of diagnostic test accuracy. Res Syn Meth. 2021;12:45–54. https://doi.org/10.1002/ jrsm.1444 21. Owen RK, Bradbury N, Xin Y, Cooper N, Sutton A. Meta- Insight: an interactive web-based tool for analyzing, interrogat- ing, and visualizing network meta-analyses using R-shiny and netmeta. Res Synth Methods. 2019;10:569-581. 22. Freeman SC, Kerby CR, Patel A, Cooper NJ, Quinn T, Sutton AJ. Development of an interactive web-based tool to
https://openalex.org/W2137383110	https://europepmc.org/articles/pmc3680401?pdf=render	English	null	The Mangrove Nursery Paradigm Revisited: Otolith Stable Isotopes Support Nursery-to-Reef Movements by Indo-Pacific Fishes	PloS one	2,013	cc-by	8,146	Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution L restricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded through WOTRO Science for Global Development (WOTRO)by the Netherlands Organization for Scientific Research (NWO). I.N. was funded by a Vidi grant from NWO. The field work was supported by a grant from Schure-Beijerinck-Popping Fonds and Western Indian Ocean Marine Science Association (WIOMSA) to I.A.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Funding: This study was funded through WOTRO Science for Global Development (WOTRO)by the Netherlands Organization for Sci was funded by a Vidi grant from NWO. The field work was supported by a grant from Schure-Beijerinck-Popping Fonds and Western In Association (WIOMSA) to I.A.K. The funders had no role in study design, data collection and analysis, decision to publish, or prepara was funded by a Vidi grant from NWO. The field work was supported by a grant from Schure-Beijerinck-Popping Fonds and Western Indian Ocean Marine Science Association (WIOMSA) to I.A.K. 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. mpeting Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ivan.nagelkerken@adelaide.edu.au The Mangrove Nursery Paradigm Revisited: Otolith Stable Isotopes Support Nursery-to-Reef Movements by Indo-Pacific Fishes ael A. Kimirei1,2, Ivan Nagelkerken1,3*, Yunus D. Mgaya4, Chantal M. Huijbers1,5 1 Radboud University Nijmegen, Institute for Water and Wetland Research, Department of Animal Ecology and Ecophysiology, Nijmegen, The Netherlands, 2 Tanzania Fisheries Research Institute, Kigoma, Tanzania, 3 Southern Seas Ecology Laboratories, School of Earth and Environmental Sciences, The University of Adelaide, Adelaide, Australia, 4 College of Natural and Applied Sciences, Department of Aquatic Science and Fisheries, University of Dar es Salaam, Dar es Salaam, Tanzania, 5 Australian Rivers Institute – Coasts and Estuaries, Griffith University, Gold Coast campus, Southport, Australia Abstract Mangroves and seagrass beds have long been perceived as important nurseries for many fish species. While there is growing evidence from the Western Atlantic that mangrove habitats are intricately connected to coral reefs through ontogenetic fish migrations, there is an ongoing debate of the value of these coastal ecosystems in the Indo-Pacific. The present study used natural tags, viz. otolith stable carbon and oxygen isotopes, to investigate for the first time the degree to which multiple tropical juvenile habitats subsidize coral reef fish populations in the Indo Pacific (Tanzania). Otoliths of three reef fish species (Lethrinus harak, L. lentjan and Lutjanus fulviflamma) were collected in mangrove, seagrass and coral reef habitats and analyzed for stable isotope ratios in the juvenile and adult otolith zones. d13C signatures were significantly depleted in the juvenile compared to the adult zones, indicative of different habitat use through ontogeny. Maximum likelihood analysis identified that 82% of adult reef L. harak had resided in either mangrove (29%) or seagrass (53%) or reef (18%) habitats as juveniles. Of adult L. fulviflamma caught from offshore reefs, 99% had passed through mangroves habitats as juveniles. In contrast, L. lentjan adults originated predominantly from coral reefs (65–72%) as opposed to inshore vegetated habitats (28–35%). This study presents conclusive evidence for a nursery role of Indo-Pacific mangrove habitats for reef fish populations. It shows that intertidal habitats that are only temporarily available can form an important juvenile habitat for some species, and that reef fish populations are often replenished by multiple coastal habitats. Maintaining connectivity between inshore vegetated habitats and coral reefs, and conserving habitat mosaics rather than single nursery habitats, is a major priority for the sustainability of various Indo Pacific fish populations. Citation: Kimirei IA, Nagelkerken I, Mgaya YD, Huijbers CM (2013) The Mangrove Nursery Paradigm Revisited: Otolith Stable Isotopes Support Nursery-to-Reef Movements by Indo-Pacific Fishes. PLoS ONE 8(6): e66320. doi:10.1371/journal.pone.0066320 Editor: Sharyn Jane Goldstien, University of Canterbury, New Zealand Received January 9, 2013; Accepted May 3, 2013; Published June 12, 2013 irei 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. pyright: 2013 Kimirei et al. June 2013 \| Volume 8 \| Issue 6 \| e66320 Nursery-to-Reef Movements by Indo-Pacific Fishes Only a couple of studies have provided unambiguous evidence of nursery-to-reef movement in the Indo-Pacific [17,21], but failed to separate the role of individual juvenile habitats (e.g. mangrove vs. seagrass vs. reef). As a result, we know little of how different nursery habitats contribute to overall replenishment of offshore adult populations. This gap of knowledge is concerning as species may show different dependencies on different nursery habitats, and this dependency may further change through ontogeny. Hence, identifying the individual contribution of multiple nursery habitats to adult populations is of critical importance for management and conservation purposes. The objective of this study, therefore, was to test to which degree a suite of Indo-Pacific reef fish species has passed through putative mangrove vs. seagrass vs. coral reef nursery habitat. We analyzed a robust dataset of otoliths from several Indo-Pacific coral reef fish species collected at nearshore and offshore reef sites in Tanzania. Our results reveal the degree of connectivity among Indo-Pacific tropical coastal habitats, and we evaluate the role of two critical, circumtropical juvenile habitats for adult reef fish populations at different distances from the shore. Studies on nursery function of tropical reef habitats have predominantly focused on the Caribbean region, while the much larger Indo-Pacific region remains largely unstudied [24]. There is no a priori reason to reject a potential importance of ecosystem connectivity for offshore productivity and replenishment of reef populations in the Indo-Pacific, and it is likely that coastal reef seascapes in the Indo-Pacific are connected in similar ways by fish movements as in the Caribbean [9,24,25]. The function of shallow-water ecosystems as juvenile habitat depends, however, on habitat availability and accessibility [26]. Unlike in the Caribbean where shallow-water habitats (especially mangroves) are perma- nently available to juvenile fish [27], Indo-Pacific mangrove systems along coastal shorelines are mostly available to fish only during high tides [9,28]. Also, the arrangement of mangroves and seagrass beds in relation to reef habitats within the coastal seascape can profoundly affect the degree and type of ecological and biological connectivity between these habitats [29,30]. In the Indo- Pacific region, clear-water vegetated habitats are often more intermixed compared to Caribbean islands where they are spatially separated, while the much larger tidal ranges in the Indo-Pacific facilitate non-ontogenetic reef fish movements [28]. Nursery-to-Reef Movements by Indo-Pacific Fishes as stable isotope signatures in fish muscle tissue and earbones (otoliths) or elemental composition of otoliths [13]. (island vs. continental coastlines). Another questionable argument that has previously been used to evaluate nursery function is that relatively few Indo-Pacific fish species appear to depend on mangrove habitats [36]. However, if these few species are of high commercial importance, are highly abundant species, or fulfill important ecological roles, then they can have important impacts on ecosystem production, functioning and resilience [11,37,38]. The application of otolith chemistry to track fish movement is based on the assumption that fish living and feeding in different environments incorporate a detectable chemical tag if they reside in environments long enough [14]. Otoliths grow continuously throughout the life of a fish and remain chemically inert once formed, and can thus provide a detailed history of a fish’s environment. The use of elemental chemistry is less suitable for non-estuarine tropical environments as the water chemistry of juvenile vs. adult marine habitats is usually more uniform [15] as opposed to those located along a gradient from fresh to marine waters in (temperate) estuarine regions. This problem does not arise when using stable isotope signatures of otoliths, such as 12C/13C ratios, which clearly differ among different vegetated habitats [16,17]. Dissolved inorganic carbon (DIC) typically contributes 70–80% to otolith carbon and varies among water bodies around major vegetation types. Oxygen isotopes are often related to variability in water temperature and salinity and can thus provide a distinct signature of habitats in shallow, warmer water like mangroves and seagrass beds compared to coral reefs in cooler water [18]. Therefore, it is a very suitable method to determine ontogenetic shifts among habitats. Although otolith chemistry is recognized as a valuable tool, and has been increasingly used over the last decade, still very few studies have used otoliths to reconstruct the environmental history of fish [19]. Only very recently a few studies have provided convincing evidence of ontogenetic movement from Caribbean mangrove/ seagrass nurseries to adult offshore habitat [20,21,22,23]. Recent studies have found that during their juvenile stage multiple species are present only in Indo-Pacific mangroves [39,40,41], and that isolated reefs show significantly lower adult densities of such species than reefs directly adjacent to these ecosystems [42]. This suggests that mangroves in this region could play a valuable role in replenishing offshore populations of some species. Nursery-to-Reef Movements by Indo-Pacific Fishes The large tidal range potentially also leads to stable isotope signatures showing more overlap among different habitats due to tidal exchange of water bodies between habitats [31,32]. Ethics Statement This study was not evaluated by an animal ethics committee because there was no such committee in Tanzania during the course of the study. We obtained written permission from the Director of Fisheries in the Ministry of Livestock and Fisheries Development (MLFD) of the United Republic of Tanzania (URT) in 2009 [43] to capture fish on the reef using spear guns and fish in the mangroves using nets. On the coral reef, fish were sacrificed under water directly after spearing by cervical dislocation. Mangrove and seagrass fish were mostly supplied dead by fishermen, but the fish we caught ourselves in the mangroves were sacrificed by hypothermia. Introduction habitats, conservation efforts should focus on protecting habitat mosaics [10]. Coastal habitats such as mangroves and seagrass beds are acknowledged as important nursery habitats for various species of reef fish, most of which are important to fisheries [1,2] and some of which are threatened [3]. These ecosystems are, however, highly affected by anthropogenic stressors like unsustainable fishing practices, habitat loss, and eutrophication [1,4]. Seagrass beds are declining globally at rates of about 7% per year [5] while mangroves are decreasing in surface area by 1–2% per year [6]. Conservation and management of these habitats and their fisheries has received increasing attention based on their importance as juvenile fish habitat and their biological connectivity that enhances coastal marine productivity and biodiversity [7]. Likewise, designation and performance of marine protected areas (MPAs) can be improved by knowledge accrued from habitat connectivity studies [8,9]. Because species that undergo ontogenetic habitat shifts cannot be conserved and managed by protecting single Until very recently, only indirect and circumstantial evidence existed in support of the paradigm that various species of coral reef fishes use mangroves or seagrass beds as essential juvenile habitat. Evidence was mostly based on higher juvenile densities and lower predation risk in these habitats as compared to the adult coral reef habitat (see review by [11]). Nurseries are defined as habitats whose ‘contribution per unit area to the production of individuals that recruit to adult populations is greater, on average, than production from other habitats in which juveniles occur’ [12]. Therefore, a habitat will only function as a productive nursery if its individuals reach adult populations, for which evidence of actual movement between habitats is of crucial importance. Long-term movement data to support ontogenetic cross-ecosystem shifts is difficult to obtain as artificial tags are expensive and not suitable for use in juvenile fishes or for long-term tracking. As a result, there has been an increasing focus on the use of natural tags such June 2013 \| Volume 8 \| Issue 6 \| e66320 1 June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org Nursery-to-Reef Movements by Indo-Pacific Fishes June 2013 \| Volume 8 \| Issue 6 \| e66320 Data and Statistical Analysis 13 y Combined stable carbon (d13C) and oxygen (d18O) signatures of the juvenile (inner) otolith sections from adult reef fish were compared to that of the outer otolith section from juvenile fish living in mangrove or seagrass habitats to determine whether adult fish had passed through either of these juvenile habitats. Fishes caught from the coral reef spanned a wide size range (Table 1), thus reflecting different birth years. We therefore collected juvenile fish from the putative mangrove and seagrass nurseries in different years, to incorporate into our analysis potential temporal variability in otolith stable isotope signatures within nursery habitats. A MANOVA showed that either d13C or d18O varied significantly (F.6.28, p,0.001) across years for the 3 species, but never both stable isotope signatures together, meaning that habitats could always be distinguished throughout time based on at least one stable isotope signature. Notwithstanding some temporal variability, there was little overlap among habitat signatures (see Fig. 2). A one-way ANOVA was used to test for differences in otolith d13C and d18O, respectively, between habitats. We tested for significant differences in otolith d13C and d18O between the juvenile signatures of mangrove and seagrass fish, and the adult signatures of coral reef fish (i.e. outer otolith margin). A Gabriel post-hoc test was used for comparison of means, while a Games-Howell post-hoc test was used when the requirement for homogeneity of variance was violated. Signifi- cance levels of p,0.05 were used in all tests. A quadratic discriminant function analysis (QDFA) using the jack-knife classification was done to examine classification success of assigning individuals to their known origin. SPSS 20 for Windows was used for all analyses [46]. Figure 1. Map of the study area. Reef contours (approx. 17 m depth) are indicated by thick black lines. Hatched area indicates location of the mangrove forest. SGK = sampling site at the shoreline seagrass bed at Kunduchi. Nearshore reefs fringe the island of Mbudya, while offshore reefs are located at ‘Far Reef’ and ‘Gold Reef’. doi:10.1371/journal.pone.0066320.g001 Three fish species, viz. Lethrinus harak, L. lentjan, and Lutjanus fulviflamma, were selected for this study. Studies based on size- frequency data suggest that these species undergo ontogenetic mangrove/seagrass-to-reef habitat shifts [40,41,44,45]. Fishes were collected from three different habitats (mangrove, seagrass, coral reef) (Table 1). An earlier visual census study showed that these habitats and locations harbored highest densities of the selected species ([40], Kimirei unpubl. Data and Statistical Analysis 13 data); nevertheless it is possible that we did not sample some minor juvenile habitat types in the area. Adults of L. lentjan and L. fulviflamma were caught from both the nearshore reef around Mbudya Island (about 3 km to mangrove) and two offshore reefs (,9 km to mangrove), whereas L. harak could only be caught from the nearshore reef due to their very low abundances on the offshore reefs [40]. Fishes from the mangrove habitat were collected with a 1610 m seine net which was dragged against the current during outgoing tide, as well as using hook and line. Fishes from the seagrass beds were all purchased from local fishermen that utilized beach seines at low tide. As fishermen operated their seine nets in front of the research institute the origin of these fish could be confirmed. Specimens from the coral reef were caught using a spear gun. All specimens were measured for total length (TL) and weight before the sagittae otoliths were removed, cleaned and stored pending analysis. A maximum likelihood analysis (MLA; estimator # 5) ‘HISEA’ developed by Millar [47] was used to determine the proportion of adult fish originating from the different habitats. For this analysis we used the combination of stable carbon and oxygen isotope signatures of the outer otolith margins of juvenile mangrove and Nursery-to-Reef Movements by Indo-Pacific Fishes Nursery-to-Reef Movements by Indo-Pacific Fishes Figure 1. Map of the study area. Reef contours (approx. 17 m depth) are indicated by thick black lines. Hatched area indicates location of the mangrove forest. SGK = sampling site at the shoreline seagrass bed at Kunduchi. Nearshore reefs fringe the island of Mbudya, while offshore reefs are located at ‘Far Reef’ and ‘Gold Reef’. doi:10.1371/journal.pone.0066320.g001 otoliths were then cross-sectioned in the transverse plane through the core. For juvenile fishes from the mangroves and seagrass bed the outer otolith margin which reflects the current habitat was analyzed. For larger fishes from the reef, both the juvenile zone (which is the area directly adjacent to the core) which reflects earlier life in putative nurseries, and the outer otolith margin reflecting the adult reef habitat, were analyzed. The location for sampling the juvenile zone in adult otoliths was based on the mean otolith width of the mangrove/seagrass juvenile fish. For large L. harak ($15 cm TL) from the seagrass beds, both the inner and outer otolith zones were analyzed to additionally determine the degree to which large juveniles from seagrass beds had spent their earlier juvenile stage in mangrove habitat and had moved to seagrass beds afterwards. The sectioned otoliths were drilled with a micromill that produced otolith CaCO3 powder from a crater with a diameter of approximately 0.35 mm. Two craters were drilled per sample on opposite sides of the cross section to provide enough otolith powder for analysis. The powder (weight $10 mg) was collected with a scalpel and put into a glass tubes for further analysis. A few drops of pure (100%) orthophosphoric acid were added to the tube containing the powder at 80uC to dissolve all CaCO3. The isotope ratios of 12C/13C and 16O/18O of the released CO2 were measured using a Gas Bench mass spectrometer equipped with an automated carbonate extraction line (Kiel device). The NIST SRM 8544 (NBS 19) was used as a carbonate standard, which was routinely monitored during sample runs. The precision of analyses based on the measurements of this standard was within 0.05%. Study Area and Species The Kunduchi area in Dar es Salaam Tanzania, where the study was conducted, has only one mangrove-lined creek (Manyema Creek), an extensive shoreline seagrass bed, two nearshore islands with fringing coral reefs which are separated from the mainland by a 15 m deep channel running almost parallel to the coastline, and several offshore submerged deep coral reefs (Figure 1). The mangroves are dominated by Sonneratia alba along the sides of the Manyema creek. The creek receives substantial freshwater input only during heavy rainfall. The seagrass bed along the shoreline of the mainland lies at 0.5–5 m depth and the nearshore reefs along the island of Mbudya lie at 2– 4 m depth, depending on the tides, while the deep offshore coral reefs are located at 15–20 m followed by mudflats at greater depths [40]. Even though the Indo-Pacific region harbors vast areas of mangroves and tropical seagrass beds, our understanding of the nursery role of these habitats in this region remains rudimentary. Based on fish density data, there has been a long standing debate of this function in this region (see reviews by [33] and [11]). The consensus from most studies is that Indo-Pacific mangroves play a minor role as critical juvenile habitat for reef or offshore fish species [34,35,36]. Nagelkerken [24] argued, however, that the apparent disparity between the two biogeographic regions is based on an invalid comparison, confounded by differences in tidal range (low vs. high), salinity (estuarine vs. marine), and spatial setting June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org 2 Otolith Analysis After cleaning with deionized water, otoliths were mounted on glass plates and embedded in Araldite resin. The embedded June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66320 3 Nursery-to-Reef Movements by Indo-Pacific Fishes Table 1. The number of individuals collected per habitat and species per year. Lethrinus harak Lethrinus lentjan Lutjanus fulviflamma Year NCR OCR SG MG NCR OCR SG MG NCR OCR SG MG 2007 - - 5 - - - - - - - 17 16 2008 1 - 3 14 1 - 13 4 - - 7 6 2009 25 - 28 - 1 - 6 - - - - - 2010 - - - - 56 - - - - 22 - - 2011 - - - - - - - - 20 - - - 2012 - - - - - 20 - - - - - - Size range (cm) 24.6–39.6 - 8.2–25.2 3.3–10.7 21.2–35.6 16.1–38.4 8.0–20.4 3.8–9.0 17.9–22.3 16.2–20.0 14.4–20.9 4.0–13.2 NCR = nearshore coral reefs; OCR = offshore coral reefs; SG = seagrass bed; MG = mangroves; size range = total fish length. doi:10.1371/journal.pone.0066320.t001 NCR = nearshore coral reefs; OCR = offshore coral reefs; SG = seagrass bed; MG = mangroves; size range = total fish length doi:10.1371/journal.pone.0066320.t001 seagrass fish and of adult coral reef fish as baseline data. We used the otoliths of adult reef fish instead of juvenile reef fish as the latter were not observed on the reef during visual surveys. Because otolith composition results from a complex interaction between physiological and environmental factors [14], a potential ontoge- netic effect on d13C and d18O cannot be completely ruled out a priori. However, we performed linear regression analyses between reef fish body size and stable carbon and oxygen isotope values, respectively, to ascertain that the coral reef signature from the adult otolith margins was reflective of fish from the ocean environment, and did not differ from that of juvenile reef fish (if they were to be found on the reef) due to growth. We tested reef fish between 16 and 40 cm in length, comprising fish from a wide range in age (between ,3 and 28 years). Results The three habitats (mangrove, seagrass, and coral reef) differed significantly based on otolith d13C and/or d18O signatures (Table 2, Fig. 2). For L. harak, otolith d13C differed significantly among all habitats, while seagrass d18O signatures differed from that of the mangrove and coral reef, respectively. For L. lentjan both otolith d13C and d18O differed between the coral reef and seagrass, but not between the coral reef and mangroves, or between seagrass and mangroves. When seagrass and mangrove signatures were combined they did significantly differ from the coral reef signature. For L. fulviflamma only otolith d18O differed among all habitats. Classification success based on a quadratic discriminant function analysis was very high for L. harak and L. lentjan (.73%) and relatively high for L. fulviflamma (61%) (Table 3). The classification was based on d13C as well as d18O otolith signatures and both isotope ratios were important in discrimina- tion of the three different habitats. This study is one of the first to present conclusive evidence that mangrove and seagrass habitats replenish reef fish populations in an Indo-Pacific locality, and is the first to identify the relative importance of multiple potential juvenile habitats in replenishing adult populations. The importance of these putative juvenile habitats differed among fish species, and among reefs located at different distances from these habitats. For adults collected on nearshore reefs, the combined contribution of mangrove and seagrass habitat was highest for L. harak (82%), followed by L. lentjan (35%) and L. fulviflamma (19%), while for offshore reefs this was 28% and 100% for the latter two species, respectively. The large contribution of coral reefs to the nearshore as well as offshore adult populations of L. lentjan (65–72%) suggests that L. lentjan populations may be largely self-replenishing by the reef habitat. In contrast, the juvenile source habitats that were important for L. fulviflamma showed a large contrast for adults collected from nearshore vs. offshore reefs. However, considering that reef fish densities for this species were 13 times higher on offshore than nearshore reefs (1.25 vs. 0.09 fish per 100 m2, respectively), most of the reef fish in this coastal area had likely originated from mangroves in terms of total population size. The observed variability in juvenile habitat contribution to adult populations indicates clear species-specific differences in nursery habitat dependency as well as presence of spatial differences in degree of population replenishment by nursery habitats [40]. Results For all three species, except L. fulviflamma from the nearshore reef, the d13C and/or d18O signatures from juvenile margins of nearshore and offshore reef fish individuals were significantly different compared to the adult margin signatures (Fig. 2), suggesting that the two life stages used different habitats. The results of the maximum likelihood analysis using both carbon and oxygen stable isotopes showed that adults from the three species had passed through different juvenile habitats. Adult L. harak fish from the reef were only collected on nearshore reefs. Over 81% of these fish originated from either mangrove (29%) or seagrass (53%) habitats, while the remainder (18%) had grown up on the reef (Table 3). Large specimens caught on the seagrass bed originated largely from the seagrass (70%) and partly from the mangrove (30%) habitat. Most adults of L. lentjan that were collected on the offshore reefs had spent their juvenile stage on coral reefs (72%) compared to a much smaller amount of fish that had passed through seagrass and mangrove habitats (28%). Nearshore adult reef fish of this species also predominantly originated from coral reefs (65%). L. fulviflamma showed contrasting results: for adults collected on offshore reefs, mangroves were the dominant juvenile The results from our otolith study supported those from field surveys for some species, but not for others. Adult populations of L. harak were replenished to a greater degree by seagrass beds than by mangroves. This result corresponds to previous visual census data which also showed that mangroves are less important as a juvenile habitat for L. harak compared to seagrass [40]. On the contrary, L. lentjan showed a higher contribution from the coral reef to both nearshore and offshore adult populations than seagrass bed and mangrove combined, whereas visual census surveys suggest that this species primarily uses seagrass beds as juvenile habitats [40]. The above indicates that visual census data should be interpreted Table 2. Results of a one-way ANOVA on otolith d13C and d18O, respectively, of Lethrinus harak, Lethrinus lentjan, and Lutjanus fulviflamma for three potential juvenile habitats. Otolith Analysis None of the regressions for either carbon or oxygen showed a significant relationship with size (p.0.11, R2,0.14) for any species, except for d13C in Lethrinus lentjan which showed a positive relationship (p = 0.01) but with a low R2 explaining only a small proportion of the overall variability (R2 = 0.32 and 0.12 for nearshore vs. offshore reef fish, respec- tively). The observation that increase in body size has little to no effect on stable isotope signatures in our fish was further supported by lack of such a relationship in the fishes collected from the seagrass beds as well, which had a sufficiently large size range to test for this potential ontogenetic effect (all separate regression for fish size vs. d13C and d18O, respectively, for all 3 species: p.0.14, R2,0.08). These results show that potential ontogenetic effects are minor compared to habitat differences. Signatures from the inner (juvenile) part of adult reef fish otoliths were used in the MLA as Figure 2. Mean (6 SE) otolith d13C and d18O from the outer otolith margins of juvenile fish collected from mangroves (Mg) and seagrass (Sg) habitats, from the inner (juvenile) parts of otolith of adult fish collected from nearshore (NCr) and offshore (OCr) reefs, and from the outer otolith margins of coral reef adults (Cr), averaged per habitat for each of the species: a) Lethrinus harak, b) Lethrinus lentjan, and c) Lutjanus fulviflamma. doi:10.1371/journal.pone.0066320.g002 Figure 2. Mean (6 SE) otolith d13C and d18O from the outer otolith margins of juvenile fish collected from mangroves (Mg) and seagrass (Sg) habitats, from the inner (juvenile) parts of otolith of adult fish collected from nearshore (NCr) and offshore (OCr) reefs, and from the outer otolith margins of coral reef adults (Cr), averaged per habitat for each of the species: a) Lethrinus harak, b) Lethrinus lentjan, and c) Lutjanus fulviflamma. doi:10.1371/journal.pone.0066320.g002 June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org 4 Nursery-to-Reef Movements by Indo-Pacific Fishes Nursery-to-Reef Movements by Indo-Pacific Fishes Nursery-to-Reef Movements by Indo-Pacific Fishes the unknown mixed dataset to estimate the origin of these fishes. Finally, to determine in which habitat large juvenile L. harak, collected in seagrass habitat, had spent their earlier juvenile stage, juvenile seagrass and mangrove signatures were used as the baseline, and adult outer margins as unknown mixed sample. Maximum likelihood estimates and standard deviations were generated in HISEA by bootstrapping with 500 simulations. Otolith Analysis habitat (99%) and none of these fish had a juvenile signature that indicated a contribution from the coral reef. In contrast, nearshore L. fulviflamma adults mainly showed a coral reef signature (81%) in the juvenile zones of their otoliths, compared to that from seagrass (19%) or mangrove (0.03%) habitat. However, L. fulviflamma densities on nearshore reefs were at least an order of a magnitude smaller compared to those on offshore reefs (Table 3). Results harak in seagrass beds. Table 3. Estimated contribution (% 6 SD) from Maximum Likelihood Analysis of different potential juvenile habitats to adult nearshore and offshore reef populations of three reef fish species, and large juvenile (.15 cm TL) L. harak in seagrass beds. Classification success is based on a quadratic discriminant function analysis using jack-knife classification to examine the success of assigning individuals to their known origin. Both analyses are based on d13C and d18O values combined. Because seagrass and mangrove habitat signatures did not differ for L. lentjan, they had to be combined for this analysis. CR = coral reef; SG = seagrass bed; MG = mangroves. doi:10.1371/journal.pone.0066320.t003 with caution, and be combined with data collected by different methods to effectively identify and assess the nursery function of putative juvenile habitats. Clearly, juvenile fish densities as obtained through visual surveys only reflect the standing stock at the time of observation. They fail to quantify, however, how much of this standing stock will continue to survive and move to reefs at some point in time. Differential mortality among juvenile habitats, for example, could subsequently lead to different population contributions by individual habitats as would be deduced from absolute abundances. depends on habitat mosaics rather than on individual habitats [10,52], which is an important consideration for species and fisheries management and conservation. The usage of coastal habitat mosaics by juveniles of some reef fish could provide an insurance effect (e.g. [53]) against natural or human perturbations, or failure of management efforts in single habitats. Coastal habitats are currently under high pressure from anthropogenic activities and climate change [4,5,6,54,55] which are threatening their existence, and pose a threat to the recruitment, persistence and sustainability of marine fish popula- tions. Coral reefs are being seriously overfished across the globe [56]. In this light, contributions of juvenile habitats with a currently lower than average contribution to adult populations could become critical in maintaining offshore fish stocks at sustainable levels. Identification of nursery habitats has tradition- ally been based on habitats that supply a higher number of recruits to adult populations than the average across all juvenile habitats [12,57]. However, without any guarantees that the most produc- tive nursery habitat can be managed effectively or guarded against natural disturbances, we should spread the risk of potential management failure by conserving seascapes containing multiple patches of connected habitats. Results This approach also incorporates multiple juvenile habitat usage throughout ontogeny as well as maintenance of habitat linkages resulting from daily feeding or shelter-seeking migrations [58,59,60]. Our study showed that about a third of the large juvenile Lethrinus harak in seagrass beds had utilized mangroves during their earlier life stage. Such niche shifts are very common in tropical coastal fish species [61,62], and effective replenishment of adult populations can only be accom- plished by incorporating all habitats that are successively used by fishes during their juvenile stage. Because of the temporary nature of the Indo-Pacific mangroves’ availability to fishes due to the tidal regime, and the relatively low number of species using them as juvenile habitats [29,34,36,48], their nursery function has long been questioned (see [11,48]). The permanent inundation of Caribbean mangroves, on the other hand, translates to high abundances of juvenile coral reef fishes that use these habitats [49]. Nevertheless, the range in degree of replenishment of reef populations by recruits from mangrove habitats does not seem to be very different between the two regions – e.g. Indo-Pacific: 88% for Lutjanus fulvus ([17]; based only on mangrove-reef migrations over short time scales as stable isotope analysis of muscle tissue was used); 29% and 99% for Lethrinus harak and Lutjanus fulviflamma, respectively (this study) vs. Caribbean: 36% for Haemulon flavolineatum [15]; 40–74% for Haemulon flavolineatum and 99% for Lutjanus apodus [20]. This indicates that Indo-Pacific mangroves can play an equally important role as juvenile habitat for some species as is the case in the Caribbean. Using three potential juvenile habitats (mangrove, seagrass, coral reef) as a source, the current study suggests that no single habitat maintains reef fish populations of these species (except perhaps L. fulviflamma on offshore reefs), but act together in replenishing adult populations. Previous studies have shown high (88–99%) contributions from single juvenile habitats to adult reef fish populations, but none of these studies included multiple juvenile habitats, or the coral reef itself was not taken into account as a possible juvenile habitat [17,20,22]. Studies showing single habitats that contribute close to 100% of recruits to adult populations are rare in general [15,22,50,51]. Results The different contributions of the three habitats in the present study indicates that maintenance of reef fish populations in the Indo-Pacific In conclusion, our results provide strong support for a nursery role of Indo-Pacific mangroves for certain species of reef fishes, but also indicate that seascape structure plays a vital role, and habitat mosaics rather than individual habitats should be conserved to maintain effective replenishment of offshore fish stocks. Results d13C d18O post-hoc post-hoc CR CR MG CR CR CR MG CR vs vs vs vs vs vs vs vs F p SG MG SG SG+MG F p SG MG SG SG+MG Lethrinus harak 27.335 ,0.001 ,0.001 ,0.001 0.002 7.741 0.001 0.002 0.845 0.002 Lethrinus lentjan 6.351 0.003 0.003 0.115 0.999 ,0.001 6.666 0.002 0.001 0.240 0.989 ,0.001 Lutjanus fulviflamma 1.155 0.320 0.947 0.572 0.371 11.039 ,0.001 0.003 ,0.001 0.011 Non-significant values (p.0.05), which indicate no differences among habitats, are indicated in bold. Due to non-significant post-hoc tests for otolith d13C and d18O of L. lentjan between mangrove and seagrass these two habitats had to be combined. CR = coral reef; SG = seagrass bed; MG = mangroves. doi:10.1371/journal.pone.0066320.t002 Non-significant values (p.0.05), which indicate no differences among habitats, are indicated in bold. Due to non-significant post-hoc tests for otolith d13C and d18O of L. lentjan between mangrove and seagrass these two habitats had to be combined. CR = coral reef; SG = seagrass bed; MG = mangroves. doi:10.1371/journal.pone.0066320.t002 June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org 5 Nursery-to-Reef Movements by Indo-Pacific Fishes Table 3. Estimated contribution (% 6 SD) from Maximum Likelihood Analysis of different potential juvenile habitats to adult nearshore and offshore reef populations of three reef fish species, and large juvenile (.15 cm TL) L. harak in seagrass beds. Juvenile habitat N Mean density (100 m22) Classification success (%) SG MG CR SG+MG Lethrinus harak Nearshore reef fish 25 0.0360.02 73.3 52.9618.6 28.9613.6 18.2614.1 Large seagrass fish 9 0.2160.18 82.0 69.7620.0 30.3620.0 - Lethrinus lentjan Nearshore reef fish 20 0.1860.05 77.4 65.3622.0 34.7622.0 Offshore reef fish 54 0.0760.11 72.4612.2 27.6612.2 Lutjanus fulviflamma Nearshore reef fish 19 0.0960.07 60.5 18.9621.2 0.0360.6 81.1621.2 Offshore reef fish 21 1.2561.45 1.266.1 98.966.2 0.060.0 Classification success is based on a quadratic discriminant function analysis using jack-knife classification to examine the success of assigning individuals to their known origin. Both analyses are based on d13C and d18O values combined. Because seagrass and mangrove habitat signatures did not differ for L. lentjan, they had to be combined for this analysis. CR = coral reef; SG = seagrass bed; MG = mangroves. doi:10.1371/journal.pone.0066320.t003 Table 3. Estimated contribution (% 6 SD) from Maximum Likelihood Analysis of different potential juvenile habitats to adult nearshore and offshore reef populations of three reef fish species, and large juvenile (.15 cm TL) L. References 25. Nakamura Y, Hirota K, Shibuno T, Watanabe Y (2012) Variability in nursery function of tropical seagrass beds during fish ontogeny: timing of ontogenetic habitat shift. Marine Biology 159: 1305–1315. 1. Blaber SJM (2009) Relationships between tropical coastal habitats and (offshore) fisheries. In: Nagelkerken I, editor. Ecological connectivity among tropical coastal ecosystems. Dordrecht: Springer Science and Business Media. 533–564. 26. Nagelkerken I, Bothwell J, Nemeth RS, Pitt JM, van der Velde G (2008) Interlinkage between Caribbean coral reefs and seagrass beds through feeding migrations by grunts (Haemulidae) depends on habitat accessibility. Marine Ecology Progress Series 368: 155–164. 2. Mumby PJ, Edwards AJ, Arias-Gonzalez JE, Lindeman KC, Blackwell PG, et al. (2004) Mangroves enhance the biomass of coral reef fish communities in the Caribbean. Nature 427: 533–536. 3. Dorenbosch M, Grol MGG, Nagelkerken I, van der Velde G (2006) Seagrass beds and mangroves as potential nurseries for the threatened Indo-Pacific humphead wrasse, Cheilinus undulatus and Caribbean rainbow parrotfish, Scarus quacamaia. Biological Conservation 129: 277–282. 27. Nagelkerken I, Blaber SJM, Bouillon S, Green P, Haywood M, et al. (2008) The habitat function of mangroves for terrestrial and marine fauna: A review. Aquatic Botany 89: 155–185. 28. Unsworth RKF, Bell JJ, Smith DJ (2007) Tidal fish connectivity of reef and seagrass habitats in the Indo-Pacific. Journal of The Marine Biological Association of the United Kingdom 87: 1287–1296. 4. Duke NC, Meynecke J-O, Dittmann S, Ellison AM, Anger K, et al. (2007) A world without mangroves? Science 317: 41–42. 5. Waycott M, Duarte CM, Carruthers TJB, Orth RJ, Dennison WC, et al. (2009) Accelerating loss of seagrasses across the globe threatens coastal ecosystems. Proceeding of the National Academy of Sciences USA 106: 12377–12381. 29. Dorenbosch M, Grol MGG, Nagelkerken I, van der Velde G (2006) Different surrounding landscapes may result in different fish assemblages in East African seagrass beds. Hydrobiologia 563: 45–60. g y 6. Valiela I, Bowen JL, York JK (2001) Mangrove forests: One of the world’s threatened major tropical environments. BioScience 51: 807–815. 30. Sheaves M, Johnston R (2009) Ecological drivers of spatial variability among fish fauna of 21 tropical Australian estuaries. Marine Ecology Progress Series 385: 245–260. threatened major tropical environments. BioScience 51: 807–815 7. Costanza R, d9Arge R, De Groot R, Farber S, Grasso M, et al. (1997) The value of the world’s ecosystem services and natural capital. Nature 287: 253–260. 31. References Mumby PJ, Hastings A (2008) The impact of ecosystem connectivity on coral reef resilience Journal of Applied Ecology 45: 854–862. 38. Gonzalez Carman V, Falabella V, Maxwell S, Albareda D, Campagna C, et al. (2012) Revisiting the ontogenetic shift paradigm: The case of juvenile green turtles in the SW Atlantic. Journal of Experimental Marine Biology and Ecology 429: 64–72. 15. Chittaro PM, Fryer BJ, Sale R (2004) Discrimination of French grunts (Haemulon flavolineatum Desmarest, 1823) from mangrove and coral reef habitats using otolith microchemistry. Journal of Experimental Marine Biology and Ecology 308: 169–183. 39. Dorenbosch M, Grol MGG, Christianen MJA, Nagelkerken I, van der Velde G (2005) Indo-Pacific seagrass beds and mangroves contribute to fish density and diversity on adjacent coral reefs. Marine Ecology Progress Series 302: 63–76. 16. Bouillon S, Connolly RM, Gillikin D (2012) Use of stable isotopes to understand food webs and ecological processes. In: Wolanski E, McClusky DS, editors. Treatise on estuarine and coastal science, Book 7. Waltham: Academic Press. 40. Kimirei IA, Nagelkerken I, Griffioen B, Wagner C, Mgaya YD (2011) Ontogenetic habitat use by mangrove/seagrass-associated coral reef fishes shows flexibility in time and space. Estuarine, Coastal and Shelf Science 92: 47– 58. 17. Nakamura Y, Horinouchi M, Shibuno T, Tanaka Y, Miyajima T, et al. (2008) Evidence of ontogenetic migration from mangroves to coral reefs by black-tail snapper Lutjanus fulvus: stable isotope approach. Marine Ecology Progress Series 355: 257–266. 41. Nakamura Y, Tsuchiya M (2008) Spatial and temporal patterns of seagrass habitat use by fishes at the Ryukyu Islands, Japan. Estuarine, Coastal and Shelf Science 76: 345–356. 18. Campana SE (1999) Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Marine Ecology Progress Series 188: 263–297. 42. Dorenbosch M, Verberk WCEP, Nagelkerken I, van der Veldel G (2007) Influence of habitat configuration on connectivity between fish assemblages of Caribbean seagrass beds, mangroves and coral reefs. Marine Ecology Progress Series 334: 103–116. 19. Campana SE (2005) Otolith science entering the 21st century. Marine and Freshwater Research 56: 485–495. 20. Mateo I, Durbin EG, Appeldoorn RS, Adams AJ, Juanes F, et al. (2010) Role of mangroves as nurseries for French grunt Haemulon flavolineatum and schoolmaster Lutjanus apodus assessed by otolith elemental fingerprints. Marine Ecology Progress Series 402: 197–212. 43. URT (2009) The Fisheries Regulations, 2009. GNNo 308 of 28/8/2009. Dar es Salaam, Tanzania: Government Printer. 44. Acknowledgments We thank the staff of the Department of Aquatic Sciences and Fisheries of the University of Dar es Salaam for their cooperation and for providing June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org 6 Nursery-to-Reef Movements by Indo-Pacific Fishes Nursery-to-Reef Movements by Indo-Pacific Fishes office space and research facilities. We express our gratitude to Suzan Verdegaal and Hubert Vonhof of the Vrije Universiteit Amsterdam for laboratory assistance during otoliths analysis. Appreciation is expressed to Michel Trommelen, Piet Blankers, Nanne van Hoytema, Niek Slooter, Estrella Tapias, Ben Griffioen, Coen Wagner, Peter Smittenaar, and Mathias Igulu for their invaluable field assistance, and Hassan and Mmanga for operating the research boat. References Kruitwagen G, Nagelkerken I, Lugendo BR, Mgaya YD, Bonga SEW (2010) Importance of different carbon sources for macroinvertebrates and fishes of an interlinked mangrove–mudflat ecosystem (Tanzania). Estuarine, Coastal and Shelf Science 88: 464–472. 8. Nagelkerken I, Grol MGG, Mumby PJ (2012) Effects of marine reserves versus nursery habitat availability on structure of reef fish communities. PLoS ONE 7: e36906. 9. Olds AD, Connolly RM, Pitt KA, Maxwell PS (2012) Habitat connectivity improves reserve performance. Conservation Letters 5: 56–63. 32. Lugendo BR, Nagelkerken I, van der Velde G, Mgaya YD (2006) The importance of mangroves, mud and sand flats, and seagrass beds as feeding areas for juvenile fishes in Chwaka Bay, Zanzibar: gut content and stable isotope analyses. Journal of Fish Biology 69: 1639–1661. 10. Sheaves M (2009) Consequences of ecological connectivity: the coastal ecosystem mosaic. Marine Ecology Progress Series 391: 107–115. 11. Nagelkerken I (2009) Evaluation of nursery function of mangroves and seagrass beds for tropical decapods and reef fishes: patterns and underlying mechanisms. In: Nagelkerken I, editor. Ecological connectivity among tropical coastal ecosystems. Dordrecht: Springer Science and Business Media. 357–400. 33. Faunce CH, Layman CA (2009) Sources of variation that affect perceived nursery function of Mangroves. In: Nagelkerken I, editor. Ecological connectivity among tropical coastal ecosystems. Dordrecht: Springer Science and Business Media. 401–421. 12. Beck MW, Heck KL, Able KW, Childers DL, Eggleston DB, et al. (2001) The identification, conservation, and management of estuarine and marine nurseries for fish and invertebrates. Bioscience 51: 633–641. 34. Laroche J, Baran E, Rasoanandrasana NB (1997) Temporal patterns in a fish assemblage of a semiarid mangrove zone in Madagascar. Journal of Fish Biology 51: 3–20. 13. Gillanders BM (2009) Tools for studying biological marine ecosystem interactions - Natural and artificial tags. In: Nagelkerken I, editor. Ecological connectivity among tropical coastal ecosystems. Dordrecht: Springer Science and Business Media. 457–492. 35. Sheaves M (2001) Are there really few piscivorous fishes in shallow estuarine habitats? Marine Ecology Progress Series 222: 279–290. 36. Thollot P (1992) Importance of mangroves for Pacific reef fish species, myth or reality? Proceedings of the 6th International Coral Reef Symposium 2: 934–941. 14. Elsdon TS, Wells BK (2008) Otolith chemistry to describe movements and life- history parameters of fishes: Hypotheses, assumptions, limitations and inferences. Oceanography and Marine Biology: An Annual Review 46: 297– 330. 37. Author Contributions Conceived and designed the experiments: IAK IN YDM. Performed the experiments: IAK. Analyzed the data: IAK IN CMH. Wrote the paper: IAK IN CMH. Nursery-to-Reef Movements by Indo-Pacific Fishes 48. Blaber SJM (2000) Tropical estuarine fishes: ecology, exploitation and conservation. Fish and Aquatic Resources Series 7. Oxford: Blackwell Science. 56. Newton K, Cote IM, Pilling GM, Jennings S, Dulvy NK (2007) Current and future sustainability of island coral reef fisheries. Current Biology 17: 655–658 57. Adams AJ, Dahlgren CP, Kellison GT, Kendall MS, Layman CA, et al. (2006) Nursery function of tropical back-reef systems. Marine Ecology Progress Series 318: 287–301. 49. Verweij MC, Nagelkerken I, Wartenbergh SLJ, Pen IR, van der Velde G (2006) Caribbean mangroves and seagrass beds as daytime feeding habitats for juvenile French grunts, Haemulon flavolineatum. Marine Biology 149: 1291–1299. 58. Dorenbosch M, Verweij MC, Nagelkerken I, Jiddawi N, van der Velde G (2004) Homing and daytime tidal movements of juvenile snappers (Lutjanidae) between shallow-water nursery habitats in Zanzibar, western Indian Ocean. Environ- mental Biology of Fishes 70: 203–209. 50. Gillanders BM (1997) Patterns of abundance and size structure in the blue groper, Achoerodus viridis (Pisces, Labridae): evidence of links between estuaries and coastal reefs. Environmental Biology of Fishes 49: 153–173. and coastal reefs. Environmental Biology of Fishes 49: 153–173. gy 51. Gillanders BM (2002) Connectivity between juvenile and adult fish populations: gy 51. Gillanders BM (2002) Connectivity between juvenile and adult fish populations: do adults remain near their recruitment estuaries? Marine Ecology Progress Series 240: 215–223. gy 59. Verweij MC, Nagelkerken I (2007) Short and long-term movement and site fidelity of juvenile Haemulidae in back-reef habitats of a Caribbean embayment. Hydrobiologia 592: 257–270. ( ) y j p p do adults remain near their recruitment estuaries? Marine Ecology Progress Series 240: 215–223. 52. Sheaves M (2005) Nature and consequences of biological connectivity in mangrove systems. Marine Ecology Progress Series 302: 293–305. 60. Lugendo BR, Nagelkerken I, Kruitwagen G, van der Velde G, Mgaya YD (2007) Relative importance of mangroves as feeding habitat for fish: a comparison between mangrove habitats with different settings. Bulletin of Marine Science 80: 497–512. 53. Yates PM, Heupel MR, Tobin AJ, Simpfendorfer CA (2012) Diversity in young shark habitats provides the potential for portifolio effects. Marine Ecology Progress Series 458: 269–281. 61. Nagelkerken I, Dorenbosch M, Verberk WCEP, Cocheret de la Moriniere E, van der Velde G (2000) Importance of shallow-water biotopes of a Caribbean bay for juvenile coral reef fishes: patterns in biotope association, community structure and spatial distribution. Marine Ecology Progress Series 202: 175–192. 54. References Lugendo BR, Pronker A, Cornelissen I, de Groene A, Nagelkerken I, et al. (2005) Habitat utilisation by juveniles of commercially important fish species in a marine embayment in Zanzibar, Tanzania. Aquatic Living Resources 18: 149– 158. 21. McMahon KW, Berumen ML, Mateo I, Elsdon TS, Thorrold SR (2011) Carbon isotopes in otolith amino acids identify residency of juvenile snapper (Family: Lutjanidae) in coastal nurseries. Coral Reefs 30: 1135–1145. 22. Verweij MC, Nagelkerken I, Hans I, Ruseler SM, Mason PRD (2008) Seagrass nurseries contribute to coral reef fish populations. Limnology and Oceanogra- phy 53: 1540–1547. 45. Unsworth RK, De Leon PS, Garrard SL, Jompa J, Smith DJ, et al. (2008) High connectivity of Indo-Pacific seagrass fish assemblages with mangrove and coral reef habitats. Marine Ecology Progress Series 353: 213–224. 23. Huijbers CM, Nagelkerken I, Debrot A, Jongejans E (2013) Geographic coupling of juvenile and adult habitat shapes spatial population dynamics of a coral reef fish. Ecology: in press. doi.org/10.1890/11-1759.1. 46. Field A (2005) Discovering statistics using SPSS; Wright DB, editor.. London: SAGE Publications. 47. Millar RB (1990) Comparison of methods for estimating mixed stock fishery composition. Canadian Journal of Fisheries and Aquatic Sciences 47: 2235– 2241. coral reef fish. Ecology: in press. doi.org/10.1890/11-1759.1. 24. Nagelkerken I (2007) Are non-estuarine mangroves connected to coral reefs through fish migration? Bulletin of Marine Science 80: 595–607. June 2013 \| Volume 8 \| Issue 6 \| e66320 7 June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org Nursery-to-Reef Movements by Indo-Pacific Fishes Baker AC, Glynn PW, Riegl B (2008) Climate change and coral reef bleaching: An ecological assessment of long-term impacts, recovery trends and future outlook. Estuarine Coastal and Shelf Science 80: 435–471. 55. Gillanders BM (2006) Seagrasses, fish, and fisheries. In: Larkum AWD, editor. Seagrasses: Biology, Ecology and Conservation. Amsterdam: Springer Science and Business Media. 503–536. p gy g 62. Kimirei IA, Nagelkerken I, Trommelen M, Blankers P, van Hoytema N, et al. (2013) What drives ontogenetic niche shifts of fishes in coral reef ecosystems? Ecosystems: in press. DOI: 10.1007/s10021-013-9645-4 PLOS ONE \| www.plosone.org June 2013 \| Volume 8 \| Issue 6 \| e66320 PLOS ONE \| www.plosone.org 8
https://openalex.org/W4233232823	https://discovery.ucl.ac.uk/10120197/13/Dutey-Magni_dlab018.pdf	English	null	Feasibility study of hospital antimicrobial stewardship analytics using electronic health records	Research Square (Research Square)	2,021	cc-by	8,086	Received 6 October 2020; accepted 27 January 2021 Background: Hospital antimicrobial stewardship (AMS) programmes are multidisciplinary initiatives to optimize antimicrobial use. Most hospitals depend on time-consuming manual audits to monitor clinicians’ prescribing. But much of the information needed could be sourced from electronic health records (EHRs). Objectives: To develop an informatics methodology to analyse characteristics of hospital AMS practice using routine electronic prescribing and laboratory records. Methods: Feasibility study using electronic prescribing, laboratory and clinical coding records from adult patients admitted to six specialities at Queen Elizabeth Hospital, Birmingham, UK (September 2017–August 2018). The study involved: (i) a review of AMS standards of care; (ii) their translation into concepts measurable from commonly available EHRs; and (iii) a pilot application in an EHR cohort study (n " 61679 admissions). Results: We developed data modelling methods to characterize antimicrobial use (antimicrobial therapy episode linkage methods, therapy table, therapy changes). Prescriptions were linked into antimicrobial therapy episodes (mean 2.4 prescriptions/episode; mean length of therapy 5.8 days), enabling several actionable ﬁnd- ings. For example, 22% of therapy episodes for low-severity community-acquired pneumonia were congruent with prescribing guidelines, with a tendency to use broader-spectrum antibiotics. Analysis of therapy changes revealed IV to oral therapy switching was delayed by an average 3.6 days (95% CI: 3.4–3.7). Microbial cultures were performed prior to treatment initiation in just 22% of antibacterial prescriptions. The proposed methods enabled ﬁne-grained monitoring of AMS practice down to specialities, wards and individual clinical teams by case mix, enabling more meaningful peer comparison. Conclusions: It is feasible to use hospital EHRs to construct rapid, meaningful measures of prescribing quality with potential to support quality improvement interventions (audit/feedback to prescribers), engagement with front-line clinicians on optimizing prescribing, and AMS impact evaluation studies. methodological skill. This hinders hospitals’ capacity to monitor prescribing on a large scale.8,10,11 JAC- Antimicrobial Resistance JAC- Antimicrobial Resistance JAC Antimicrob Resist doi:10.1093/jacamr/dlab018 V C The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 of 10 Feasibility study of hospital antimicrobial stewardship analytics using electronic health records Downloaded from https://academic.oup.com/jacamr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 1Institute of Health Informatics, University College London, London, UK; 2University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK; 3Institute of Epidemiology & Health Care, University College London, London, UK 1Institute of Health Informatics, University College London, London, UK; 2University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK; 3Institute of Epidemiology & Health Care, University College London, London, UK Corresponding author. E-mail: p.dutey-magni@ucl.ac.uk †Members are listed in the Acknowledgements section. Corresponding author. E-mail: p.dutey-magni@ucl.ac.uk †Members are listed in the Acknowledgements section. Received 6 October 2020; accepted 27 January 2021 AMS metrics Relevant deﬁnitions and standards of care were identiﬁed from internation- al hospital antimicrobial stewardship and infection treatment guidelines using a list systematically compiled in 2018,26 alongside four UK-speciﬁc reference sources.2,9,27,28 We narrowed down a list of measures (Table 1) on the basis of (i) their relevance to inform a hospital AMS strategy and (ii) the availability of sufﬁcient information to measure them within commonly encountered EHRs. These measures characterize the following: g g p g y The aim of the present paper was to assess the feasibility of auditing antimicrobial stewardship practices using routinely col- lected EHRs in order to provide relevant information to different AMS stakeholders including clinicians, hospital managers and policy- makers. Key objectives were to: (i) infer the indication of antibiotics prescribed to inpatients; (ii) assess the congruence of individual pre- scriptions with local prescribing guidelines, particularly in relation to empirical therapy; (iii) compute metrics of stewardship beyond con- sumption of antibiotics; and (iv) compare these metrics between specialities and between consultant teams within specialities. • Antimicrobial consumption (dose and duration). Deﬁned daily doses, days of therapy (DOT), and length of therapy (LOT: duration of the epi- sode, irrespective of the number of antimicrobials administered concur- rently) were calculated and aggregated by ward, speciality, consultant teams, and clinical indication as per deﬁnitions by Ibrahim et al.29 emic.oup.com/jacamr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 • Changes of therapy tracked changes in antibacterial treatment choices across a ‘therapy episode’ (Data modelling section) and their timing relative to microbiological outcomes and clinical progression. One such change, de-escalation, is recommended when microbial culture and susceptibilities are available, or when there is limited evidence of infec- tion. It is most easily measured in antibiotics with the broadest spec- trum of activity, where only a small number of other drugs would have equivalent spectrum. Conversion from IV therapy to oral therapy is an- other commonly recommended change of therapy intervention, which can facilitate discharge and reduce some adverse effects of injec- tions.30 We computed the time by which criteria for switching from IV to oral regimens were met, based on a set of ‘ABCD’ criteria listed in QEHB’s antimicrobial prescribing guidelines (Table 2), some of which are in common with the Glasgow Audit Tool.28 Out of these, ability to take oral medication (criterion B) could not be assessed from records, but other criteria could be measured continuously. Ethics This research was approved by University College London’s Research Ethics Committee (REC reference 16765/002). Informed consent was not sought for the secondary analysis of pseudonymized EHRs. • Congruence of practice with prescribing guidelines. Prescriptions starting a therapy episode were compared with ﬁrst-line choice of empirical therapy recommended in local prescribing guidelines. • Adherence to microbial sampling guidelines recommending submission of bacterial cultures prior to initiation of empirical treat- ment.27 We computed the proportion of prescriptions with a micro- bial sample taken in the 3 days leading up to antibacterial therapy initiation. Introduction The aims of antimicrobial stewardship (AMS) are ‘ﬁrst, to ensure ef- fective treatment of patients with infection, and second, to minim- ize collateral damage from antimicrobial use’.1 Hospital AMS guidelines2–7 recommend regular clinical audits of prescriptions and feedback of results to prescribers by infection specialists. Yet, doing so is labour-intensive and dependent on specialist expert- ise,8 as it involves reviewing what diagnostic tests were performed and assessing the compliance with local prescribing guidelines. Similarly, point prevalence surveys conducted for infection surveil- lance9 can be prohibitive both in terms of professional time and Electronic health records (EHRs) collected routinely by hospital information systems offer potential solutions to this problem. King et al.12 and Hand et al.13 scoped the potential role of electronic pre- scribing software in supporting prescribers across the full antibiotic lifecycle (prescription initiation, review, discontinuation and dispensing of discharge medications). Other studies have demon- strated the feasibility of using computerized laboratory results, including microbial cultures and sensitivities, to guide the choice of antimicrobial agent in empirical therapy14 and increase the proportion of cases treated with effective antimicrobials.15 V C The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 of 10 1 of 10 Dutey-Magni et al. EHRs thus have the potential to enable a range of functions rec- ommended in AMS guidelines,2 particularly: audit of practice, feed- back to prescribers, and infection surveillance (tracking syndromes, pathogens and susceptibility). Despite this, the use of EHRs to drive AMS programmes remains ‘underexploited’.16 Extraction of records is challenging,17 resulting in very limited secondary use for evidence-based medicine.18 In response, the UK’s Antimicrobial Resistance National Action Plan set goals for a comprehensive use of EHRs to ‘support and drive good antimicrobial stewardship by coding, auditing and providing feedback for surveillance’ by 2025.16 EUCAST interpretative criteria,23 and classiﬁed bacterial isolates by MDR proﬁle (multiple, extensive and pan-drug resistance) according to rules set out by Magiorakos et al.24 CURB-65,25 an important risk stratiﬁcation score for community-acquired pneumonia (CAP), was computed without the confusion score due to lack of reliable data. Study design and population We conducted a retrospective cohort study of records corresponding to epi- sodes of care in six specialities (general medicine, respiratory medicine, geriatric medicine, cardiology, general surgery, urology) at Queen Elizabeth Hospital Birmingham (QEHB) for adult inpatients admitted between 1 September 2017 and 31 August 2018 (n " 61679 admissions). QEHB is a specialist teaching hospital in Birmingham, UK with over 1000 general and acute inpatient beds. Underlying concepts are deﬁned and mapped to relevant SNOMED CT concept codes31 in Table S1. AMS metrics This feasibility study followed three steps. First, we synthesized concepts relevant to antimicrobial stewardship performance from clinical guidelines and infection surveillance protocols and trans- lated them into operational deﬁnitions applicable to EHRs. Second, we modelled and visualized records to reﬁne deﬁnitions that could be applied to data from one specialist hospital in Birmingham, UK. Third, we computed AMS metrics and reviewed compliance of clinical practices with AMS guidelines. JAR ABCD criteria: considerations for IV to oral switch (see detailed criteria in Appendix S3) Markers Temperature 36C–38C for 48 h Functional gastrointestinal tract No malabsorption No interaction with other medications Enteral drug form available Patient can swallow and tolerate oral ﬂuids via a tube No unexplained tachycardia (heart rate less than 90 beats/min in the past 12 h) Blood pressure stable in the past 24 h Respiratory rate less than 20 breaths/min in the past 24 h White cell count 4–12 % 109 cells/L OR a high white cell count that is falling Falling C-reactive protein Liver abscess Osteomyelitis, septic arthritis Inadequately drained abscesses or empyema Cavitating pneumonia Staphylococcus aureus bacteraemia Severe necrotizing soft tissue infections Severe infections during chemotherapy related neutropenia Infected implants/prosthesis Meningitis/encephalitis Intracranial abscesses Mediastinitis Endocarditis Exacerbation of cystic ﬁbrosis/bronchiectasis mr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 JAR Feasibility of hospital antimicrobial stewardship analytics Table 1. Overview of antimicrobial stewardship metrics Domain Measures Antimicrobial consumption Proportion of hospital admissions with at least one antimicrobial prescription Mean DOT (total duration of all prescriptions, including where there is overlap, e.g. combination therapy) Mean LOT (time elapsed between the ﬁrst and the last drug administration in an episode) Rate of DOT and LOT per 1000 admissions29 Change of therapy (stop, switch, continue) Proportion of ﬁrst-line monotherapy or combination therapy leading to a different choice of therapy, continu- ation, or discontinuation IV to oral administration switch Proportion of antimicrobial therapy episodes initiated by IV route being subsequently converted in full to oral route Mean time elapsed between IV therapy initiation and its complete conversion to oral therapy Congruence with guidelines Proportion of antimicrobial therapy episodes initiated with one of the ﬁrst-line treatment options listed in the local empirical prescribing guidelines Microbial culture taking Proportion of prescriptions belonging to a therapy episode initiated within 3 h of a blood, urine, skin or sterile site microbial sample being taken Table 2. ABCD criteria: considerations for IV to oral switch (see detailed criteria in Appendix S3) Criteria Markers A Afebrile for at least 24 h Temperature 36C–38C for 48 h B Able to take oral medication (not measured) Functional gastrointestinal tract No malabsorption No interaction with other medications Enteral drug form available Patient can swallow and tolerate oral ﬂuids via a tube C Clinically improving No unexplained tachycardia (heart rate less than 90 beats/min in the past 12 h) Blood pressure stable in the past 24 h Respiratory rate less than 20 breaths/min in the past 24 h White cell count 4–12 % 109 cells/L OR a high white cell count that is falling Falling C-reactive protein D Not suffering from certain deep- seated/high-risk infections Liver abscess Osteomyelitis, septic arthritis Inadequately drained abscesses or empyema Cavitating pneumonia Staphylococcus aureus bacteraemia Severe necrotizing soft tissue infections Severe infections during chemotherapy related neutropenia Infected implants/prosthesis Meningitis/encephalitis Intracranial abscesses Mediastinitis Endocarditis Exacerbation of cystic ﬁbrosis/bronchiectasis om/jacamr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 Table 2. Variables Graph theory principles were used to construct periods of uninterrupted antibiotic therapy (therapy episodes) by linking related prescription records. Rule deﬁnitions underpinning this linkage are available along with this paper (Appendix S1, Table S2). This enabled identiﬁcation of sequences of drug administration making up therapy episodes, particularly transition from one class of antimicrobials to another. Pseudonymized EHRs consisted of patient demographics, clinical diagnosis codes (ICD-10,19 reclassiﬁed as shown in Table S3, available as Supplementary data at JAC-AMR Online), clinical procedure codes (OPCS- 4),20 episodes of care (pseudonymized consultant code, consultant special- ity), ward movements, and key investigation results (blood counts, vital signs, blood pressure, organ function). For each antimicrobial therapy episode, a dynamic table could be con- structed with an hourly resolution capturing changes in therapy in relation to clinical parameters (Table 3). It is used as the basis for analysing changes of therapy in relation to clinical response to treatment (including tracking the timing of conversion from IV to oral therapy administration). Antibacterial drug prescription and administration records were extracted from QEHB’s locally developed Prescribing, Information and Communication System (PICS).21 PICS follows the common UK ‘dose- based’ prescribing approach,22 in which prescribers issue a request contain- ing one or more drug names (Trade Family), dose, route and frequency. Data processing software was written in Structured Query Language (SQL), R and tidyverse32–34 and served as a prototype for the Ramses package.35,36 Microbial culture results, including no-growth results and cultures ordered by general practitioners were extracted from PICS. We applied 2 of 10 JAR Prescribing indication inference (supervised classiﬁcation) been collected during an audit of antibacterial prescribing conducted by pharmacists between 2012 and 2017. This dataset included 4200 prescrip- tions issued for 2712 patients in the following specialities: general medicine, respiratory medicine, geriatric medicine and general surgery. Pharmacists classiﬁed each prescription into 21 possible indications including ‘not speci- ﬁed’ and ‘other’ using data in PICS and paper medical records. A total of 463 prescriptions did not have a valid clinical indication, and 364 could not be PICS captures drug prescription indications as free text. Such information was not made available to researchers as it contained patient identiﬁable information. It was also affected by a high prevalence of missing data (in the region of 50%). In order to demonstrate our approach, drug indications were instead classiﬁed retrospectively using a training dataset that had 3 of 10 3 of 10 Dutey-Magni et al. Table 3. Example structure of a therapy table Patient Time Mode Last WBC WBC trend 72 h Peak CRP in last 72 h Last CRP . . . ABCD criteria met? X 2018-07-31 18:49:51 IV 11.0 #0.05 151 100 . . . yes X 2018-07-31 19:49:51 IV 8.2 #0.02 151 100 . . . yes X 2018-07-31 20:49:51 oral 8.2 #0.02 151 40 . . . yes . . . . . . . . . . . . . . . . . . . . . . . . . . . WBC, white blood cell count; CRP, C-reactive protein concentration. Table 3. Example structure of a therapy table converted into oral therapy, with a median and mean duration of IV treatment of 2.4 days and 3.5 days, respectively. On the con- trary, 11210 IV sequences (64%) continued with injections until end of therapy, with a median duration of 1.3 days and a mean duration of 3.5 days. As shown in Figure 2, variation in the conver- sion to oral therapy across clinical teams and specialities was evi- dent and can be attributed, at least in part, to case mix. For instance, a likely explanation for cardiology’s lower conversion rate (8%) is that prolonged IV therapy is recommended for deep- seated infections such as endocarditis. linked to electronic prescription records, restricting the analysis to a total of 3228 prescriptions corresponding to 2901 therapy episodes. Indication cat- egories with fewer than 50 episodes (endocarditis, bronchiectasis, diabetic foot and/or osteomyelitis, surgical prophylaxis) were reclassiﬁed as ‘other’. Antimicrobial consumption descriptive characteristics A basic characterization of prescribing requires linking prescriptions into episodes of therapy, to describe their duration in relation to patient demographics or type of infection treated. Between 1 September 2017 and 31 August 2018, there were 61679 adult admissions (46 853 distinct patients) across the six specialities. Table 4 presents key metrics characterizing antibacterial use (prevalence, duration, quantity) by age group. The mean length of admission was 4.2 days, and 21757 admissions (35%) contained at least one antibacterial prescription. A total of 59884 antibacter- ial prescriptions were issued, corresponding to 24511 antibacterial therapy episodes, 141 of which spanned more than one admis- sion. The mean length of antibacterial therapy episodes (LOT) was 5.8 days, equivalent to a mean 8.7 days of therapy (DOT) per ad- mission. The mean DOT increased with age and was signiﬁcantly higher (9.9 days) in emergency admissions than in elective admis- sions (4.3 days, Figure 1). Congruence with prescribing guidelines We take the example of CAP. In addition to being the most com- mon indication for therapy initiation, CAP prescribing guidelines revolved around a widely adopted risk stratiﬁcation score (CURB- 65) which could be measured from EHRs. Of 4222 therapy epi- sodes initiated for CAP, 4109 (97%) could be linked with a CURB-65 severity score in the 48h before or after antibiotic initiation (assuming a mental confusion score of 0, as this information was not recorded electronically). At the time of prescribing, QEHB guidelines recommended: Results We sought to analyse the timeliness of conversion from IV to oral therapy based on ABCD criteria (Table 2). Out of 6404 IV sequences successfully switched, 2670 (42%) met A, C and D crite- ria before oral conversion occurred. Out of 11210 sequences never switched, 2682 (21%) met A, C and D criteria before end of therapy. Across both sets, the delay between criteria being met and end/ conversion of therapy had a median of 2.1 days, a mean of 3.6 days [95% CI: 3.4–3.7], and a standard deviation of 5.7 days, suggesting considerable variation. Figure 3 presents team- and speciality-level mean delays, suggesting once again some differ- ences between consultant teams within specialities. Prescribing indication inference (supervised classiﬁcation) These records were used as training data to predict the clinical indication across all antimicrobial prescriptions, using random forest classiﬁcation with a moderate-to-low balanced accuracy of 59% overall. Predictive ana- lytics were estimated using repeated 5-fold stratiﬁed cross-validation and are reported in Appendix S2 and Table S4. mic.oup.com/jacamr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 Changes of therapy Changes of therapy could be analysed from the structure of ther- apy episodes to identify escalation or de-escalation. For instance, therapy episodes initiated with meropenem (n " 969) were most commonly: (i) stopped (33%) after a mean duration of 3.0 days; (ii) continued (28%) after a mean duration of 2.0 days; (iii) switched to piperacillin/tazobactam (12%) after a mean duration of 1.1 days; or (iv) switched to co-amoxiclav (9.1%) after a mean duration of 1.9 days. Outcomes (i), (iii) and (iv) can be regarded as de- escalation in this particular instance. • CURB-65 score 0 or 1: amoxicillin; doxycycline (penicillin allergy). gy • CURB-65 score 2: amoxicillin/clarithromycin; benzylpenicillin/ clarithromycin; moxiﬂoxacin (penicillin allergy). • CURB-65 score 3!: co-amoxiclav/clarithromycin; moxiﬂoxacin (penicillin allergy). Table 5 reports antibiotics initiated as ﬁrst-line therapy in 2569 low-severity CAP episodes, that is, episodes with a CURB-65 score of 0 or 1. Out of 927 patients whose CURB-65 score can conﬁdently be assumed to be at the most 1 (score of 0 when omitting the missing mental confusion score), just 207 (22%) therapy episodes JAR Feasibility of hospital antimicrobial stewardship analytics Table 4. Characteristics of admissions and antibacterial therapy by age group in six selected specialities (September 2017–August 2018) Age group (years) All admissions Admissions with 1 prescription(s) unique patients, N admissions, N LOS, mean (SD) LOS, IQR DOT per 1000 bed-days [95% CI] admissions, N (% total admissions) LOS, mean (SD) LOS, IQR prescriptions, N therapy episodes, N LOT, mean (SD) LOT, IQR DOT, mean (SD) 18–24 3088 3937 1.9 (6.0) 0.2–1.7 788 [787–788] 1020 (26) 4.6 (10.6) 0.8–4.7 2294 1069 4.1 (7.5) 1.0–4.4 5.7 (15.5) 25–34 4455 5626 2.2 (6.8) 0.2–1.8 789 [789–790] 1439 (26) 5.8 (12.0) 0.9–5.9 3523 1541 5.1 (10.5) 1.0–5.2 6.9 (16.2) 35–44 5056 6389 2.5 (6.9) 0.2–2.0 795 [794–795] 1617 (25) 6.6 (11.7) 1.0–7.4 4176 1748 5.7 (9.8) 1.0–6.4 7.8 (15.2) 45–54 6596 8423 2.7 (7.2) 0.2–2.3 827 [827–827] 2307 (27) 7.1 (12.0) 1.2–8.2 5965 2523 5.8 (11.7) 1.0–6.3 8.3 (21.1) 55–64 7627 9977 3.6 (8.4) 0.2–3.5 834 [833–834] 3281 (33) 8.5 (12.5) 1.6–9.8 8989 3707 6.2 (10.9) 1.1–7.0 9.2 (18.0) 65–74 8448 11 230 4.4 (9.5) 0.2–4.7 740 [740–740] 4277 (38) 8.9 (13.2) 1.8–10.3 11 620 4868 5.6 (7.8) 1.3–7.0 8.5 (15.1) 75–84 7196 9815 6.3 (11.6) 0.4–7.4 674 [674–674] 4440 (45) 10.7 (14.8) 2.1–13.5 12 900 5164 5.9 (7.1) 2.0–7.4 9.4 (12.9) 85–94 4082 5668 8.6 (13.1) 0.8–11.0 618 [617–618] 2986 (53) 12.9 (15.4) 2.8–17.0 9196 3558 6.2 (6.1) 2.0–8.1 10.1 (12.2) 95! 464 614 10.2 (13.5) 1.0–14.7 607 [606–608] 390 (64) 13.4 (14.9) 2.6–19.0 1221 475 5.8 (5.3) 2.0–7.3 9.7 (11.2) All ages 46 853a 61 679 4.2 (9.5) 0.2–4.1 726 [726–726] 21 757 (35) 9.1 (13.6) 1.6–10.7 59 884 24 653 5.8 (8.8) 1.3–7.0 8.7 (15.6) were initiated with the recommended drug, while 331 (36%) received therapy recommended for higher CURB-65 scores, demonstrating a preference for broad-spectrum antibiotics in prescribers. Principal ﬁndings This single-site study demonstrates a pragmatic approach to com- puting meaningful measures of AMS from electronic prescription, laboratory and hospital care records to support stewardship teams in rapidly identifying areas of prescribing behaviour where there is scope for improved stewardship. In addition to measuring vari- ation in antibiotic use, we demonstrate the feasibility of using rou- tine data to assess overall compliance with guidelines (using the example of CAP) and show how these datasets can be used to compute a range of prescribing metrics (Table 1) built around inter- national AMS recommendations. This can be used to monitor performance; inform the design of stewardship interventions; evaluate their impact; and engage clinical teams in audit and feed- back interventions to optimize their prescribing.16 OS, length of stay (days); LOT, total LOT per admission (days). olumn does not add up to total as patients may change age group during the year. Microbial culture taking Across a total of 59696 prescriptions ordered by six selected spe- cialities, 22% (n " 13210) were issued after at least one specimen was sampled from blood, drains, respiratory tract, intravascular devices, CNS, aspirates or other tissue or bone samples. Narrowing the criterion to blood samples only, 18% (n " 10906) of all pre- scriptions and 38% (1174/3107) of prescriptions for meropenem (mainly used to treat bloodstream infections), could be linked to such a sample. Figure 4 reports ﬁndings broken down by speciality and consultant team. Considerable variation can be observed, which could be further examined in relation to variations in therapy indication and compliance with guidelines across specialities/ team. Switch from IV to oral therapy Within 16688 out of the 24510 antibacterial therapy episodes, we identiﬁed 17614 sequences consisting of one or more IV prescrip- tions. Overall, 6404 (36%) of such the IV sequences were 4 of 10 4 of 10 JAR Study strengths and limitations This study is novel in attempting to measure clinical constructs that normally require manual audits or point prevalence surveys using routinely collected data.28,37 We outlined ways of measuring stewardship performance in clinical practice beyond antimicrobial consumption, the main indicator currently used in stewardship surveillance.38 National surveillance systems for prescribing and resistance in secondary care provide high-quality measures of re- sistance and prescribing for policy-makers, but they do not address the needs of front-line clinicians who require more detailed metrics to identify opportunities to improve their performance. This feasi- bility study demonstrates the potential for locally developed analytics to address the local needs and stewardship priorities of clinicians using routinely collected EHRs. Future iterations of our approach could be expanded to report on the effective and timely use of surgical prophylaxis (and its congruence with guidelines), timely initiation of antimicrobial therapy and adequate empirical therapy coverage of microbial isolates. py g Outside of intensive care research, existing literature contains few examples of EHR research simultaneously analysing electronic prescribing, laboratory and care records. To our knowledge, only 5 of 10 5 of 10 Dutey-Magni et al. ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 18–24 25–34 35–44 45–54 55–64 65–74 75–84 85–94 95–104 All 1 2 3 4 5 6 7 8 9 10 11 Mean DOT per admission (days) Age (years) Admission method ● ● Elective Emergency Figure 1. Mean and 95% CI of the total DOT per admission in patients receiving antimicrobials at any point during an admission (September 2017– August 2018). ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 18–24 25–34 35–44 45–54 55–64 65–74 75–84 85–94 95–104 All 1 2 3 4 5 6 7 8 9 10 11 Mean DOT per admission (days) Age (years) Admission method ● ● Elective Emergency Figure 1. Mean and 95% CI of the total DOT per admission in patients receiving antimicrobials at any point during an admission (September 2017– August 2018). Admission method ● ● Elective Emergency Admission method Mean DOT per admission (days) m/jacamr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 Figure 1. Study strengths and limitations Mean and 95% CI of the total DOT per admission in patients receiving antimicrobials at any point during an admission (September 2017– August 2018) ●●●●●●●●●●●●●●●●●●●●● ● ●●●●●●●● ●● ●●●●●●●●●●●●●●●●●●●●●● ● ● ●● ●●●●●●●●●● ● ●● ●●●● ●●●●●●●●● ●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●● ●●● ● ● ● ● ● ● ● ● ● ● ● Medical CARDIOLOGY Medical GENERAL MEDICINE Medical GERIATRIC MEDICINE Medical RESPIRATORY MEDICINE Surgical GENERAL SURGERY Surgical UROLOGY 0 5 10 15 20 0 10 20 30 4 8 12 5 10 0 10 20 30 40 50 2.5 5.0 7.5 10.0 0 20 40 60 80 Consultant team rank Percentage switch to oral before end of therapy Figure 2. Point and 95% CI estimates of the proportion of IV therapy converted into oral therapy ranked by consultant team by speciality (September 2017–August 2018). The horizontal line indicates the point estimate for the entire speciality. ●●●●●●●●●●●●●●●●●●●●● ● Medical CARDIOLOGY 0 5 10 15 20 0 20 40 60 80 Percentage switch to oral before end of therapy ●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●● ●●● ● ● ● ● ● ● ● ● ● ● ● Surgical GENERAL SURGERY Surgical UROLOGY 0 10 20 30 40 50 2.5 5.0 7.5 10.0 Medical RESPIRATORY MEDICINE 12 5 Consultant team rank Figure 2. Point and 95% CI estimates of the proportion of IV therapy converted into oral therapy rank (September 2017–August 2018). The horizontal line indicates the point estimate for the entire speciality. Figure 2. Point and 95% CI estimates of the proportion of IV therapy converted into oral therapy ranked by consultant team by speciality (September 2017–August 2018). The horizontal line indicates the point estimate for the entire speciality. ● ● ● ● ● ● ● ● ● ● ● ●●● ●●●●●● ● ●●●●●● ● ●●●●●●●●●●●●●● ●●●● ●●●●●●●● ● ●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ● ● ● ● ● ● Medical CARDIOLOGY Medical GENERAL MEDICINE Medical GERIATRIC MEDICINE Medical RESPIRATORY MEDICINE Surgical GENERAL SURGERY Surgical UROLOGY 3 6 9 5 10 15 5 10 5 10 0 10 20 30 40 2 4 6 0 5 10 15 Consultant team rank Mean time to oral switch after ABCD criteria met (days) Figure 3. Point and 95% CI estimates of the mean time (days) elapsed between ABCD criteria being met and conversion to oral therapy, ranked by consultant team by speciality (September 2017–August 2018). The horizontal line indicates the point estimate for the entire speciality. Implications Findings from this feasibility study are now informing the develop- ment of an open-source software package35 designed to enable hospitals to build their own stewardship analytics using routinely collected EHRs. This has the potential to transform the delivery of stewardship in hospitals by making detailed information on pre- scribing patterns and resistance widely available in the context of increasing use of electronic prescription, laboratory and hospital care record systems in high-income nations. As of 2020, half of England’s acute hospitals had adopted electronic prescribing.42 International guidelines2–7 recommend local investment into sur- veillance and analytics to rationalize the use of antimicrobials. In particular, the UK’s Antimicrobial Resistance National Action Plan16 aims to complete the introduction of electronic prescribing systems across England by 2025, alongside the adoption of inter- national clinical terminology in computerized laboratory sys- tems.43 Strong evidence supports the use of feedback to prescribers,2,44 but feedback needs to be relevant, targeted (team or individual level), reliable and timely to inﬂuence prescribing be- haviour.45 Further research is needed to statistically adjust those measures for case mix in the same way as consumption meas- ures.46 User-centred research47 is also needed to tailor these measures to individual clinical teams, AMS teams and hospital managers. There is also a need for research to develop evidence- based standards of care for antimicrobial stewardship, for instance to support decisions around de-escalation.48 This could be facili- tated by observational studies of routine care records. analytical models. Such platforms are neither feasible in most hos- pitals, nor justiﬁed for simple surveillance of antibiotic use, stew- ardship performance and pathogen susceptibility. The pragmatic approach described in the present paper would be accessible to a wider range of hospitals, particularly if inter- operable software35,36 and code lists/vocabularies are made widely available. In those conditions, a modest proportion of an information analyst’s time would be sufﬁcient to validate and map local data to standardized vocabularies and generate comprehensive reports. Metrics speciﬁed in Table 1 are designed to be feasible independently of variation in EHRs and vocabularies across hospitals. JAR JAR Feasibility of hospital antimicrobial stewardship analytics Table 5. First-line therapy choice in CAP episodes in patients with a CURB-65 score of 0 or 1 First-line therapy Therapy episodes, n (% column total) URB-65 " 0 URB-65 " 1 Amoxicillin 205 (22.1) 249 (15.5) Amoxicillin, clarithromycin 56 (6.0) 104 (6.5) Azithromycin 4 (0.4) 6 (0.4) Benzylpenicillin 2 (0.2) 3 (0.2) Benzylpenicillin, clarithromycin 14 (1.5) 31 (1.9) Benzylpenicillin, metronidazole 1 (0.1) 0 (0.0) Ciproﬂoxacin 2 (0.2) 9 (0.6) Clarithromycin 76 (8.2) 75 (4.7) Clarithromycin, co-amoxiclav 261 (28.2) 541 (33.8) Co-amoxiclav 104 (11.2) 211 (13.2) Meropenem 9 (1.0) 24 (1.5) Piperacillin/tazobactam 2 (0.2) 2 (0.1) Ceftriaxone 10 (1.1) 2 (0.1) Clarithromycin, moxiﬂoxacin 2 (0.2) 4 (0.2) Clarithromycin, piperacillin/ tazobactam 4 (0.4) 9 (0.6) Meropenem, vancomycin 7 (0.8) 10 (0.6) Other 168 (18.1) 322 (20.1) Total 927 (100) 1602 (100) URB-65, severity score based on CURB-65,25 with mental confusion item set to 0: urea (blood urea nitrogen .7 mmol/L) (1 point), respirations per min .30 (1 point), systolic blood pressure ,90 mmHg (1 point), age 65 years (1 point). Table 5. First-line therapy choice in CAP episodes in patients with a CURB-65 score of 0 or 1 prescribing systems which may make it feasible to assess con- gruence with prescribing using EHRs alone. Similarly, the lack of access to dispensing records prevented analysis of ‘to take away’ medications issued at discharge, which can signiﬁcantly prolong the total LOT. Finally, prescription records were obtained from a snapshot source and did not include a history of changes made to prescriptions’ intended duration. This pre- vented analysis of how frequently prescriptions were stopped early. All analyses were restricted to structured data and did not attempt to derive information that may have been recorded in free text in medical notes. Study strengths and limitations Consultant team rank gure 3. Point and 95% CI estimates of the mean time (days) elapsed between ABCD criteria being met and co onsultant team by speciality (September 2017–August 2018). The horizontal line indicates the point estimate for Figure 3. Point and 95% CI estimates of the mean time (days) elapsed between ABCD criteria being met and conversion to oral therapy, ranked by consultant team by speciality (September 2017–August 2018). The horizontal line indicates the point estimate for the entire speciality. large bespoke data engineering platforms have achieved this.39–41 Unlike the present study, such platforms exploit electronic mes- sages streaming from hospital information systems in real-time: these contain dynamic information, unlike the retrospective view provided from EHRs commonly curated in hospital warehouses. This noteworthy difference has implications: the structure and content of electronic messages tend to be system-speciﬁc and re- quire signiﬁcant investment into developing dedicated data and provided from EHRs commonly curated in hospital warehouses. This noteworthy difference has implications: the structure and content of electronic messages tend to be system-speciﬁc and re- quire signiﬁcant investment into developing dedicated data and 6 of 10 Conclusions This study shows it is feasible to draw on electronic prescription, la- boratory and hospital care records to provide meaningful meas- ures of AMS, by: p This feasibility study reveals the challenges associated with assessing congruence with local prescribing guidelines and the complexity of prescribing decisions. This is partly due to limitations of routine data, but it also reﬂects a lack of consensus around when and how to de-escalate antibiotics. Manual review of indi- vidual prescribing records led authors to conclude that there is too much ambiguity in EHRs to conﬁdently assess the appropri- ateness of individual prescribing decisions. Prescribing indica- tion data were not available and made it necessary to rely upon statistical classiﬁcation. This introduced error into the ﬁndings: for example, the classiﬁer precision for CAP was 80% (Table S4), indicating that one in ﬁve episodes classiﬁed as CAP were likely to have a different indication. However, it is increasingly common for prescribing indication to be recorded in electronic (i) Reconstructing ‘therapy episodes’, which link all relevant prescription records and enable analyses of the length, changes and discontinuation of antimicrobial therapy. (i) Reconstructing ‘therapy episodes’, which link all relevant prescription records and enable analyses of the length, changes and discontinuation of antimicrobial therapy. (ii) Inferring the clinical intent and indication of prescriptions (for both monotherapy and combination therapy). We have illustrated the use of supervised classiﬁcation in general medicine specialities with moderate accuracy for the most common infection categories. (iii) Computing stewardship performance and quality metrics. Examples include conversion of IV therapy to oral therapy when patients show signs of resolution, microbial culture sampling and congruence with guidelines. 7 of 10 7 of 10 Dutey-Magni et al. Conclusions ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Medical CARDIOLOGY Medical GENERAL MEDICINE Medical GERIATRIC MEDICINE Medical RESPIRATORY MEDICINE Surgical GENERAL SURGERY Surgical UROLOGY Meropenem Piperacillin/Tazobactam Co−amoxiclav Other 0 10 20 10 20 30 40 50 4 8 12 16 5 10 0 20 40 3 6 9 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 Consultant team rank Percentage with blood sampled up to 3 days before initiation Figure 4. Funding This study was carried out as part of Preserving Antibiotics through Safe Stewardship (PASS), a programme grant funded by the Economic & Social Research Council (grant reference ES/P008321/1). A.H. is a National Institute for Health Research (NIHR) Senior Investigator. L.S. is funded by a NIHR Clinician Scientist Award (CS-2016-007). 9 March 2021 Members of the PASS research group Niall Anderson, Elise Crayton, Gillian Forbes, Arnoupe Jhass, Emma Richardson, Michelle Richardson, Patrick Rockenschaub, Catherine Smith, Elizabeth Sutton, Rosanna Traina (Investigation); Lou Atkins, Anne Conolly, Spiros Denaxas, Ellen Fragaszy, Rob Horne, Patty Kostkova, Conclusions Point and 95% CI estimates of the proportion of prescriptions initiated with a blood culture sampled in the 3 days leading up to initiation of prescription and/or therapy by consultant team by speciality by drug type in six selected specialities (September 2017–August 2018). Consultant teams are ranked by percentage with a sample for ‘other’ antibiotic class. The horizontal line indicates the point estimate for the entire speciality. Downloaded from https://academic.oup.com/jacamr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 Downloaded from https://academic.oup.com/jacamr/article/3/1/dlab018/6158273 by Figure 4. Point and 95% CI estimates of the proportion of prescriptions initiated with a blood culture sampled in the 3 days leading up to initiation of prescription and/or therapy by consultant team by speciality by drug type in six selected specialities (September 2017–August 2018). Consultant teams are ranked by percentage with a sample for ‘other’ antibiotic class. The horizontal line indicates the point estimate for the entire speciality. Fabiana Lorencatto, Susan Michie, Jennifer Mindell, John Robson, Claire Royston, Carolyn Tarrant, James Thomas, Jonathan West (Conceptualization, Funding Acquisition and Resources); Haydn Williams (Resources); Nadia Elsay, Chris Fuller (Project Administration). However, one of the most signiﬁcant obstacles hindering hospitals’ stewardship efforts lies the difﬁculty in extracting and analysing EHRs from a range of diverse systems.18 Reproducible analytical tools are now available to assist microbiology culture and susceptibility analytics.49 Software development is underway to support other hospitals in adopting the approach tested in the present study.35,36 Acknowledgements This work uses pseudonymized data provided by patients and collected by the NHS as part of their care and support. The authors also acknow- ledge the contribution of staff of the Pharmacy Department at University Hospital Birmingham NHS Foundation Trust, who collected high-quality antimicrobial review outcome data. Transparency declarations None to declare. p None to declare. References 1 Davey P, Sneddon J, Nathwani D. Overview of strategies for overcoming the challenge of antimicrobial resistance. Expert Rev Clin Pharmacol 2010; 3: 667–86. 19 WHO. The ICD-10 Classiﬁcation of Mental and Behavioural Disorders. Clinical Descriptions andDiagnostic Guidelines. https://www.who.int/classiﬁca tions/icd/en/bluebook.pdf. 2 NICE. Antimicrobial Stewardship: Systems and Processes for Effective Antimicrobial Medicine Use. NICE guideline NG15. https://www.nice.org.uk/ guidance/ng15. 20 Health and Social Care Information Centre. OPCS Classiﬁcation of Interventions and Procedures Version 4.8 (SCCI0084 Amd 105/2015). https:// digital.nhs.uk/data-and-information/information-standards/information- standards-and-data-collections-including-extractions/publications-and-noti ﬁcations/standards-and-collections/scci0084-opcs-classiﬁcation-of-interven tions-and-procedures. 3 SARI Hospital Antimicrobial Stewardship Working Group. Guidelines for Antimicrobial Stewardship in Hospitals in Ireland. https://www.hpsc.ie/a-z/ microbiologyantimicrobialresistance/infectioncontrolandhai/guidelines/ File,4116,en.pdf. 4 Haute Autorite´ de Sante´. Antibiotic Therapy and Prevention of Bacterial Resistance in Healthcare Organisations. Clinical Practice Guideline 2008. https://www.has-sante.fr/jcms/c_665169/fr/strategie-d-antibiotherapie- et-prevention-des-resistances-bacteriennes-en-etablissement-de-sante. 21 Nightingale PGG, Adu D, Richards NT et al. Implementation of rules based computerised bedside prescribing and administration: intervention study. BMJ 2000; 320: 750–3. 22 UK Health and Social Care Information Centre. dm!d Implem Guide (Secondary Care) V5.0. https://www.nhsbsa.nhs.uk/sites/defa 2017-02/Secondary_Care_Electronic_Prescribing_Implemen Guidance_5_0.pdf. 5 Pulcini C, Binda F, Lamkang AS et al. Developing core elements and check- list items for global hospital antimicrobial stewardship programmes: a con- sensus approach. Clin Microbiol Infect 2019; 25: 20–5. 6 Dellit TH, Owens RC, McGowan JE et al. Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America guidelines for developing an institutional program to enhance antimicrobial steward- ship. Clin Infect Dis 2007; 44: 159–77. 23 EUCAST. EUCAST Expert Rules Version 3.1. Intrinsic Resistanc Exceptional Phenotypes Tables. http://www.eucast.org/ﬁleadmi media/PDFs/EUCAST_ﬁles/Expert_Rules/Expert_rules_intrinsic_e tional_V3.1.pdf. 7 Stichting Werkgroep Antibiotica Beleid. A Teams Practical Guide. Antimicrobial Stewardship in the Netherlands. https://swab.nl/en/download- practice-guide. 24 Magiorakos A-P, Srinivasan A, Carey RB et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard deﬁnitions for acquired resistance. Clin 8 Chung GWW, Wu JEE, Yeo CLL et al. Antimicrobial stewardship: a review of prospective audit and feedback systems and an objective evaluation of out- comes. Virulence 2013; 4: 151–7. 25 Lim WS. Deﬁning community acquired pneumonia severity on presenta- tion to hospital: an international derivation and validation study. Thorax 2003; 58: 377–82. 9 ECDC. Point Prevalence Survey of Healthcare-Associated Infections and Antimicrobial Use in European Acute Care Hospitals – Protocol Version 5.3. https://ecdc.europa.eu/en/publications-data/point-prevalence-survey-health i d i f i d i i bi l 26 EU-JAMRAI. Work Package 7. List of revised Guidelines; Tools and Implementation Methods for Antibiotic Stewardship: Hospital Care. https:// web.archive.org/web/20201201115136/https://eu-jamrai.eu/wp-content/ uploads/2019/04/EUjamrai_D7.1_RevisedGuidelinesATBS_HOSPITALCare_ WP7_2018.10.31_Rev01.pdf. care-associated-infections-and-antimicrobial-use-3. Disclaimer The views expressed in this article are those of the authors and not ne- cessarily those of the NIHR or the Department of Health and Social Care. 16 UK Department of Health and Social Care. Tackling Antimicrobial Resistance 2019-2024. The UK’s Five-Year National Action Plan. https://www. gov.uk/government/publications/uk-5-year-action-plan-for-antimicrobial-re sistance-2019-to-2024. Supplementary data 17 Baysari MT, Lehnbom EC, Li L et al. The effectiveness of information tech- nology to improve antimicrobial prescribing in hospitals: a systematic review and meta-analysis. Int J Med Inform 2016; 92: 15–34. pp y Tables S1 to S4 and Appendices S1 to S4 are available as Supplementary data at JAC-AMR Online. 18 Micallef C, Chaudhry NT, Holmes AH et al. Secondary use of data from hospital electronic prescribing and pharmacy systems to support the quality and safety of antimicrobial use: a systematic review. J Antimicrob Chemother 2017; 72: 1880–5. Feasibility of hospital antimicrobial stewardship analytics survey of 142 UK infection specialists. J Antimicrob Chemother 2017; 72: 1206–16. survey of 142 UK infection specialists. J Antimicrob Chemother 2017; 72: 1206–16. prescription review data. P.F.D.-M programmed and conducted analyses and drafted the manuscript. M.J.G., L.S., A.H. and P.F.D.-M. reviewed visu- alizations of results and interpreted the ﬁndings. All authors read and approved the submitted manuscript. prescription review data. P.F.D.-M programmed and conducted analyses and drafted the manuscript. M.J.G., L.S., A.H. and P.F.D.-M. reviewed visu- alizations of results and interpreted the ﬁndings. All authors read and approved the submitted manuscript. 14 Ca´novas-Segura B, Campos M, Morales A et al. Development of a clinical decision support system for antibiotic management in a hospital environ- ment. Prog Artif Intell 2016; 5: 181–97. 15 Curtis CE, Al Bahar F, Marriott JF. The effectiveness of computerised deci- sion support on antibiotic use in hospitals: a systematic review. PLoS One 2017; 12: e0183062. Author contributions D.M. extracted data for the study team. M.J.G. extracted and classiﬁed microbial culture and susceptibility records. G.S. led the collection of the 8 of 10 8 of 10 JAR References 10 Howard P, Pulcini C, Levy HG et al. An international cross-sectional survey of antimicrobial stewardship programmes in hospitals. J Antimicrob Chemother 2014; 70: 1245–55. 27 PHE. Start Smart–Then Focus. Antimicrobial Stewardship Toolkit for English Hospitals, https://www.gov.uk/government/publications/antimicro bial-stewardship-start-smart-then-focus. 11 Van Gastel E, Costers M, Peetermans WEE et al. Nationwide implementa- tion of antibiotic management teams in Belgian hospitals: a self-reporting survey. J Antimicrob Chemother 2010; 65: 576–80. 28 Seaton RA, Nathwani D, Burton P et al. Point prevalence survey of anti- biotic use in Scottish hospitals utilising the Glasgow Antimicrobial Audit Tool (GAAT). Int J Antimicrob Agents 2007; 29: 693–9. 12 King A, Cresswell KM, Coleman JJ et al. Investigating the ways in which health information technology can promote antimicrobial stewardship: a conceptual overview. J R Soc Med 2017; 110: 320–9. 29 Ibrahim OM, Polk RE. Antimicrobial use metrics and benchmarking to im- prove stewardship outcomes: methodology, opportunities, and challenges. Infect Dis Clin North Am 2014; 28: 195–214. 13 Hand KS, Cumming D, Hopkins S et al. Electronic prescribing system design priorities for antimicrobial stewardship: a cross-sectional 9 of 10 9 of 10 Dutey-Magni et al. 30 Athanassa Z, Makris G, Dimopoulos G et al. Early switch to oral treatment in patients with moderate to severe community-acquired pneumonia. Drugs 2008; 68: 2469–81. JA, Barrenechea E et al. eds. Advances in Artiﬁcial Intelligence. CAEPIA 2016. Lecture Notes in Computer Science, Vol 9868. Springer, 2016; 261–270. doi: 10.1007/978-3-319-44636-3_24. 31 UK Health and Social Care Information Centre. UK SNOMED CT Drug Extension, RF2: Full, Snapshot & Delta. https://isd.digital.nhs.uk. 42 Wilkinson E. ‘A Blessing and a Curse’: The Struggle to Introduce e- Prescribing. The Pharmaceutical Journal. https://www.pharmaceutical-jour nal.com/news-and-analysis/features/a-blessing-and-a-curse-the-struggle- to-introduce-e-prescribing/20206818.article. 32 R Core Team. R: A Language and Environment for Statistical Computing. https://www.r-project.org/. 43 NHS Digital. BETA - Clinical Information Standards. https://digital.nhs.uk/ about-nhs-digital/our-work/nhs-digital-data-and-technology-standards/clin ical-information-standards. 33 Wickham H. tidyverse: Easily Install and Load the ‘Tidyverse’, https:// cran.r-project.org/package"tidyverse. 34 Wickham H, Franc¸ois R, Henry L et al. dplyr: A Grammar of Data Manipulation, https://cran.r-project.org/package"dplyr. 44 Ivers N, Jamtvedt G, Flottorp S et al. Audit and feedback: effects on pro- fessional practice and healthcare outcomes. Cochrane Database Syst Rev 2012; issue 6: CD000259. 35 Dutey-Magni PF, Shallcross L. Ramses: R Package for Antimicrobial Stewardship & Surveillance [software]. https://github.com/ramses-antibiot ics/ramses-package/. doi:10.5281/zenodo.4428900. 45 Brehaut JC, Colquhoun HL, Eva KW et al. Practice feedback inter- ventions: 15 suggestions for optimizing effectiveness. Ann Intern Med 2016; 164: 435. References 36 Dutey-Magni PF, Shallcross L. Ramses: R Package for Antimicrobial Stewardship & Surveillance [software documentation]. https://ramses-antibi otics.web.app/. 46 van Santen KL, Edwards JR, Webb AK et al. The Standardized Antimicrobial Administration Ratio: a new metric for measuring and compar- ing antibiotic use. Clin Infect Dis 2018; 67: 179–85. emic.oup.com/jacamr/article/3/1/dlab018/6158273 by University College London user on 19 March 2021 37 Charani E, de Barra E, Rawson TM et al. Antibiotic prescribing in general medical and surgical specialties: a prospective cohort study. Antimicrob Resist Infect Control 2019; 8: 151. 47 Yardley L, Morrison L, Bradbury K et al. The person-based approach to intervention development: application to digital health-related behavior change interventions. J Med Internet Res 2015; 17: e30. 38 Vlahovic-Palcevski V, Gyssens IC et al. Quality indicators and quantity metrics of antibiotic use. In: Pulcini C, Ergo¨nu¨l O¨, Can F, eds. Antimicrobial Stewardship. Elsevier, 2017; 29–37. 48 Tabah A, Bassetti M, Kollef MH et al. Antimicrobial de-escalation in critical- ly ill patients: a position statement from a task force of the European Society of Intensive Care Medicine (ESICM) and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Patients Study Group (ESGCIP). Intensive Care Med 2020; 46: 245–65. 39 Beaudoin M, Kabanza F, Nault V et al. An antimicrobial prescription sur- veillance system that learns from experience. AI Mag 2014; 35: 15. 40 Lovis C, Colaert D, Stroetmann VNN. DebugIT for patient safety - improv- ing the treatment with antibiotics through multimedia data mining ofhetero- geneous clinical data. Stud Health Technol Inform 2008; 136: 641–6. 49 Berends MS, Luz CF, Friedrich AW et al. AMR – an R package for working with antimicrobial resistance data. bioRxiv 2019; doi: 10.1101/810622. 41 Morales A, Ca´novas-Segura B, Campos M et al. Proposal of a big data plat- form for intelligent antibiotic surveillance in a hospital. In: Luaces O, Ga´mez 10 of 10 10 of 10
https://openalex.org/W2145165627	https://biblio.ugent.be/publication/1172758/file/1172759.pdf	Dutch	null	De grote school van de natie. Legerartsen over drankmisbruik en geslachtsziekten in het Belgisch leger (circa 1850-1950)	Bijdragen en mededelingen betreffende de geschiedenis der Nederlanden	2,000	cc-by	23,274	De grote school van de natie. Legerartsen over drankmisbruik en geslachtsziekten in het Belgisch leger (circa 1850-1950)1 De grote school van de natie. Legerartsen over drankmisbruik en geslachtsziekten in het Belgisch leger (circa 1850-1950)1 3 Zie bijvoorbeeld A. Mooij, Geslachtsziekten en besmettingsangst. Een historisch-sociologische studie 1850-1990 (Amsterdam, 1993)35, 111-112. 2 P. Scholliers schaart zich wat de alcoholbestrijding in België betreft uitdrukkelijk achter deze moraliserings- en disciplineringsthese. Zie: "Een vijand dien men kennen moet'. Jenever in België in de negentiende en vroege twintigste eeuw', in: E. van Schoonenberghe, ed., Jenever in de lage landen (Brugge, 1996) 138-157. A. M. Brandt omschrijft de strijd tegen de geslachtsziekten in de Verenigde Staten in hoofdzaak als een moraliseringsoffensief. Volgens hem werden de venerische ziekten de voorbije twee eeuwen vooral aangewend voor de regulering van het seksueel gedrag. De venerische ziekten werden verbonden met illegitieme seksualiteit en de oplossing voor het geslachtsziektenprobleem werd louter in gedragsveranderingen gezocht. Zie: A. M. Brandt, No magie bullet. Asocial history of venereal disease in the United States since 1800 (New York, Oxford, 1987). Vgl. K. Velle, 'De syfiliskwestie in België in de negentiende en het begin van de twintigste eeuw', Tijdschrift voor sociale wetenschappen, IV (1987) 331- 363; R. Davidson, 'Venereal disease, sexual morality, and public health in interwar Scotland', Journal of the history of sexuality, XXI (1994) 267-294. LIESBET NYS De geschiedenis van het alcoholisme in de tweede helft van de negentiende en de eerste helft van de twintigste eeuw wordt, net als de geschiedenis van de geslachts- ziekten in die periode, vaak in termen van moralisering en disciplinering beschreven. De strijd tegen het drankmisbruik en de venerische ziekten wordt geïnterpreteerd als een (al dan niet weloverwogen en bewuste) poging van de gevestigde klassen om hun restrictieve (seksuele) moraal aan de arbeidersklasse op te dringen, om middenklasse- waarden als matigheid en zelfcontrole te verdedigen tegen de losbandigheid van het proletariaat2. Het valt echter op dat bijvoorbeeld de strijd die in het leger tegen de geslachtsziekten werd gevoerd, veel minder vanuit dit moraliseringsperspectief wordt bekeken. Vaak wordt beweerd dat legerartsen in hun strijd tegen de venerische ziekten bij soldaten niet zozeer door morele als wel door sanitaire motieven werden gedreven, dat hun offensief tegen de geslachtsziekten vooral door een bekommernis om de volksgezondheid was ingegeven3. Wat de Belgische legerartsen betreft, lijkt het inderdaad zo dat hun offensief tegen de geslachtsziekten in aanzienlijke mate door sanitaire motieven was geïnspireerd. In ieder geval hebben zij hun venerisch offensief vaak met sanitaire argumenten gele- gitimeerd en een aantal onder hen stelde zelfs expliciet dat zij het venerisch probleem niet 'moralistisch', maar wel vanuit een louter sanitair-medisch standpunt benaderden. Toch geeft ook hun vertoog over de venerische ziekten overduidelijk blijk van een bezorgdheid om de moraal. Het Belgisch leger had in de negentiende en het begin van de twintigste eeuw op moreel vlak een bijzonder kwalijke reputatie. In de maat- schappij leefde de idee dat de kazerne een oord van verderf en losbandigheid was, 1 Dit artikel kwam tot stand in het kader van een project over het degeneratiedenken in wetenschap en cultuur in België ( 1850-1950), dat wordt gefinancierd door het Fonds voor Wetenschappelijk Onderzoek Vlaanderen. Dank ben ik verschuldigd aan J. Tollebeek en aan K. Wils voor hun kritische opmerkingen bij een eerdere versie van dit artikel. 2 P. Scholliers schaart zich wat de alcoholbestrijding in België betreft uitdrukkelijk achter deze moraliserings- en disciplineringsthese. Zie: "Een vijand dien men kennen moet'. Jenever in België in de negentiende en vroege twintigste eeuw', in: E. van Schoonenberghe, ed., Jenever in de lage landen (Brugge, 1996) 138-157. A. M. Brandt omschrijft de strijd tegen de geslachtsziekten in de Verenigde Staten in hoofdzaak als een moraliseringsoffensief. De grote school van de natie. Legerartsen over drankmisbruik en geslachtsziekten in het Belgisch leger (circa 1850-1950)1 De grote school van de natie. Legerartsen over drankmisbruik en geslachtsziekten in het Belgisch leger (circa 1850-1950)1 5 De opzet van het tijdschrift wordt uiteengezet in: A. Meynne, 'A nos collègues de l'armée', Archives de médecine militaire, I (januari 1848) i-vi. Zie ook K. Veile, 'Bronnen voor de medische geschiedenis. De Belgische medische pers (begin XIXde eeuw-1940)', Annalen van de Belgische vereniging voorde geschie- denis van de hospitalen en de volksgezondheid, XXIII-XXIV (1985-1986) 83; R. François, 'Geschiedenis van het militaire medische tijdschrift', in: E. Evrard, J. Mathieu, ed.,Asklepios onder de wapens. 500 Jaar militaire geneeskunde in België (Brussel, 1997) 459-469. Het tijdschrift verschijnt vandaag nog steeds, zij het als Annales medicinae militaris Belgicae. Onder meer tijdens de twee wereldoorlogen werd de publicatie ervan wel kortstondig (niet voor de volledige duur van de oorlogen) gestaakt. François geeft een overzicht van de vele naamsveranderingen die het tijdschrift heeft ondergaan (460). Het eerste nummer droeg bijvoorbeeld de titel Archives de médecine militaire, vanaf 1849 heette het Archives belges de médecine militaire, vanaf 1863 Archives médicales belge enzovoort. In wat volgt geef ik de titel van het tijdschrift in alle gevallen verkort als Archives weer. LIESBET NYS Volgens hem werden de venerische ziekten de voorbije twee eeuwen vooral aangewend voor de regulering van het seksueel gedrag. De venerische ziekten werden verbonden met illegitieme seksualiteit en de oplossing voor het geslachtsziektenprobleem werd louter in gedragsveranderingen gezocht. Zie: A. M. Brandt, No magie bullet. Asocial history of venereal disease in the United States since 1800 (New York, Oxford, 1987). Vgl. K. Velle, 'De syfiliskwestie in België in de negentiende en het begin van de twintigste eeuw', Tijdschrift voor sociale wetenschappen, IV (1987) 331- 363; R. Davidson, 'Venereal disease, sexual morality, and public health in interwar Scotland', Journal of the history of sexuality, XXI (1994) 267-294. BMGN, 115 (2000) afl.3, 392-42S De grote school van de natie 393 een plaats waar alcoholische dranken rijkelijk vloeiden en onzedelijke praktijken tot de dagelijkse realiteit behoorden. Juist in de radicale bestrijding van de geslachtsziekten en ook van het drankmisbruik bij soldaten zagen de Belgische legerartsen een efficiënte manier om de morele toestand in het leger te verbeteren, om het leger moreel te ver- heffen. In dit opstel wil ik dan ook betogen dat de Belgische militaire artsen met hun offensief tegen de venerische ziekten en het drankmisbruik bij soldaten zeker niet alleen een verbetering van de gezondheidstoestand in de kazerne beoogden, maar dat zij daarmee ook ontegensprekelijk morele doeleinden nastreefden. Bij de strijd tegen de venerische ziekten en het alcoholisme in het leger gingen sanitaire en morele drijfveren hand in hand. Ze raakten bovendien verweven met andere motieven, die niet altijd even helder van het sanitair-morele motivatiecomplex te onderscheiden zijn: het vergroten van de faam en het prestige van het leger, het verhogen van de militaire en nationale efficiëntie, de regeneratie van het ras. Een tweede opzet van dit opstel bestaat erin om ook de rol van deze motieven bij het drank- en venerisch offensief van de Belgische legerartsen te verduidelijken. Als belangrijkste bron voor dit opstel diende het tijdschrift van de gezondheidsdienst van het Belgisch leger. In januari 1848 kregen de artsen van de Belgische strijdmacht een eigen forum: voor het eerst verschenen de Archives de médecine militaire (in het vervolg kortweg de Archives genoemd). Voor hoofdredacteur Armand Meynne4, een sociaalvoelende legerarts en hygiënist die in 1865 met zijn Topographie médicale de Belgique internationale faam zou verwerven, kondigde het tijdschrift een nieuw tijdperk voor de militaire geneeskunde in België aan. 4 Over Meynne: K. Veile, 'Armand Joseph Meynne. Legerarts en sociaal denker', in: J. de Maeyer, e. a., ed., Er is leven voor de dood. Tweehonderd jaar gezondheidszorg in Vlaanderen (Kapellen, 1998) 108-110. GESLACHTSZIEKTEN EN ALCOHOLISME IN DE ARCHIVES Het venerisch probleem en het alcoholisme, in de tweede helft van de negentiende en de eerste helft van de twintigste eeuw brandende kwesties in de medische wereld, konden ook bij de Belgische legerartsen op bijzondere belangstelling rekenen. Er rustte in dc Archives in geen geval een taboe op de geslachtsziekten. Al ging de aandacht in de eerste jaargangen vooral uit naar cholera en oftalmie6, toch werden ook de venerische ziekten van bij het begin onbeschroomd besproken7. De Archives volgden het onderzoek over de geslachtsziekten op de voet, vaak met originele bijdragen, soms aan de hand van artikelen die — eventueel in samenvatting — uit buitenlandse (vooral Franse) medische tijdschriften werden overgenomen. Het tijdschrift biedt dan ook een mooi overzicht van de frustraties en successen die de Venerologie in de tweede helft van de negentiende en de eerste helft van de twintigste eeuw beleefde. In de tweede helft van de negentiende eeuw waren het vooral frustraties, want ondanks het uitgebreid wetenschappelijk onderzoek werd er toen nauwelijks enige vooruitgang in de kennis over de geslachtsziekten geboekt. Ook de therapeutische bestrijding van de ziekten bevond zich duidelijk in een impasse. Voor de behandeling van gonorroe werd vooral naar spoelingen met kaliumpermanganaat of zilvernitraat gegrepen, maar echte genezing van de ziekte werd daarmee zelden bereikt. Aan syfilispatiënten werd meestal een kwikbehandeling voorgeschreven, maar sommige artsen beweerden dat patiënten daar meer schade van ondervonden dan dat ze er baat bij hadden. De syfi- lisatiepogingen van de Franse arts Auzias-Turenne, die in het begin van de jaren vijf- tig in de Archives ruime aandacht kregen, liepen met een sisser af8. De hele tweede helft van de negentiende eeuw zochten artsen koortsachtig maar tevergeefs naar een specifiek geneesmiddel tegen de verschillende geslachtsziekten. In de Archives werd bij wijlen heftig gediscussieerd over de heilzaamheid van bepaalde therapeutische middeltjes die aan venerische patiënten werden toegediend. 6 Oftalmie (oogontsteking) vormde tot het laatste kwart van de negentiende eeuw een echte plaag in het Belgisch leger. Soms zochten legerartsen de oorzaak van deze aandoening in geslachtsziekten als syfilis of gonorroe. Zie D. D. Edwards, 'Microbiology of the eye and ophthalmia', in: D. M. Albert, D. D. Edwards, ed., The history ofophthalmology (Cambridge, 1996) 147-183. 7 In het prestigieuze artsenblad Le scalpel verschenen in de jaren vijftig ook geregeld artikelen over de diagnose en behandeling van geslachtsziekten. In het hygiënistisch tijdschrift Le mouvement hygiénique (MH) daarentegen werd het onderwerp doodgezwegen. LIESBET NYS Al te lang was de gezondheids- dienst van het leger door artsen gebruikt als een opstapje voor een carrière in de burgerlijke samenleving. In de toekomst zouden artsen hun ambities binnen de gezond- heidsdienst van de strijdmacht kunnen realiseren. De Archives moesten de profes- sionele belangen van de legerartsen verdedigen. Maar daarnaast had het blad ook uit- drukkelijk wetenschappelijke oogmerken. Het publiceerde zowel artikelen over medi- sche problemen die eigen waren aan het militaire milieu (zoals oorlogsblessures) als bijdragen van algemeen geneeskundige aard. De Archives ontpopten zich in de tweede helft van de negentiende en het begin van de twintigste eeuw tot één van de meest toonaangevende geneeskundige tijdschriften van België5. 394 Liesbet Nys Liesbet Nys GESLACHTSZIEKTEN EN ALCOHOLISME IN DE ARCHIVES 6 Oftalmie (oogontsteking) vormde tot het laatste kwart van de negentiende eeuw een echte plaag in het Belgisch leger. Soms zochten legerartsen de oorzaak van deze aandoening in geslachtsziekten als syfilis of gonorroe. Zie D. D. Edwards, 'Microbiology of the eye and ophthalmia', in: D. M. Albert, D. D. Edwards, ed., The history ofophthalmology (Cambridge, 1996) 147-183. 6 Oftalmie (oogontsteking) vormde tot het laatste kwart van de negentiende eeuw een echte plaag in het Belgisch leger. Soms zochten legerartsen de oorzaak van deze aandoening in geslachtsziekten als syfilis of gonorroe. Zie D. D. Edwards, 'Microbiology of the eye and ophthalmia', in: D. M. Albert, D. D. Edwards, ed., The history ofophthalmology (Cambridge, 1996) 147-183. 7 In het prestigieuze artsenblad Le scalpel verschenen in de jaren vijftig ook geregeld artikelen over de diagnose en behandeling van geslachtsziekten. In het hygiënistisch tijdschrift Le mouvement hygiénique (MH) daarentegen werd het onderwerp doodgezwegen. Tot 1899, het jaar van de eerste internationale conferentie over de venerische ziekten, wijdde het nauwelijks meer dan enkele regels aan syfilis of de daarmee verbonden prostitutiekwestie. Hoofdredacteur Belval was zich van die leemte bewust. Hij wijtte ze aan 'la difficulté à traiter ce sujet scabreux entre tous'. Zie: MH, lil (december 1887) 478. Le mouvement hygiénique richtte zich dan ook op een breed geïnteresseerd publiek, terwijl het lezerspubliek van de Ar- chives en Le scalpel uitsluitend uit artsen bestond. 8 Auzias-Turenne geloofde dat men gezonde mensen immuun kon maken tegen syfilis door ze met het syfilisvirus in te enten. De Franse Academie voor geneeskunde veroordeelde in 1852 de syfilisatie. In de Archives verschenen in de jaren vijftig bijna maandelijks fragmenten uit de 'Cours de syphilisation' van Auzias-Turenne. Ook na de veroordeling van de syfilisatie in Frankrijk zetten de Archives de publicatie hiervan verder. Toch bleef de syfilisatietheorie in de Archives zeker niet vrij van kritiek. Zie bijvoorbeeld Binard, 'Sur la syphilisation'. Archives, X (mei-juni 1857) 399-405 en Verheyen, 'Considérations sur trois faits de la médecine contemporaine, présentées à l'Académie royale de médecine', Archives, XI (januari 1858)28-53. 8 Auzias-Turenne geloofde dat men gezonde mensen immuun kon maken tegen syfilis door ze met het syfilisvirus in te enten. De Franse Academie voor geneeskunde veroordeelde in 1852 de syfilisatie. In de Archives verschenen in de jaren vijftig bijna maandelijks fragmenten uit de 'Cours de syphilisation' van Auzias-Turenne. Ook na de veroordeling van de syfilisatie in Frankrijk zetten de Archives de publicatie hiervan verder. Toch bleef de syfilisatietheorie in de Archives zeker niet vrij van kritiek. Zie bijvoorbeeld Binard, 'Sur la syphilisation'. Archives, X (mei-juni 1857) 399-405 en Verheyen, 'Considérations sur trois faits de la médecine contemporaine, présentées à l'Académie royale de médecine', Archives, XI (januari 1858)28-53. 7 In het prestigieuze artsenblad Le scalpel verschenen in de jaren vijftig ook geregeld artikelen over de diagnose en behandeling van geslachtsziekten. In het hygiënistisch tijdschrift Le mouvement hygiénique (MH) daarentegen werd het onderwerp doodgezwegen. Tot 1899, het jaar van de eerste internationale conferentie over de venerische ziekten, wijdde het nauwelijks meer dan enkele regels aan syfilis of de daarmee verbonden prostitutiekwestie. Hoofdredacteur Belval was zich van die leemte bewust. Hij wijtte ze aan 'la difficulté à traiter ce sujet scabreux entre tous'. Zie: MH, lil (december 1887) 478. Le mouvement hygiénique richtte zich dan ook op een breed geïnteresseerd publiek, terwijl het lezerspubliek van de Ar- chives en Le scalpel uitsluitend uit artsen bestond. GESLACHTSZIEKTEN EN ALCOHOLISME IN DE ARCHIVES Tot 1899, het jaar van de eerste internationale conferentie over de venerische ziekten, wijdde het nauwelijks meer dan enkele regels aan syfilis of de daarmee verbonden prostitutiekwestie. Hoofdredacteur Belval was zich van die leemte bewust. Hij wijtte ze aan 'la difficulté à traiter ce sujet scabreux entre tous'. Zie: MH, lil (december 1887) 478. Le mouvement hygiénique richtte zich dan ook op een breed geïnteresseerd publiek, terwijl het lezerspubliek van de Ar- chives en Le scalpel uitsluitend uit artsen bestond. 8 Auzias-Turenne geloofde dat men gezonde mensen immuun kon maken tegen syfilis door ze met het syfilisvirus in te enten. De Franse Academie voor geneeskunde veroordeelde in 1852 de syfilisatie. In de Archives verschenen in de jaren vijftig bijna maandelijks fragmenten uit de 'Cours de syphilisation' van Auzias-Turenne. Ook na de veroordeling van de syfilisatie in Frankrijk zetten de Archives de publicatie hiervan verder. Toch bleef de syfilisatietheorie in de Archives zeker niet vrij van kritiek. Zie bijvoorbeeld Binard, 'Sur la syphilisation'. Archives, X (mei-juni 1857) 399-405 en Verheyen, 'Considérations sur trois faits de la médecine contemporaine, présentées à l'Académie royale de médecine', Archives, XI (januari 1858)28-53. 395 De grote school van de natie In het begin van de twintigste eeuw kwam het onderzoek over de geslachtsziekten echter in een stroomversnelling terecht. In 1905 slaagden Fritz Schaudinn en Erich Hoffmann erin de bacterie die syfilis veroorzaakt, de treponema pallidum, af te zonderen9. In 1906 werd de serodiagnostiek van Bordet-Wassermann op punt gesteld en in 1910 maakte de ontdekking door Paul Ehrlich van salvarsan als medicijn tegen syfilis een eind aan de langdurige therapeutische impasse. Al deze wetenschappelijke successen werden in de Archives op voorzichtig enthousiasme onthaald. Legerartsen deden in de militaire hospitalen onderzoek naar de zenuwaandoeningen die soms na het toedienen van salvarsan optraden, en zochten naar de beste verhouding tussen salvarsan en kwik of salvarsan en bismuth voor de zogenaamde gemengde behandeling van syfilis, die dit soort zenuwaandoeningen moest voorkomen. Na de Tweede Wereld- oorlog verschenen in de Archives de eerste berichten over de penicilline. Die leek een volledige genezing van de geslachtsziekten te garanderen en zou dan ook weldra op algemene schaal worden ingevoerd10. Naar het alcoholprobleem ging in de Archives proportioneel gezien minder aandacht uit. Toch bracht het tijdschrift geregeld verslag uit over het onderzoek dat in binnen- en buitenland werd verricht naar de gevolgen van alcoholische dranken op het men- selijk lichaam. 9 In 1879 had A. Neisser al de bacterie die gonorroe veroorzaakt, de gonokok, ontdekt. 10 Voor het negentiende- en twintigste-eeuwse onderzoek over de geslachtsziekten: E. Bäumler, Amors vergifteter Pfeil. Kulturgeschichte einer verschwiegenen Krankheit (Hamburg, 1976); J. T. Crissey, L. C. Parish, The dermatology and syphilology of the nineteenth century (New York, 1981 ); C. Quétel, Le mal de Naples. Histoire de la syphilis (Parijs, 1986). Voor België: Craps, Jacqué, 'La dermatologie et la syphili- graphie en Belgique pendant un siècle', in: Cent ans de médecine en Belgique (1830-1930) (Brussel, 1931) 193-268; P. Rebmann, 'Syfilis, 1830-1860. Een historisch en wetenschapskritisch onderzoek naar de medische constructie van een epidemie' (Onuitgegeven licentiaatsverhandeling KU Leuven; Leuven, 1989); Idem, 'Syfilis 1834-1850. De geboorte van een epidemie', Belgisch tijdschrift voor nieuwste ge- schiedenis (BTNG), XXII (1991) 569-623. 12 Van Vyve, 'Ulcère de l'estomac chez un alcoolique. Sclérose du pancréas', Archives, XXX (oktober 1877)247-252. 13 Daarnaast was bij artsen ook het verband tussen drankmisbruik en tuberculose populair. Legerartsen 11 Zie hiervoor D. Nourrisson, Alcoolisme et antialcoolisme en France sous la troisième république. L'exemple de la seine inférieure (Parijs, 1988); Idem, Le buveur au 19e siècle (Parijs, 1990). Vgl. G. John- stone, 'From vice to disease? The concepts of dipsomania and inebriety, 1860-1908', Social and legal studies. An international journal, V (1996) 37-56. 9 In 1879 had A. Neisser al de bacterie die gonorroe veroorzaakt, de gonokok, ontdekt. 10 Voor het negentiende- en twintigste-eeuwse onderzoek over de geslachtsziekten: E. Bäumler, Amors vergifteter Pfeil. Kulturgeschichte einer verschwiegenen Krankheit (Hamburg, 1976); J. T. Crissey, L. C. Parish, The dermatology and syphilology of the nineteenth century (New York, 1981 ); C. Quétel, Le mal de Naples. Histoire de la syphilis (Parijs, 1986). Voor België: Craps, Jacqué, 'La dermatologie et la syphili- graphie en Belgique pendant un siècle', in: Cent ans de médecine en Belgique (1830-1930) (Brussel, 1931) 193-268; P. Rebmann, 'Syfilis, 1830-1860. Een historisch en wetenschapskritisch onderzoek naar de medische constructie van een epidemie' (Onuitgegeven licentiaatsverhandeling KU Leuven; Leuven, 1989); Idem, 'Syfilis 1834-1850. De geboorte van een epidemie', Belgisch tijdschrift voor nieuwste ge- schiedenis (BTNG), XXII (1991) 569-623. 11 Zie hiervoor D. Nourrisson, Alcoolisme et antialcoolisme en France sous la troisième république. L'exemple de la seine inférieure (Parijs, 1988); Idem, Le buveur au 19e siècle (Parijs, 1990). Vgl. G. John- stone, 'From vice to disease? The concepts of dipsomania and inebriety, 1860-1908', Social and legal studies. An international journal, V (1996) 37-56. 12 Van Vyve, 'Ulcère de l'estomac chez un alcoolique. Sclérose du pancréas', Archives, XXX (oktober 1877)247-252. 15 Zie bijvoorbeeld E. Henrotay, 'Considérations sur les écoulements de l'urètre et leur traitement'. Archives, III (september 1850) 184; Semet, 'Hôpital militaire de Liège. Note sur un cas de syphilis maligne observé dans le service de M. le médecin de régiment de Ire classe Hymblet', Archives, L (april I897) 245. 16 A. Bellens, 'Het alcoholprobleem en de strijdkrachten', Acta médicinalia militaria Belgica, CXXXVII (1985)124-126. schreven het hoge aantal tuberculoselijders in het leger ten dele toe aan de alcoholexcessen van soldaten. Zie bijvoorbeeld G. Martin, 'La lutte contre la tuberculose. Les sanatoria populaires de l'empire d'Allemagne', Archives, L1V (april 1901) 331; Lejeune, 'La lutte contre la tuberculose dans l'armée', Archives, LVII (1904) 202-204. Vgl. D. Nourrisson, 'Tuberculose et alcoolisme ou du bon usage d'un aphorisme', in: J.-P. Bardet, e. a.. Peurs et terreurs face à la contagion. Choléra, tuberculose, syphilis XlXe-XXe siècle (Parijs, 1988) 199-217; D. S. Barnes, The making of a social disease. Tuberculosis in nineteenth-century France (Berkeley, Los Angeles, Londen, 1995) 138-173. Verder werden dronkaards ook erg vatbaar geacht voor cholera. Zie onder andere E. Chevalier, Recueil des règlements, instructions, lois, arrêtés et circulaires concernant le service de santé de l'armée (Charleroi, Brussel, 1891) 100. 14 Ch. Petithan, 'Note sur le traitement de l'uréthrite', Archives, XXXI (maart 1863) 226-227. Vgl. F. van den Branden, 'Importance des complications de la blennorrhagie', Archives, LXXII1 (februari 1920) 100. 17 Tussen het soldatenberoep en syfilis werd al vele eeuwen een verband gelegd. Zie O. Temkin, 'On the history of 'morality and syphilis", in: O. Temkin, The double face of Janus and other essays in the history of medicine (Baltimore, Londen, 1977) 477. GESLACHTSZIEKTEN EN ALCOHOLISME IN DE ARCHIVES In de tweede helft van de negentiende eeuw ontdekten artsen het alco- holisme als 'ziekte'. Drankmisbruik evolueerde van een zonde naar een pathologisch verschijnsel, een medisch studieobject11. Artsen gingen minutieus na welke schade alcohol in het menselijk organisme kon aanrichten. Ook een aantal legerartsen hield zich met dergelijk onderzoek bezig. Assistent-arts Van Vyve bijvoorbeeld beschreef in 1878 in de Archives de ravages die alcoholexcessen in het lichaam van een drieën- vijftigjarige brigadier hadden aangericht en uiteindelijk zelfs tot de dood van de onver- beterlijke jeneverdrinker hadden geleid12. Alcoholmisbruik kwam in de Archives echter vooral ter sprake als oorzaak en versnel- lende factor van allerhande ziekten, bijvoorbeeld van de geslachtsziekten. Tussen alcoholmisbruik en venerische ziekten werd in de tweede helft van de negentiende en het begin van de twintigste eeuw een nauw verband verondersteld13. Aangenomen Liesbet Nys 396 werd dat geslachtsziekten bijna altijd in benevelde toestand werden opgelopen. Leger- artsen waren ervan overtuigd dat ook de venerische ziekten bij soldaten meestal aan excessief drankgebruik te wijten waren. Daarom raadde legerarts Charles Petithan zijn rekruten steevast aan om nooit in beschonken toestand een vrouw te ontmoeten14. Mensen die al aan een venerische ziekte leden, werd ten zeerste afgeraden nog alco- holische dranken te consumeren. Eén enkel alcoholexces kon — aldus sommige legerartsen — het ziekteproces drastisch versnellen of ernstige complicaties veroor- zaken15. Het alcoholvraagstuk en het geslachtsziektenprobleem konden beide op grote belang- stelling van de legerartsen rekenen en ze werden vaak zo sterk met elkaar verweven dat er eigenlijk nog slechts sprake was van één en hetzelfde probleem. Het feit dat in de kringen van militaire geneeskundigen zoveel aandacht naar de venerische ziekten en het drankmisbruik uitging, hield niet alleen verband met de bezorgdheid die in het algemeen over beide 'ziekten' in de medische wereld leefde, maar vindt ook een ver- klaring in het beeld dat de maatschappij over het soldatenberoep had. 14 Ch. Petithan, 'Note sur le traitement de l'uréthrite', Archives, XXXI (maart 1863) 226-227. Vgl. F. van den Branden, 'Importance des complications de la blennorrhagie', Archives, LXXII1 (februari 1920) 100. 21 Bigot, Daumerie, Canstatt, Mémoires couronnés par le congrès médical de Belgique, sur la question suivante, proposée pour sujet de prix. Exposer et déterminer les moyens médicaux et les mesures administratives et réglementaires propres à arrêter ou à modérer la propagation de la syphilis, suivis du rapport général sur le concours (SA., s. a.) 90. De idee dat soldaten in belangrijke mate bijdroegen tot de verspreiding van de geslachtsziekten op het platteland, werd door vele negentiende en vroeg-twintigste- eeuwse artsen aangehangen. Zie bijvoorbeeld ook: Lamelle, 'Conférence internationale pour la prophylaxie de la syphilis et des maladies vénériennes', MH, XV (1899) 293-294. EEN RISICOBEROEP In 1985 publiceerde geneesheer majoor Bellens in de Acta médicinalia militaria Belgica, de opvolger van de Archives, een bijdrage over het alcoholprobleem bij de strijdkrachten, maar niet — zo stelde hij uitdrukkelijk — omdat drankmisbruik in het leger meer zou voorkomen dan in de burgerlijke samenleving16. In de negentiende en het begin van de twintigste eeuw werd daarentegen wél algemeen aangenomen dat het gevaar voor drankmisbruik bij militairen groter was dan bij gewone stervelingen. Ook hun kans op geslachtsziekten werd hoger geraamd dan die van de modale mens17. Soldaten werden in de negentiende en het begin van de twintigste eeuw vaak als een De grote school van de natie 397 risicogroep voor alcoholisme en venerische ziekten beschouwd. Het leger werd door velen aangezien als een leerschool voor alcohol- en venerische excessen18. Het was een gemeenplaats dat jongemannen tijdens hun kazernejaren in een leven van ontucht en losbandigheid verzeild raakten. De populaire literatuur staat bol van de anekdotes over jonge rekruten die in het leger hun kuisheid voor een zedeloze levensstijl inruilden en daaraan één of andere geslachtsziekte overhielden19. Vooral in katholieke kringen werd vaak hard geklaagd over de belabberde moraal in het leger20. Ook de medische wereld verdacht het soldatenvolk van een decadente en liederlijke levenswandel, die zich wreekte met de meest gruwelijke venerische ziekten. On sait qu'aussitôt que les militaires ont recouvré toute leur indépendance, ordinairement le premier usage qu'ils en font c'est de s'enivrer, et le second c'est d'aller se vautrer dans les maisons de débauche, d'où ils sortent souvent avec la maladie qu'ils emportent alors avec eux pour la répandre dans la campagne, schreef een commissie van artsen in de jaren 183021. De commissie moest een oordeel vellen over de verhandelingen die waren ingestuurd als antwoord op de prijsvraag van het Brussels medisch congres van 1835. De organisatoren van dat congres wilden achterhalen met welke maatregelen het syfilisprobleem het best kon worden bestreden. In bijna alle ingestuurde verhandelingen werd bijzondere aandacht gevraagd voor het 'risicovolle' soldatenberoep. schreef een commissie van artsen in de jaren 183021. De commissie moest een oordeel vellen over de verhandelingen die waren ingestuurd als antwoord op de prijsvraag van het Brussels medisch congres van 1835. De organisatoren van dat congres wilden achterhalen met welke maatregelen het syfilisprobleem het best kon worden bestreden. In bijna alle ingestuurde verhandelingen werd bijzondere aandacht gevraagd voor het 'risicovolle' soldatenberoep. 18 Dat was niet alleen in België het geval. Ook in vele andere landen leefde bij de publieke opinie de idee dat het leger een oord van verderf was. Zie hiervoor onder andere H. L. Wesseling, Soldaat en krijger. Franse opvattingen over leger en oorlog aan de vooravond van de Eerste Wereldoorlog (Amsterdam, 1988) 33-34; E. W. R. van Roon, 'De dienstplicht op de markt gebracht. Het fenomeen dienstvervanging in de negentiende eeuw', Bijdragen en mededelingen betreffende de geschiedenis der Nederlanden (BMGN), CIX (1994) 617; D. M. Peers, 'Soldiers, surgeons and the campaigns to combat sexually transmitted diseases in colonial India, 1805-1860', Medical history, XLII (1998) 139-140, 147. 19 Bijvoorbeeld A. van lersel, De kanker der samenleving. Siphilis. Realistische zedenschets (Antwerpen, s. a.j. 20 Zie onder andere de tussenkomst van de aalmoezenier van het militair kamp van Beverloo op het katholieke Congrès des oeuvres sociales. In: Congrès des oeuvres sociales à Liège 26-29 septembre 1886 (Luik, 1886)483-491. 19 Bijvoorbeeld A. van lersel, De kanker der samenleving. Siphilis. Realistische zedenschets (Antwerpen, s. a.j. EEN RISICOBEROEP Zelfs de legerartsen timmerden vlijtig mee aan de topos van de losbandige soldaat. Ook zij wezen op de bijzondere vatbaarheid van (vooral jonge) rekruten voor drank- misbruik en geslachtsziekten en ze haalden hiervoor een resem verklaringen aan. In de eerste plaats maakte de leeftijd van de soldaten dat zij zich sterk tot het vrouwelijk geslacht aangetrokken voelden. Bovendien viel in de kazerne elke moraliserende invloed van familieleden weg. Oudere soldaten gaven vaak het slechte voorbeeld aan de jongere rekruten. Daarnaast bracht ook heimwee naar het ouderlijke nest jonge Liesbet Nys 398 militairen volgens de legerartsen soms van het rechte pad. In vredestijd dreef de verveling de soldaten naar de drankgelegenheden in de buurt van de kazerne, in oorlogstijd meenden militairen kracht en moed uit de drank te putten. Soldaten grepen ook gemakkelijk naar de fles om een militaire overwinning te vieren. Maar vooral hun volstrekte onwetendheid op seksueel gebied maakte de jonge rekruten, vooral die van het platteland, in de ogen van de legerartsen tot een bijzonder gemakkelijke prooi voor immoreel en bandeloos gedrag. De al genoemde militaire arts Petithan schreef in 1863: On peut affirmer, pour l'honneur ou plutôt la vertu de nos conscrits campagnards, que beaucoup d'entre eux arrivent au service vierges encore, sinon tout à fait innocents. Ils commencent à servir Cupidon sous les drapeaux, et comme on ne leur apprend guère l'exercice du milieu malin, il arrive qu'ils le servent mal et qu'ils blessent souvent leur propre arme22. Ook de legerartsen droegen zonder twijfel bij tot de verspreiding van het beeld dat het leger een kweekschool van zedeloze en verdorven mannen was, waar drankmis- bruik en geslachtsziekten een vruchtbare voedingsbodem vonden. De vraag of het alcohol- en geslachtsziektenprobleem in het leger ook daadwerkelijk veel dramatischer proporties aannam dan daarbuiten, wordt verder in dit opstel beantwoord. 22 Petithan, 'Note sur le traitement de l'uréthrite', 227. DE PLAATSVERVANGERS Er was één categorie soldaten die door de legerartsen in het bijzonder verantwoordelijk werd gesteld voor de slechte morele reputatie van het leger: die van de plaatsvervan- gers. De Belgische troepen werden in de negentiende eeuw door loting samengesteld, maar vaak lieten gegoede (en steeds meer ook minder gegoede) ingelote mannen zich tegen betaling vervangen door jongelui uit de laagste klassen van de samenleving. Heel wat legerartsen verweten deze plaatsvervangers dat ze door hun baldadig en ongedisciplineerd gedrag de moraal in het leger ondermijnden. Volgens hen spen- deerden de plaatsvervangers al hun vrije tijd en geld in louche drankgelegenheden in de buurt van de kazernes en spoorden zij de andere soldaten aan tot navolging van hun losbandige levensstijl. Regimentsarts Auguste Jansen, bekend om zijn niet aflatende strijd tegen het alco- holisme, gaf in 1876 zijn misprijzen voor de plaatsvervangers bijzonder weinig om- floerst te kennen: Les remplaçants, dont la moralité laisse tant à désirer, ont le plus souvent déjà avant leur entrée au service des habitudes d'ivrognerie. Les marchands d'hommes les recrutent dans la lie de la société. Pour les découvrir, ils parcourent les quartiers pauvres et populeux, faisant dans les cabarets de bas étage un appel aux fainéants, aux déclassés et concluent le 22 Petithan, 'Note sur le traitement de l'uréthrite', 227. 399 De grote school van de natie marché entre deux verres d'eau-de-vie. A partir de ce moment jusqu'au jour de l'incor- poration, le vendu passe son temps dans l'orgie et il y perd souvent ce qui lui reste d'argent, de santé et d'honnêteté. Dès leur arrivée au dépôt, ils deviennent les hôtes assidus des cabarets voisins du quartier et de la cantine de l'agent du casernement. Ils y dépensent en peu de jours leur prime d'engagement. Ces hommes vicieux sont toujours de mauvais soldats et l'exemple qu'ils donnent aux jeunes troupiers est des plus pernicieux23. Legerartsen wezen de plaatsvervangers niet alleen aan als de schuldigen voor het alcoholprobleem, maar ook bijvoorbeeld voor het hoge aantal geslachtsziekten in het leger. Hun beperkte scholing en hun ontuchtige levenswandel maakten hen op dit vlak uiterst verdacht24. Voor de legerartsen was de demoraliserende invloed van de plaatsvervangers een voldoende argument om voor de afschaffing van de plaatsvervanging en de invoering van de persoonlijke dienstplicht te pleiten. 23 A. Jansen, 'De l'usage et de l'abus des boissons alcooliques dans l'armée', Bulletin de la société de médecine d'Anvers, XXXVII (1876) 502-503. 24 Zie bijvoorbeeld Ch. Petithan. 'Prophylaxie des maladies vénériennes', Archives, XXVII (november 1885) 357. De meeste officieren en vele dienstplichtigen deelden deze misprijzende houding tegenover de plaatsvervangers. Ook zij uitten frequent hun ongenoegen over de geringe intellectuele ontwikkeling van deze soldaten, over hun immoreel en ongedisciplineerd gedrag, over het hoog aantal criminele daden en deserties in hun rangen enzovoort. Zie hiervoor: L. de Vos, Het effectief van de Belgische krijgsmacht en de militiewetgeving, 1830-1914 (Brussel, 1985)64, 183-184. Vgl. B. Schnappet, Le remplacement militaire en France. Quelques aspects politiques, économiques et sociaux du recrutement au XIXe siècle (Parijs, 1968) 150-153, 280; Van Roon, 'De dienstplicht op de markt gebracht', 617; R. Spork, 'De discussie over het remplaçantenstelsel in Nederland 1873-1898', De negentiende eeuw, IX (1985) 56-57. 25 Heel wat tegenstanders van de persoonlijke dienstplicht (in hoofdzaak katholieken) verwezen evenzeer naar de immorele toestand in het leger om hun standpunt te bepleiten. Zij hoopten met het behoud van de plaatsvervanging zoveel mogelijk jongemannen uit de betere klassen te behoeden voorde nefaste moraal die volgens hen in de kazernes heerste. Zie bijvoorbeeld C. Verfaillie, ' Katholiek verweer tegen de persoon- lijke dienstplicht (1886-1887)' (Onuitgegeven licentiaatsverhandeling KU Leuven; Leuven, 1983) 39-40; De Vos, Het effectief van de Belgische krijgsmacht, 184-186. 26 Congres des oeuvres sociales, 505-512. Vgl. 'De la prostitution. (Extraits de rapports)'. Archives, XXXIV (november 1888) 349; V. de Vaucleroy, De l'alcoolisme dans l'armée et des moyens de le combattre (Brussel, 1900) 3. Slechts één keer vond ik in de Archives kritiek op de stigmatisering van de plaatsvervangers en van bepaalde vrijwilligers in het leger. In 1878 reageerde assistent-arts Van Vy ve kribbig op de verwijten die Petithan tegen de 'mauvais volontaires' had gespuid: 'S'il est vrai qu'une certaine catégorie de soldats abandonnent leur prime dans les cabarets, nous devons reconnaître aussi que les volontaires avec primes 23 A. Jansen, 'De l'usage et de l'abus des boissons alcooliques dans l'armée', Bulletin de la société de médecine d'Anvers, XXXVII (1876) 502-503. 24 Zie bijvoorbeeld Ch. Petithan. 'Prophylaxie des maladies vénériennes', Archives, XXVII (november 1885) 357. De meeste officieren en vele dienstplichtigen deelden deze misprijzende houding tegenover de plaatsvervangers. Ook zij uitten frequent hun ongenoegen over de geringe intellectuele ontwikkeling van deze soldaten, over hun immoreel en ongedisciplineerd gedrag, over het hoog aantal criminele daden en deserties in hun rangen enzovoort. Zie hiervoor: L. de Vos, Het effectief van de Belgische krijgsmacht en de militiewetgeving, 1830-1914 (Brussel, 1985)64, 183-184. Vgl. B. Schnappet, Le remplacement militaire en France. Quelques aspects politiques, économiques et sociaux du recrutement au XIXe siècle (Parijs, 1968) 150-153, 280; Van Roon, 'De dienstplicht op de markt gebracht', 617; R. Spork, 'De discussie over het remplaçantenstelsel in Nederland 1873-1898', De negentiende eeuw, IX (1985) 56-57. 25 Heel wat tegenstanders van de persoonlijke dienstplicht (in hoofdzaak katholieken) verwezen evenzeer naar de immorele toestand in het leger om hun standpunt te bepleiten. Zij hoopten met het behoud van de plaatsvervanging zoveel mogelijk jongemannen uit de betere klassen te behoeden voorde nefaste moraal die volgens hen in de kazernes heerste. Zie bijvoorbeeld C. Verfaillie, ' Katholiek verweer tegen de persoon- lijke dienstplicht (1886-1887)' (Onuitgegeven licentiaatsverhandeling KU Leuven; Leuven, 1983) 39-40; De Vos, Het effectief van de Belgische krijgsmacht, 184-186. 26 Congres des oeuvres sociales, 505-512. Vgl. 'De la prostitution. (Extraits de rapports)'. Archives, XXXIV (november 1888) 349; V. de Vaucleroy, De l'alcoolisme dans l'armée et des moyens de le combattre (Brussel, 1900) 3. Slechts één keer vond ik in de Archives kritiek op de stigmatisering van de plaatsvervangers en van bepaalde vrijwilligers in het leger. In 1878 reageerde assistent-arts Van Vy ve kribbig op de verwijten die Petithan tegen de 'mauvais volontaires' had gespuid: 'S'il est vrai qu'une certaine catégorie de soldats abandonnent leur prime dans les cabarets, nous devons reconnaître aussi que les volontaires avec primes DE PLAATSVERVANGERS Heel wat legerartsen beweerden dat de afschaffing van de plaatsvervanging het alcoholmisbruik en de venerische ziekten in het leger drastisch zou terugdringen en tot een moreel herstel van de strijdkrachten zou leiden25. Petithan bijvoorbeeld hield eind september 1886 op het katholieke Congrès des oeuvres sociales in Luik, vanuit zijn bekende bekommernis om de moraal in het leger, een krachtig pleidooi voor de afschaffing van de plaatsvervanging en de invoering van de persoonlijke dienstplicht. Om te verhinderen dat jonge militairen in het leger nog langer in contact zouden komen met 'des hommes tarés' wilde hij niet alleen de plaatsvervangers uit het leger zien verdwijnen, maar ook alle 'mauvais vo- lontaires', de jongemannen die slechts naar het leger werden gestuurd omdat er verder niets met hen viel aan te vangen. Volgens Petithan was het uiterst gevaarlijk om het leger louter uit de laagste klassen van de samenleving te rekruteren26. 400 Liesbet Nys De strijd die de artsen van het Belgisch leger — met de steun van de meeste officieren trouwens — op het einde van de negentiende eeuw tegen de plaatsvervanging voerden, was in belangrijke mate een strijd om de morele verheffing van het leger. Legerartsen schoven de slechte morele reputatie van de strijdmacht volledig in de schoenen van de plaatsvervangers. Hun abominabele moraal, onder meer af te lezen uit het hoog aantal alcoholici en venerische patiënten dat zich in hun rangen bevond, vormde volgens de militaire artsen een smet op het blazoen van het leger. Alleen de invoering van de persoonlijke dienstplicht, die de ingelote jongemannen uit de hogere klassen van de samenleving zou verplichten om zelf hun legerdienst te vervullen, kon aldus de legerartsen tot een moreel herstel van de strijdmacht leiden. De welopgevoede en degelijk geschoolde mannen uit de betere milieus van het land zouden volgens hen zonder twijfel het moreel gedrag van de soldaten van lagere komaf in gunstige zin beïnvloeden. Het vertoog van de militaire artsen over de plaatsvervangers illustreert dat de strijd tegen de geslachtsziekten en het alcoholisme in het leger zeker niet alleen door sanitaire, maar duidelijk ook door morele aspiraties was ingegeven. Daarnaast toont dat vertoog aan dat ook in het leger klasse-elementen een rol speelden bij de definiëring van het alcohol- en geslachtsziektenprobleem. Zowel het alcoholisme als de venerische ziekten werden tot een moreel probleem van de laagste rangen van het leger herleid. 28 België schafte de plaatsvervanging met een ruime vertraging op de andere Europese landen af. In Frankrijk was dat bijvoorbeeld al in 1872 gebeurd. Nederland voerde in 1898 de persoonlijke dienstplicht in. Voor de eindeloze debatten die tussen 1830 en 1914 in België over de rekruteringswijze voor het leger werden gevoerd, zie De Vos, Het effectief van de Belgische krijgsmacht. Vgl. Schnapper, Le replacement militaire; V. G. Kiernan, 'Conscription and society in Europe before the war of 1914-1918', in: H. J. Kaye, ed., History, classes and nation-states. Selected writings of V. G. Kiernan (Oxford, 1988) 166-185; W. Klinkert, Het vaderland verdedigd. Plannen en opvattingen over de verdediging van Nederland 1874- 1914 (Den Haag, 1992); H. te Velde, Gemeenschapszin en plichtsbesef. Liberalisme en nationalisme in Nederland 1870-1918 (Den Haag, 1992) 41-49, 53-55, 97-99. sont, dans quelques corps, victimes d'injustes préjugés. Sans doute, la plupart de ces volontaires sont loin de constituer l'élite de la société, mais c'est un motif de plus pour que l'influence moralisatrice, qu'on attribue à l'armée, s'exerce sur ces intelligences. Méprisés, repoussés a priori comme de mauvais sujets, ces hommes cherchent dans l'ivresse de l'alcool l'oubli de leurs peines et finissent par y prendre ce qui leur restait de dignité', in: 'Des moyens de combattre l'ivrognerie dans l'armée', Archives, XIV (december 1878)455. 27 Petithan, 'Prophylaxie des maladies vénériennes', 352. Petithan, 'Prophylaxie des maladies vénériennes', 352. 29 Al vele eeuwen werd er een verband gelegd tussen de soldaat en de prostituee. Zie Quétel, Le mal de Naples, 283-284. 30 Hierbij dachten de toenmalige legerartsen niet in de eerste plaats aan verspreiding van de venerische ziekten onder de soldaten via homoseksuele contacten, maar wel via allerlei niet-venerische vormen van besmetting (bijvoorbeeld door besmette drinkbekers of pijpen), die in de negentiende en het begin van de twintigste eeuw ook door artsen buiten het leger als een reëel gevaar werden beschouwd. 31 J.-R. Marinus, Essai sur l'hygiène du soldat ou exposé des moyens propres à l'entretien de la santé des gens de guerre (Brussel, 1841) 17-19. Een soortgelijke bepaling werd opgenomen in de wet van 14 augustus 1887: 'Loi relative au logement des troupes en marches et en cantonnement et aux prestations militaires', Journal militaire officiel (JMO) (1887) 258-264. 32 Rulot, Sacré, La vie du soldat Belge. Considérations et conseils relatifs à l'éducation et à l'hygiène militaires (Brussel, [1913]) 129. DE PLAATSVERVANGERS Al wees af en toe wel eens een arts op het feit dat ook officieren niet 'à l'abri des blessures de Vénus' zijn27, toch lag de klemtoon op de groep van arme en ongeschoolde soldaten die uit geldnood gedwongen werd om als plaatsvervanger in het leger een bron van inkomsten te vinden. De legerartsen verdedigden de afschaffing van de plaatsvervanging soms met 'Ie grand principe de l'égalité de tout citoyen devant la loi' en ze beoogden met de invoering van de persoonlijke dienstplicht een soort 'verbroedering' van de verschillende sociale klassen in het leger, maar tegelijker- tijd gingen zij duidelijk uit van de morele superioriteit van de hogere standen. Pas in 1909 haalden de legerofficieren in het debat over de rekruteringswijze van het leger hun slag thuis. Een nieuwe militiewet voerde toen de persoonlijke dienstplicht in. Het tijdperk van de plaatsvervanging werd afgesloten28. 401 De grote school van de natie 29 Al vele eeuwen werd er een verband gelegd tussen de soldaat en de prostituee. Zie Quétel, Le mal de Naples, 283-284. 34 Standaardwerken over de geschiedenis van de prostitutie en het reglementarisme zijn: A. Corbin, Les filles de noce. Misère sexuelle et prostitution (19e siècle) (Parijs, 1978); J. R. Walkowitz, Prostitution and Victorian society. Women, class, and the state (Cambridge, 1980). Voor de prostitutiekwestie in België, zie onder andere: S. de Schaepdryver, 'De zonde in banen geleid. Gereglementeerde prostitutie in Brussel, 1844-1877. Onderzoek naar houd(st)ers van getolereerde bordelen' (Onuitgegeven licentiaatsverhandeling VUB; Brussel, 1983); Idem, 'Reglementering van prostitutie, 1844-1877: opkomst en ondergang van een experiment', BTNG, XVI (1985) 473-506; C. Huberty, L. Keunings, 'La prostitution à Bruxelles au XIXe siècle', Les cahiers de la fonderie, Il (1987) 3-22; M.-S. Dupont-Bouchat, 'Verdraagzaamheid en repressie. Fascinatie en weerzin. Elkaar dwarsende blikken op de prostitutie in België (15de-20ste eeuw)', in: Van badhuis tot eroscentrum. Prostitutie en vrouwenhandel van de Middeleeuwen lot heden (Brussel, 1995) 51-87; K. Pittomvils, 'Tussen repressie en permessiviteit. Socialisme, socialisten, prostitutie en geslachts- ziekten (einde 19de eeuw-1997)', in: D. de Weerdt, ed., Begeerte heeft ons aangeraakt. Socialisme, sekse en seksualiteit (Gent, 1999) 208-235. Over de prostitutiekwestie in Nederland: P. de Vries, Kuisheid voor mannen, vrijheid voor vrouwen. De reglementering en bestrijding van prostitutie in Nederland, 1850-1911 (Hilversum, 1997); M. Bossenbroek, J.H. Kompagnie, Het mysterie van de verdwenen bordelen. Prostitutie in Nederland in de negentiende eeuw (Amsterdam, 1998). DE PROSTITUEE In het garnizoen werden de plaatsvervangers verantwoordelijk gesteld voor het alcohol- en geslachtsziektenprobleem bij de soldaten, maar de hoofdschuldige zochten de le- gerartsen buiten de muren van de kazerne: de prostituee29. De prostituee — zo luidde het cliché — lokte de soldaten met haar provocerende blik en haar uitdagende kledij mee naar één van de vele bordelen of verdachte drankgelegenheden in de buurt van de kazerne. Daar voerde ze de soldaten in een snel tempo dronken. Geïnfecteerd met een venerische ziekte verlieten die enige tijd later het gore etablissement. Van dat ogenblik af vormden ze volgens de militaire geneesheren een ernstige bedreiging voor het hele garnizoen30. In de ogen van de legerartsen betekende de prostituee zowel een sanitair als een moreel gevaar voor de soldaten. Aan de ene kant leefde bij die artsen de idee dat de prostituee de belangrijkste haard van besmetting voor geslachts- ziekten bij soldaten was. Aan de andere kant bevatten de geschriften van negentiende- en vroeg-twintigste-eeuwse legerartsen ook eindeloze reeksen klachten over het wanvoeglijk en aanstootgevend gedrag van de publieke vrouwen, waaruit blijkt dat voor hen de prostituees, net zoals de plaatsvervangers, ook als zondebok fungeerden voor de verdorven morele toestand in het leger. In 1841 becommentarieerde de arts Marinus als volgt een Koninklijk Besluit van 1 mei 1838 dat bepaalde dat militairen de nacht niet mochten doorbrengen in ontuchthuizen: 'On ne peut qu'applaudir à cette mesure toute dans l'intérêt de la morale et de la santé du soldat'31. Pleitbezorgers voor prostitutiereglementering z g v p g g Legerartsen hadden meestal weinig oog voor de sociale omstandigheden die vrouwen in de prostitutie dreef. Prostituees werden omschreven als 'êtres dégradés' of 'viles créatures', als gedegenereerde vrouwen die uit wellust of winstbejag hun eigen lichaam verkochten. In 1913 bijvoorbeeld schreven de militaire artsen Rulot en Sacré naar aanleiding van het geslachtsziektenprobleem in een handboek voor jonge soldaten: 'Le danger, la source du mal, c'est la fille publique, la femme qui se donne à tous par bestialité ou par intérêt'32. Met dit soort brute formuleringen hoopten ze de soldaten blijkbaar een instinctieve afkeer voor de prostitutie bij te brengen. Rulot en Sacré wilden de rekruten immers aansporen tot seksuele onthouding. De idee dat seksuele onthouding voor mannen mogelijk en niet schadelijk was, had op het einde van de 402 Liesbet Nys negentiende eeuw al ruime ingang gevonden bij civiele artsen. 33 Zie bijvoorbeeld het rapport dat Petithan in 1890 voorstelde aan de commissie die in het parlement over de prostitutiekwestie was aangesteld: Documents parlementaires. Recueil des pièces imprimées par ordre de la chambre des représentants, nr. 136, séance du 5 avril 1892. Commission chargée de préparer un projet de loi sur la police des moeurs, procès-verbaux des séances de la section d'hygiène (S. 1., s. a.) 140. DE PROSTITUEE Met enige vertraging drong deze idee in het begin van de twintigste eeuw ook door tot de kringen van de militaire geneeskundigen. Die hadden zich in de tweede helft van de negentiende eeuw echter bijzonder verknocht getoond aan de idee dat het celibaat voor mannen onmogelijk (en zelfs ongezond) was. Legerartsen namen toen aan dat mannen die geen 'natuurlijke' seksuele betrekkingen konden hebben, hun toevlucht zochten in pervers seksueel gedrag. Om te vermijden dat mannen hun natuurlijke seksuele driften in masturbatie of in de aanranding van eerbare vrouwen zouden botvieren, was het volgens hen beter om prostitutie toe te laten. Legerartsen zagen in de tweede helft van de negentiende eeuw (en velen ook nog in het begin van de twintigste eeuw) prostitutie als een noodzakelijk en onuitroeibaar kwaad. Ze wierpen zich op als heftige pleitbezorgers voor de gere- glementeerde prostitutie33. Het systeem van het reglementarisme hield in dat de prostitutie niet werd bestreden, maar wel werd onderworpen aan een systematische controle van de zedenpolitie. Prostituees moesten zich laten registreren en geregeld een medisch onderzoek op geslachtsziekten ondergaan. Werd tijdens zo'n onderzoek een geslachtsziekte vastgesteld, dan werd de betreffende prostituee geïsoleerd. Het negentiende-eeuwse reglementarisme was een uitvinding van de Parijse hygiënist Parent-Duchâtelet. Hij werkte in 1836 een prostitutiereglement voor de Franse hoofd- stad uit. Dit reglement werd in de loop van de negentiende eeuw door verschillende Europese steden overgenomen34. Net als in vele andere landen oefenden ook in België de legerartsen sterke druk uit om een strikte vorm van prostitutiereglementering ingevoerd te krijgen. In de jaren vóór 1850 liet vooral chirurg Louis-Joseph Seutin zich op dit vlak niet onbetuigd. Seutin was in 1831 aangesteld tot opperarts van het Belgisch leger en maakte — on- danks de afschaffing van dit ambt in 1840 — tot 1854 deel uit van het kader van de De grote school van de natie 403 strijdmacht. Hij zorgde er onder meer voor dat de Société des sciences médicales et naturelles de Bruxelles in 1834 en het al vermelde Brussels medisch congres in 1835 een prijsvraag over het syfilisprobleem uitschreven, waardoor de problematiek van de prostitutiereglementering in de medische wereld tot een geliefd gespreksonderwerp werd. In 1842 bracht Seutin de prostitutiekwestie zelfs tot op de agenda van de Ko- ninklijke Academie voor geneeskunde, de meest prestigieuze medische instantie van het land. 35 Zie Des mesures propres à restreindre la maladie syphilitique par une commission composée de MM. Gaux. Lebreau, Seutin, Tallois et Vleminckx, Uittreksel uit Bulletin de l'Académie royale de médecine de Belgique (BARMB) (Brussel, 1843). Over Seutin: Evrard, Mathieu, ed., Asklepios onder de wapens, 114- 117; V. Jacques, 'Seutin (baron Louis-Joseph)', Biographie nationale, XXII (1914-1921) 324-339; Rebmann, Syfilis 1830-1860, 108-165. 37 Zie bijvoorbeeld 'Rapports semestriels des hôpitaux et infirmeries', Archives, I (januari 1848) 58; 'Rapports semestriels des hôpitaux et infirmeries', Archives, I (april 1848) 227-228. 35 Zie Des mesures propres à restreindre la maladie syphilitique par une commission composée de MM. Gaux. Lebreau, Seutin, Tallois et Vleminckx, Uittreksel uit Bulletin de l'Académie royale de médecine de Belgique (BARMB) (Brussel, 1843). Over Seutin: Evrard, Mathieu, ed., Asklepios onder de wapens, 114- 117; V. Jacques, 'Seutin (baron Louis-Joseph)', Biographie nationale, XXII (1914-1921) 324-339; Rebmann, Syfilis 1830-1860, 108-165. 36 Huberty, Keunings, "La prostitution à Bruxelles', 5. 37 Zie bijvoorbeeld 'Rapports semestriels des hôpitaux et infirmeries', Archives, I (januari 1848) 58; 'Rapports semestriels des hôpitaux et infirmeries', Archives, I (april 1848) 227-228. 36 Huberty, Keunings, "La prostitution à Bruxelles', 5. 36 Huberty, Keunings, "La prostitution à Bruxelles', 5. DE PROSTITUEE Seutin overtuigde de Academie ervan om bij het ministerie van binnen- landse zaken aan te dringen op legislatieve maatregelen tegen de prostitutie35. Maar de minister van binnenlandse zaken ondernam voorlopig helemaal niets tegen de prostitutie en de venerische ziekten. De bevoegdheid om op te treden tegen de prostitutie was door de gemeentewet van 1836 uitdrukkelijk aan de gemeentelijke overheden toegekend. Brussel voerde in 1844 voor het eerst een officieel prostitutie- reglement in. Officieren van het Brusselse garnizoen drongen al van in het begin van de negentiende eeuw bij het gemeentebestuur aan op reglementering, omdat het aantal venerische ziekten in het garnizoen volgens hen snel toenam36. Het strikte reglement van Brussel, meestal als hyperreglementarisme getypeerd, kende in Europa een grote uitstraling en werd de basis van vele reglementen die in andere Belgische steden — vaak ook onder druk van het plaatselijke garnizoen — werden ingesteld. De medische kringen van het leger speelden een belangrijke rol bij de invoering van het reglementarisme in België en ze bleven ook de hele tweede helft van de negentiende eeuw de meest heftige verdedigers van het systeem. In de Archives verschenen met regelmaat rapporten van legerartsen over de gezondheidstoestand van de soldaten in verschillende militaire hospitalen van het land. In deze rapporten werd de toename van geslachtsziekten in een bepaald hospitaal bijna altijd toegeschreven aan het slecht toepassen van de prostitutiereglementen of aan het ontbreken van elke reglementering in de stad waar het hospitaal zich bevond37. Legerartsen drongen steevast aan op striktere maatregelen tegen de prostitutie en een nauwkeuriger uitvoering ervan. Het liefst van al zagen zij in het hele land een uniforme en strenge reglementering ingevoerd, zodat geen enkele gemeente het probleem nog zou kunnen veronachtzamen. Ook meenden ze dat ze zelf nauwer bij de medische onderzoeken van de prostituees moesten worden betrokken, omdat het syfilisprobleem volgens hen toch vooral in het leger acuut was. In 1886-1887 debatteerde de Koninklijke Academie voor geneeskunde maandenlang over de zin en onzin van het reglementarisme. In Engeland was op het einde van de jaren zestig en bij het begin van de jaren zeventig, uit protest tegen de Contagious diseases acts die in een aantal Engelse havensteden en garnizoenen een vorm van Liesbet Nys 404 prostitutiereglementering hadden ingevoerd, onder leiding van Josephine Butler een abolitionistische beweging tot ontwikkeling gekomen. 41 Zie bijvoorbeeld de teleurstelling van legerarts Melis: 'Conférence internationale pour la prophylaxie de la syphilis et des maladies vénénennes', Archives, Lil (januari 1900) 71. Voor de commissie en haar wetsvoorstel: Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée...; E. Béco, 'État de la législation belge concernant la prostitution", in: Dubois-Havénith, Enquêtes sur l'état de la prostitution et la fréquence de la syphilis et des maladies vénériennes dans les différents pays (Brussel, 1899) 866-871; M. Vincineau, La débauche en droit et le droit à la débauche (Brussel, 1985)51-52,273-289. 38 In geen enkel land was het reglementarisme zo nauw verbonden met het leger als in Groot-Brittannië. De Contagious diseases acts waren op verzoek van de militaire overheden ingevoerd en golden alleen voor soldaten en zeelui. De abolitionisten konden verhinderen dat de acts werden uitgebreid naar de hele bevolking. Zie onder andere M. Trustram, Women of the regiment. Marriage and the Victorian army (Cambridge, 1984) 116-137. 39 Zie A. Moeller, Réglementation de la prostitution. Discours prononcé à l'Académie, le 27 novembre 1886, Uittreksel uit BARMB (Brussel, 1886); Idem, Réglementation de la prostitution. Deuxième discours prononcé à l'Académie, Uittreksel uit BARMB (Brussel, 1887). DE PROSTITUEE Die beweging internatio- naliseerde zich snel en bestond uit een amalgaam van mensen die om uiteenlopende redenen tegen het reglementarisme waren gekant: van feministen die het als een vrouw- vijandelijk systeem beschouwden tot moralisten die het als een officiële erkenning van de ontucht bestempelden38. In België was vooral de arts Alphonse Moeller een vurig verdediger van het abolitionistisch gedachtegoed. Tijdens het debat in de Academie ging hij onbedaarlijk tekeer tegen het reglementarisme39. In de ogen van legerarts Titeca, die voor de Archives verslag uitbracht over het debat, waren de abo- litionisten utopisten, die niet wilden beseffen dat de prostitutie onuitroeibaar was en daarom maar beter rigoureus kon worden gecontroleerd40. De uiteindelijke conclusie van de Academie dat het reglementarisme moest behouden blijven, werd door de legerartsen met grote tevredenheid onthaald. Een jaar na het debat in de Academie, in 1888, werd onder impuls van de katholieke minister van justitie Jules Le Jeune in het parlement een speciale commissie aangesteld over de prostitutiekwestie. Die commissie, waarvan onder meer Petithan (intussen op rust) deel uitmaakte, diende een wetsvoorstel over de prostitutie uit te werken. Het voorstel dat uiteindelijk op 21 maart 1891 door de commissie werd aangenomen, be- viel Le Jeune allerminst. De commissie stelde voor. om het reglementarisme op bepaalde punten bij te schaven maar het zeker niet af te schaffen, terwijl Le Jeune het systeem liefst zo snel mogelijk in zijn geheel zag verdwijnen. Het wetsvoorstel van de commissie werd nooit bediscussieerd in het parlement en bleef — tot spijt van heel wat legerartsen — zonder enig gevolg41. In het kader van de commissie over de prostitutiekwestie werd een specifiek onder- zoek gedaan naar het venerisch probleem in het leger. Er werd een vragenlijst opgesteld die naar verschillende Belgische garnizoenen werd verstuurd. In de Archives versche- nen fragmenten uit de binnengekomen antwoorden. Ook deze fragmenten suggereren dat de legerartsen nog steeds unaniem achter het reglementarisme stonden. Alle artsen benadrukten het belang van regelmatige en secuur uitgevoerde medische onderzoeken van de prostituees. In bijna alle fragmenten werd gesmeekt om een hardere bestrijding 38 In geen enkel land was het reglementarisme zo nauw verbonden met het leger als in Groot-Brittannië. De Contagious diseases acts waren op verzoek van de militaire overheden ingevoerd en golden alleen voor soldaten en zeelui. De abolitionisten konden verhinderen dat de acts werden uitgebreid naar de hele bevolking. Zie onder andere M. Trustram, Women of the regiment. p 40 Titeca, 'Hygiène sociale. Réglementation de la prostitution', Archives, XL (juni 1887) 402-411. De Contagious diseases acts waren op verzoek van de militaire overheden ingevoerd en golden alleen voor soldaten en zeelui. De abolitionisten konden verhinderen dat de acts werden uitgebreid naar de hele bevolking. Zie onder andere M. Trustram, Women of the regiment. Marriage and the Victorian army (Cambridge, 1984) 116-137. 39 Zie A. Moeller, Réglementation de la prostitution. Discours prononcé à l'Académie, le 27 novembre 1886, Uittreksel uit BARMB (Brussel, 1886); Idem, Réglementation de la prostitution. Deuxième discours prononcé à l'Académie, Uittreksel uit BARMB (Brussel, 1887). 40 Titeca, 'Hygiène sociale. Réglementation de la prostitution', Archives, XL (juni 1887) 402-411. 41 Zie bijvoorbeeld de teleurstelling van legerarts Melis: 'Conférence internationale pour la prophylaxie de la syphilis et des maladies vénénennes', Archives, Lil (januari 1900) 71. Voor de commissie en haar wetsvoorstel: Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée...; E. Béco, 'État de la législation belge concernant la prostitution", in: Dubois-Havénith, Enquêtes sur l'état de la prostitution et la fréquence de la syphilis et des maladies vénériennes dans les différents pays (Brussel, 1899) 866-871; M. Vincineau, La débauche en droit et le droit à la débauche (Brussel, 1985)51-52,273-289. 42 Zie Logie, 'De la prostitution', Archives, XLI (september 1888) 199-215; Spruyt, e. a., 'De la prostitution', Archives, XLI (oktober 1888) 265-286 ; 'De la prostitution. (Extraits de rapports)', 348-358. 43 Mélis, 'Conférence internationale pour la prophylaxie de la syphilis et des maladies vénériennes', Archives, LI (december 1899) 404-421, Lil (januari 1900) 56-72. 44 Stainforth, 'Ile Conférence internationale pour la prophylaxie de la syphilis et des maladies vénériennes, sous le patronage du gouvernement belge. (Bruxelles, Ire au 6 sept. 1902)', Archives, LIV (december 1902) 403-421,LV (januari 1903) 41-57, LV (februari 1903) 109-121, LV (maart 1903) 192-206, LV (april 1903) 264-274 en LV (mei 1903) 340-347. 45 Rulot, Sacré, La vie du soldat Belge, 130. DE PROSTITUEE a., tit ti ' A hi XLI ( kt b 1888) 265 286 'D l tit ti (E t it d t )' 34 405 De grote school van de natie van de clandestiene prostituees, de publieke vrouwen die zich aan de reglementering trachtten te onttrekken42. Deze prostituees waren al geruime tijd een doorn in het oog van de legerartsen. Ze werden op het vlak van de geslachtsziekten als bijzonder onveilig beschouwd. Legerartsen zagen het als zorgwekkend dat soldaten om financiële redenen juist vaker bij deze clandestiene prostituees hun toevlucht zochten dan bij de duurdere geregistreerde publieke vrouwen. In 1899 en 1902 stonden reglementaristen en abolitionisten tegenover elkaar op de eerste twee internationale conferenties over venerische ziekten, die in Brussel door de Belgische veneroloog Dubois-Havénith werden georganiseerd. Legerarts Melis bracht voor de Archives verslag uit over de eerste conferentie en schaarde zich daarbij — hoe kon het anders? — uitdrukkelijk aan de kant van de reglementaristen. In de uiteindelijke resolutie van het congres werd trouwens ook voor een reglementaristisch standpunt geopteerd43. Bij het begin van de twintigste eeuw begon de twijfel omtrent de efficiëntie van het reglementarisme echter te groeien. De tweede conferentie in Brussel erkende unaniem het falen van het politionele reglementarisme in de strijd tegen de venerische ziekten. Legerarts Stainforth, verslaggever voor de Archives van deze conferentie, kon zich hiermee blijkbaar wel verzoenen. Uit zijn verslag spreekt alvast geen echte ontevredenheid over het resultaat van de conferentie44. Blijkbaar gingen ook legerartsen zich in het begin van de twintigste eeuw langzaamaan kritischer opstellen tegenover het van kracht zijnde reglementarisme. Aan de vooravond van de Eerste Wereldoorlog kantten Rulot en Sacré zich in hun handboek voor jonge rekruten zelfs erg expliciet tegen de prostitutiereglementering, die volgens hen de soldaten een gevoel van schijnveiligheid gaf. Naar hun mening was prostitutie — geregle- menteerd of niet — altijd gevaarlijk, was het une sorte de loterie où l'on gagne presque à tout coup. Et que gagne-t-on? Triste gain que celui d'une maladie souvent incurable ou la perspective de la mort dans le désespoir ou le gâtisme45. De Eerste Wereldoorlog: reglementering of beheersing? 45 Rulot, Sacré, La vie du soldat Belge, 130. DE PROSTITUEE Marriage and the Victorian army (Cambridge, 1984) 116-137. De grote school van de natie van de clandestiene prostituees, de publieke vrouwen die zich aan de reglement trachtten te onttrekken42. Deze prostituees waren al geruime tijd een doorn in he van de legerartsen. Ze werden op het vlak van de geslachtsziekten als bijzonder onv beschouwd. Legerartsen zagen het als zorgwekkend dat soldaten om financiële red juist vaker bij deze clandestiene prostituees hun toevlucht zochten dan bij de duu geregistreerde publieke vrouwen. In 1899 en 1902 stonden reglementaristen en abolitionisten tegenover elkaar eerste twee internationale conferenties over venerische ziekten, die in Brussel de Belgische veneroloog Dubois-Havénith werden georganiseerd. Legerarts M bracht voor de Archives verslag uit over de eerste conferentie en schaarde zich da — hoe kon het anders? — uitdrukkelijk aan de kant van de reglementaristen. uiteindelijke resolutie van het congres werd trouwens ook voor een reglementaris standpunt geopteerd43. Bij het begin van de twintigste eeuw begon de twijfel om de efficiëntie van het reglementarisme echter te groeien. De tweede conferen Brussel erkende unaniem het falen van het politionele reglementarisme in de tegen de venerische ziekten. Legerarts Stainforth, verslaggever voor de Archive deze conferentie, kon zich hiermee blijkbaar wel verzoenen. Uit zijn verslag sp alvast geen echte ontevredenheid over het resultaat van de conferentie44. Blijk gingen ook legerartsen zich in het begin van de twintigste eeuw langzaamaan kriti opstellen tegenover het van kracht zijnde reglementarisme. Aan de vooravond v Eerste Wereldoorlog kantten Rulot en Sacré zich in hun handboek voor jonge rek zelfs erg expliciet tegen de prostitutiereglementering, die volgens hen de sol een gevoel van schijnveiligheid gaf. Naar hun mening was prostitutie — ger menteerd of niet — altijd gevaarlijk, was het une sorte de loterie où l'on gagne presque à tout coup. Et que gagne-t-on? Triste gai celui d'une maladie souvent incurable ou la perspective de la mort dans le désespoir gâtisme45. De Eerste Wereldoorlog: reglementering of beheersing? De Eerste Wereldoorlog bracht in de ogen van de legerartsen zedenverwilderin toegenomen promiscuïteit. In zulke tijden achtten velen onder hen het bete gereglementeerde bordelen in de buurt van het leger toe te laten om het risico o nerische ziekten bij soldaten te beperken. Al hadden heel wat legerartsen in het b van de twintigste eeuw hun grenzeloos vertrouwen in de efficiëntie van het r 42 Zie Logie, 'De la prostitution', Archives, XLI (september 1888) 199-215; Spruyt, e. 48 F. Daels, Voor onze jongens (Leiden, 1918) 12. Over Frans Daels: B. de Wever, 'Daels, Frans', Nieuwe encyclopedie van de Vlaamse beweging, I (Tielt, 1998) 836-839; L. Vandeweyer, 'Frans Daels. Arts bij het ziekbed van zijn volk', in: J. de Maeyer, e. a., ed., Er is leven voor de dood, 265-267. 49 Zie F. Bertrand,La presse francophone de tranchée au front belge, 1914-1918 (Brussel, 1971)78-79; G. Bulthé, De Vlaamse loopgravenpers tijdens de Eerste Wereldoorlog (Brussel, 1971) 57-63, 108. 46 Zie bijvoorbeeld B. Dujardin, 'La lutte antivénérienne à l'Armée', in: Congrès international de médecine et de pharmacie militaires au palais mondial du 15 au 20 juillet 1921 (Brussel, 1921) 11-12; L. Wilmaers, 'Lutte antivénérienne à l'Armée', in: ibidem, 95. De Duitsers voerden in de door hen bezette delen van België een strikte vorm van prostitutiereglementering in. Zie Craps, 'Règlements, législation et contributions médico-sociales belges dans le domaine de la lutte antivénérienne depuis la Tin du XVIIIe siècle jusqu'à nos jours', Archives belges de médecine sociale, hygiène, médecine du travail et médecine légale, VII (1959) 485; C. van Hooreweghe, 'Het prostitutioneel kader te Gent in de periode 1910-1932', Handelingen der maatschappij voor geschiedenis en oudheidkunde te Gent, XLIV (1990) 151-167. Ook in de rest van België werd volgens vele legerartsen tijdens de oorlog de controle op de prostituees opgedreven. 47 Zie J.-R.Leconte Aumôniers militaires belges de la guerre 1914-1918 (Brussel, 1969)64. In Frankrijk kwamen er wel gereglementeerde bordelen in de zone van het front. Dit was niet naar de zin van de Verenigde Staten. Voor de Amerikaanse troepen vormden die bordelen verboden terrein. Ook de Britten waren geen voorstanders van het Franse systeem van prostitutiereglementering. Zie E. H. Beardsley, 'Allied against sin. American and British responses to venereal disease in World War I', Medical history, XX (1976) 189-202; Brandt, No magic bullet, 100-106; M. Harrison, 'The British army and the problem of venereal disease in France and Egypt during the First World War', Medical history, XXXIX (1995) 142- 146. De Eerste Wereldoorlog: reglementering of beheersing? Wilmaers, 'Lutte antivénérienne à l'Armée', in: ibidem, 95. De Duitsers voerden in de door hen bezette delen van België een strikte vorm van prostitutiereglementering in. Zie Craps, 'Règlements, législation et contributions médico-sociales belges dans le domaine de la lutte antivénérienne depuis la Tin du XVIIIe siècle jusqu'à nos jours', Archives belges de médecine sociale, hygiène, médecine du travail et médecine légale, VII (1959) 485; C. van Hooreweghe, 'Het prostitutioneel kader te Gent in de periode 1910-1932', Handelingen der maatschappij voor geschiedenis en oudheidkunde te Gent, XLIV (1990) 151-167. Ook in de rest van België werd volgens vele legerartsen tijdens de oorlog de controle op de prostituees opgedreven. 47 Zie J.-R.Leconte Aumôniers militaires belges de la guerre 1914-1918 (Brussel, 1969)64. In Frankrijk kwamen er wel gereglementeerde bordelen in de zone van het front. Dit was niet naar de zin van de i d S d A ik d di b d l b d i O k d i 46 Zie bijvoorbeeld B. Dujardin, 'La lutte antivénérienne à l'Armée', in: Congrès international de médecine et de pharmacie militaires au palais mondial du 15 au 20 juillet 1921 (Brussel, 1921) 11-12; L. Wilmaers, 'Lutte antivénérienne à l'Armée', in: ibidem, 95. De Duitsers voerden in de door hen bezette delen van België een strikte vorm van prostitutiereglementering in. Zie Craps, 'Règlements, législation et contributions médico-sociales belges dans le domaine de la lutte antivénérienne depuis la Tin du XVIIIe siècle jusqu'à nos jours', Archives belges de médecine sociale, hygiène, médecine du travail et médecine légale, VII (1959) 485; C. van Hooreweghe, 'Het prostitutioneel kader te Gent in de periode 1910-1932', Handelingen der maatschappij voor geschiedenis en oudheidkunde te Gent, XLIV (1990) 151-167. Ook in de rest van België werd volgens vele legerartsen tijdens de oorlog de controle op de prostituees opgedreven. 47 Zie J.-R.Leconte Aumôniers militaires belges de la guerre 1914-1918 (Brussel, 1969)64. In Frankrijk kwamen er wel gereglementeerde bordelen in de zone van het front. Dit was niet naar de zin van de Verenigde Staten. Voor de Amerikaanse troepen vormden die bordelen verboden terrein. Ook de Britten waren geen voorstanders van het Franse systeem van prostitutiereglementering. Zie E. H. Beardsley, 'Allied against sin. American and British responses to venereal disease in World War I', Medical history, XX (1976) 189-202; Brandt, No magic bullet, 100-106; M. De Eerste Wereldoorlog: reglementering of beheersing? De Eerste Wereldoorlog bracht in de ogen van de legerartsen zedenverwildering en toegenomen promiscuïteit. In zulke tijden achtten velen onder hen het beter om gereglementeerde bordelen in de buurt van het leger toe te laten om het risico op ve- nerische ziekten bij soldaten te beperken. Al hadden heel wat legerartsen in het begin van de twintigste eeuw hun grenzeloos vertrouwen in de efficiëntie van het regle- Liesbet Nys 406 mentarisme verloren, in oorlogstijd bleven zij erg gewonnen voor het systeem46. Er kwamen in België echter geen getolereerde bordelen in de zone van het front. Onder meer de aalmoezeniers van het leger hebben zich daar heftig tegen verzet. Zij riepen de militairen op tot beheersing47. Dat deed ook (de latere flamingantische leider) Frans Daels, die tijdens de oorlog vrijwillig militair arts was aan het front. Hij schreef een aantal pamfletten waarin hij zijn afkeur van elke vorm van prostitutie duidelijk liet blijken. Prostitutie leidde volgens hem tot lichaamsontering en zielsverbastering. In erg pathetische woorden richtte hij zich tot de jongemannen aan het front: Aan u, jongens van den Yser, die hier in 't gelid staat, overkome dit niet! Gij zijt de ridders van ons land. Uw eerste plicht, na 's lands verdediging, is ridderlijkheid tegenover onze vrouwen. Onteert u niet, duwt geen zwakke vrouw in het verderf of houdt ze niet in ontucht. Ontwikkelt de krachten van uw geslachtsdrift tot kloeke mannelijkheid, tot mannenfierheid en ridderlijkheid. Gij zult u het vurigste, volste liefdegenot voor het leven verzekeren. Jongens, de harten hoog! Mannenadel uit fierheid van het jong geslacht48. Ook in de gretig gelezen frontblaadjes verschenen geregeld soortgelijke aanma- ningen49. Ook in de gretig gelezen frontblaadjes verschenen geregeld soortgelijke aanma- ningen49. Maar al deze oproepen tot seksuele beheersing ten spijt leidde de Eerste Wereldoorlog volgens de meeste legerartsen tot een enorme toename van de prostitutie. Heel wat vrouwen die voorheen een 'eerbaar' bestaan hadden geleid, werden door de oorlogs- omstandigheden tot prostitutie gedwongen om de kost te verdienen. Bij sommige le- gerartsen leek er hierdoor iets meer begrip voor de prostituee te groeien. Het besef drong langzaam door dat vrouwen niet noodzakelijk uit geilheid of genotzucht maar vaak uit armoede en ellende in de prostitutie terechtkwamen. Dit lichtjes toegenomen 46 Zie bijvoorbeeld B. Dujardin, 'La lutte antivénérienne à l'Armée', in: Congrès international de médecine et de pharmacie militaires au palais mondial du 15 au 20 juillet 1921 (Brussel, 1921) 11-12; L. 53 Een vaak geuite kritiek was bijvoorbeeld dat er in België zowel aan het front als achterin een te gering aantal venerologische centra voor soldaten werd opgericht en dat er te weinig uniformiteit bestond in de aanpak van die centra. Over het geslachtsziektenbeleid van het Belgisch leger tijdens de Eerste Wereldoorlog: F. Derbraudrenghien, 'De la conspiration du silence à la propagande. Trente-six ans de lutte antivénérienne à Liège (1912-1948)' (Onuitgegeven licentiaatsverhandeling Université de Liège; Luik, 1998) 36-48. Zie ook J. Gaudy, 'La Guerre et les maladies vénériennes', Le scalpel, LXXII (21 september 1919)425. De Eerste Wereldoorlog: reglementering of beheersing? Harrison, 'The British army and the problem of venereal disease in France and Egypt during the First World War', Medical history, XXXIX (1995) 142- 146. 407 De grote school van de natie begrip voor de beroepsprostituee ging echter gepaard met een totaal nieuw fenomeen van de oorlogsjaren: het afgrijzen van legerartsen voor promiscue meisjes die hun lichaam gratis ter beschikking stelden van de soldaten. Deze meisjes vormden volgens de legerartsen een erg belangrijke besmettingshaard voor de venerische ziekten van militairen. Zij waren — net als de clandestiene prostituees — totaal onwetend op het vlak van geslachtsziekten en stonden niet onder een regelmatige medische controle50. Het reglementarisme in het defensief 50 Ook na de oorlog bleef de angst voor deze frivole meisjes een belangrijk element in het bestrij- dingsdiscours over de geslachtsziekten. Zie bijvoorbeeld D. O. L., Neem uw plaats in! (Brussel, 1948) 42- 43. Vgl. Mooij, Geslachtsziekten en besmettingsangst, 122-169. 51 Zie onder andere J. Gaudy, 'Les maladies vénériennes à l'armée', Archives, LXX (juni 1917) 509; B. Dujardin, 'La lutte antivénérienne à l'armée', 4; L. Mélis, Contribution à l'histoire du service de santé à l'armée au cours de la guerre 1914-1918 (Brussel, 1932) 224-226. Zie ook R. Christens, K. de Clercq, Frontleven 14/18. Het dagelijks leven van de Belgische soldaat aan de IJzer (Tielt, 1987) 119. 52 L. Wilmaers, 'Lutte antivénérienne à l'armée', 80. Ook in vredestijd gingen legerartsen ervan uit dat soldaten hun geslachtsziekten vooral tijdens hun verlofperiodes opliepen. Zie bijvoorbeeld Fromont, 'Des moyens de prévenir la propagation de la syphilis dans l'armée, et spécialement dans la garnison d'Anvers. Extrait d'un rapport adressé à M. l'Inspecteur-général du service de santé de l'armée', Archives, XXIII (februari 1872)92. 53 Een vaak geuite kritiek was bijvoorbeeld dat er in België zowel aan het front als achterin een te gering aantal venerologische centra voor soldaten werd opgericht en dat er te weinig uniformiteit bestond 50 Ook na de oorlog bleef de angst voor deze frivole meisjes een belangrijk element in het bestrij- dingsdiscours over de geslachtsziekten. Zie bijvoorbeeld D. O. L., Neem uw plaats in! (Brussel, 1948) 42- 43. Vgl. Mooij, Geslachtsziekten en besmettingsangst, 122-169. 51 Zie onder andere J. Gaudy, 'Les maladies vénériennes à l'armée', Archives, LXX (juni 1917) 509; B. Dujardin, 'La lutte antivénérienne à l'armée', 4; L. Mélis, Contribution à l'histoire du service de santé à l'armée au cours de la guerre 1914-1918 (Brussel, 1932) 224-226. Zie ook R. Christens, K. de Clercq, Frontleven 14/18. Het dagelijks leven van de Belgische soldaat aan de IJzer (Tielt, 1987) 119. 52 L. Wilmaers, 'Lutte antivénérienne à l'armée', 80. Ook in vredestijd gingen legerartsen ervan uit dat soldaten hun geslachtsziekten vooral tijdens hun verlofperiodes opliepen. Zie bijvoorbeeld Fromont, 'Des moyens de prévenir la propagation de la syphilis dans l'armée, et spécialement dans la garnison d'Anvers. Extrait d'un rapport adressé à M. l'Inspecteur-général du service de santé de l'armée', Archives, XXIII (februari 1872)92. Het reglementarisme in het defensief Na de Eerste Wereldoorlog heerste er in legerkringen regelrechte paniek over het sterk toegenomen aantal venerische ziekten bij soldaten. De legerartsen waren ervan overtuigd dat de meeste soldaten hun geslachtsziekte niet aan het front, maar wel achterin, tijdens de 'verblinding van dronkenmakende verlofdagen' hadden opgelopen. Vele Belgische soldaten brachten tijdens de oorlog hun verlof door in Parijs, dat bekend stond om zijn wufte zeden en door de legerartsen al snel als de belangrijkste infectieplaats voor geslachtsziekten bij soldaten werd beschouwd51. 'Paris, centre des congés, a été aussi le grand foyer de la contagion', concludeerde legerarts Wilmaers in 192152. Toen vond in Brussel het eerste internationaal congres over militaire genees- kunde en farmacie plaats. Legerartsen bezonnen er zich over de politiek die tijdens de voorbije oorlogsperiode door de verschillende landen met betrekking tot de geslachtsziekten was gevoerd. Het beleid van het Belgisch leger werd op verschil- lende vlakken ontoereikend bevonden53. Ook probeerden legerartsen op dit congres de krijtlijnen voor een nieuwe aanpak van het venerisch probleem bij de strijdmacht uit te tekenen. Het is opvallend hoe weinig aandacht de Belgische legerartsen tijdens dit congres nog hadden voor de prostitutiereglementering. Voortaan zochten zij de oplossing voor het geslachtsziektenprobleem veel meer in individuele profylactische middelen en in seksuele voorlichting van de rekruten. g Ondanks het feit dat de prostitutiereglementering in het Interbellum in België duidelijk 408 Liesbet Nys aan sympathie en aanhang had ingeboet en dat het toezicht op de uitvoering ervan in vele steden danig was verzwakt, bleef het systeem officieel verder in voege. In de jaren dertig werd in het parlement verschillende keren een wetsvoorstel voor de afschaffing van het reglementarisme ingediend, maar steeds zonder resultaat. De Twee- de Wereldoorlog bracht nog een tijdelijke heropleving van het systeem. De Duitsers voerden, net als tijdens de Eerste Wereldoorlog, in de door hen bezette gebieden een strikte vorm van prostitutiereglementering in54. Na de Tweede Wereldoorlog ondernam de socialiste Isabelle Blume nog maar eens een poging om officieel een punt te zetten achter meer dan een eeuw reglementarisme. Haar wetsvoorstel werd wel aangenomen; in de zomer van 1948 kwam bij wet een einde aan het systeem. De ontdekking van de penicilline leidde in de daaropvolgende jaren tot een drastische afname van het aantal venerische patiënten. 54 Voor het geslachtsziektenbeleid van de Duitsers in bezet België: Craps, 'Règlements, législation et contributions médico-sociales belges', 490-493 en F. Derbraudrenghien, 'De la conspiration du silence', 121-125. Het reglementarisme in het defensief Legerartsen en het reglementarisme: een balans Legerartsen en het reglementarisme: een balans Het is eigenaardig dat legerartsen in de negentiende eeuw zo rotsvast geloofden in het systeem van het reglementarisme. Uit de medische discussies die toen over de geslachtsziekten werden gevoerd, blijkt immers duidelijk dat de diagnose van deze ziekten nog helemaal niet feilloos was. Daardoor is het zeer de vraag of de medische onderzoeken van de prostituees, hoe nauwkeurig ze ook werden uitgevoerd, wel effi- ciënt konden zijn. Maar misschien waren de steunbetuigingen van de militaire genees- kundigen aan het reglementarisme niet uitsluitend door sanitaire motieven geïnspireerd, zoals sommigen onder hen wel wilden laten uitschijnen en zoals ook vaak in de histo- riografie wordt gesuggereerd. Wellicht gingen achter hun pleidooien voor de gere- glementeerde prostitutie, net als bij vele civiele artsen, ook morele drijfveren schuil. Het onophoudelijk geklaag van de legerartsen over het obsceen gedrag dat prostituees in het openbaar tentoonspreidden, wekt bijvoorbeeld de indruk dat zij de prostitutie niet alleen wilden reglementeren omdat ze een belangrijke infectiehaard voor geslachts- ziekten was, maar ook omdat ze in hun ogen een bedreiging vormde voor de publieke moraal. Legerartsen zagen in het reglementarisme, naast een efficiënt systeem om het venerisch probleem bij soldaten te bestrijden, ook een manier om de maatschappij een voortdurende confrontatie met het onbetamelijk gedrag van de publieke vrouwen te besparen. Het reglementarisme moest de prostitutie immers uit het gewone straat- beeld doen verdwijnen en binnen de muren van een aantal weinig zichtbare getole- reerde bordelen terugdringen. Een volledige afschaffing van de prostitutie achtten de meeste legerartsen in de tweede helft van de negentiende en het begin van de twintigste eeuw niet mogelijk en zelfs vanuit moreel standpunt niet wenselijk, want mannen die hun seksuele driften niet op een natuurlijke manier vrij spel konden geven, zochten volgens hen zonder twijfel hun toevlucht in seksuele aberraties. Legerartsen waren grote aanhangers van wat tegenwoordig de leer van de dubbele moraal wordt genoemd, de opvatting dat 409 De grote school van de natie seksuele onthouding wel voor vrouwen, maar niet voor mannen mogelijk is. Het re- glementarisme, dat volledig op de leer van de dubbele moraal was gestoeld, wordt daarom dikwijls geïnterpreteerd als een vertaling van de negentiende-eeuwse mysogi- nie. Ook het feit dat alleen prostituees en niet hun mannelijke klanten aan een verne- derend medisch onderzoek werden onderworpen, wordt hiervoor vaak als argument ingeroepen. 55 Niet door Petithan. Die was er wel voorstander van dat alle mannelijke klanten van prostituees aan een medisch onderzoek op geslachtsziekten zouden worden onderworpen. Zie Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée, procès-verbaux des séances plénières, 255. Het reglementarisme in het defensief Maar het leger was (naast de marine) de enige plaats waar ook mannen een medisch onderzoek op geslachtsziekten moesten ondergaan. Soldaten werden geregeld door de militaire artsen op venerische ziekten onderzocht. Voorstellen om alle mannelijke bordeelbezoekers aan zo'n onderzoek te onderwerpen werden echter ook door de meeste legerartsen als volkomen onrealiseerbaar weggelachen55. 56 Geciteerd door A. Jansen, 'De l'usage et de l'abus des boissons alcooliques', 560. 57 A. Jansen, Précis d'hygiène (S. 1„ 1883) 285. Voor de idee van het leger als morele en intellectuele leerschool van de natie, zie onder andere R. Höhn, Die Armee als Erziehungsschule der Nation. Das Ende einer Idee (Bad Hardzburg, 1963); Wesseling, Soldaat en krijger, F. Snapper, 'Het negentiende-eeuwse Nederlandse leger. Een school der natie?', Mededelingen van de sectie militaire geschiedenis landmachtstaf, Vil ( 1984) 37-56; L. de Vos, 'De Belgische krijgsmacht als natievormende factor, 1830-1885', BTNG, XV (1984) 421-460; W. Klinkert, Het vaderland verdedigd, 356-359. 58 V. de Vaucleroy, De l'alcoolisme dans l'armée, 3. 59 Over Vleminckx: F. de Block, 'Jean-François Vleminckx, patriote de 1830', Archives, LXXV (augustus 1922) 712-743; G. Leboucq, 'Vleminckx (Jean-François)', Biographie nationale, XXIX (1957) 847-849; Evrard, Mathieu, ed., Asklepios onder de wapens, passim. EEN RADICAAL OFFENSIEF Legerartsen waren zelf medeplichtig aan de bedroevende morele reputatie die het leger in de samenleving had. Zij droegen bij tot het beeld van het leger als een oord van obsceen en bandeloos gedrag. Niet alleen benadrukten zij dat de soldaten door hun leeftijd en door de uitzonderlijke omstandigheden waarin zij leefden een risico- groep vormden voor drankmisbruik en venerische ziekten, zij wezen ook voortdurend op de extreme dreiging die de moraal in het leger vanuit twee hoeken ondervond: intern legden de plaatsvervangers een zware hypotheek op het handhaven van de goede zeden, van buiten af ondermijnden de prostituees het moreel niveau van de strijdkrachten. Maar al hielpen de legerartsen bij het creëren en verspreiden van het sombere beeld van de moraal in het leger, tegelijkertijd zaten zij ook bijzonder verveeld met deze slechte reputatie die hun beroepseer niet bepaald ten goede kwam. In een radicaal offensief tegen drankmisbruik en geslachtsziekten bij soldaten, dat zich niet alleen uitte in een aanhoudende steun aan het reglementarisme maar ook in een reeks verregaande maatregelen binnen het leger zelf, zagen zij een manier om het leger moreel te verheffen. Al beoogden de legerartsen met deze maatregelen zeker ook een verbetering van de salubriteit in de kazerne, daarnaast moesten ze het leger tot een moreel voorbeeld voor de burgerlijke samenleving maken. De Belgische legerartsen zagen het leger trouwens als een plaats waar de strijd tegen het alcoholisme en de geslachtsziekten het efficiëntst kon verlopen. De discipline en de strikte controle in de kazerne maakten een repressieve aanpak mogelijk. Dit bood volgens de legerartsen ook gunstige perspectieven voor de burgerlijke samenleving. Het leger kon een bron worden van morele regeneratie in een maatschappij die aan drankmisbruik en venerische ziekten ten onder dreigde te gaan. Meynne bijvoorbeeld verwachtte van een alcoholverbod in het leger positieve gevolgen voor de hele samenleving: 410 Liesbet Nys L'armée poserait ici un exemple de haute moralisation; elle renverrait anuellement dans les rangs de la nation plusieurs milliers de ses enfants qui, pendant leur temps de service, auraient abandonné, d'abord par soumission et plus tard par conviction, cet usage funeste, et qui seraient un enseignement pour leur proches ou leurs voisins56. Legerartsen waren ervan overtuigd dat het kazerneleven niet noodzakelijk tot zeden- bederf moest leiden, maar dat het integendeel ook een moraliserende invloed op de soldaten kon hebben. EEN RADICAAL OFFENSIEF Men moest de weerbare mannen van het land in de kazerne mo- reel hoogstaand gedrag aanleren, na afloop van hun diensttijd zouden die mannen dat gedrag dan tot voorbeeld stellen van hun familieleden en vrienden. Deze visie op de moreel-opvoedende rol van het leger sloot aan bij de ook buiten België wijd verspreide opvatting dat het leger een leerschool van de natie moest zijn, een plaats waar jonge- mannen niet alleen goede manieren werden bijgebracht, maar waar zij verder bijvoorbeeld ook leerden lezen en schrijven, waar hun vaderlandsliefde werd versterkt en waar zij zin voor hygiëne, orde, discipline en gehoorzaamheid konden ontwikkelen. 'L'armée doit être une école d'instruction et de moralisation', schreef Auguste Jansen in 1883 in zijn handboek voor militairen57. Volgens De Vaucleroy, die hygiëne onder- wees aan de militaire school, had de afschaffing van de plaatsvervanging in vele Europese landen het leger al omgevormd tot 'une véritable école d'éducation virile qui fortifie le corps, forme l'intelligence, trempe le caractère, élève le coeur et déve- loppe chez lesjeunes citoyens les sentiments du patriotisme'58. Meldingsplicht Meldingsplicht In 1842 nam Jean-François Vleminckx als inspecteur-generaal van de gezondheids- dienst van het Belgisch leger de eerste verregaande maatregelen tegen geslachtsziekten bij soldaten59. Vleminckx bepaalde dat geen enkele venerische patiënt, hoe licht zijn aandoening ook leek, nog in de kazerne mocht worden behandeld. Elke patiënt die aan een geslachtsziekte leed, moest voortaan naar het hospitaal. Daar zou hij naar de naam en de verblijfplaats worden gevraagd van de publieke vrouw die hem had besmet. Deze gegevens werden doorgespeeld aan de gemeentelijke autoriteiten, in de hoop dat die de nodige maatregelen tegen de betreffende prostituee zouden nemen. Soldaten die weigerden de informatie vrij te geven, moesten worden gestraft, net als diegenen die hun ziekte te lang voor de legerartsen verborgen hadden gehouden. De soldaten die hun ziekte onmiddellijk hadden opgebiecht, moesten daarentegen van elke straf 411 De grote school van de natie en vernedering worden vrijgesteld60. Deze maatregelen, die in het leger de verplichte melding van de venerische ziekten invoerden, getuigen overduidelijk van de tendens tot culpabilisering van de prostituee61. De radicale maatregelen van 1842 werden in de daaropvolgende jaren nader gespecificeerd. Op 18 april 1846 werd bij Koninklijk Besluit bepaald dat de venerische patiënten (en de schurftlijders) in de militaire hospitalen recht hadden op dezelfde behandeling als alle andere patiënten. Zij moesten evenveel voedsel en kleding krijgen en konden, indien zij hun ziekte binnen de vier dagen na het verschijnen van de eerste symptomen hadden gemeld, ook aanspraak op een ziekenhuissoldij maken. Vleminckx wilde met dit K. B. een einde maken aan de discriminerende behandeling die syfïli- tische (en schurftige) soldaten tot dan toe in de militaire hospitalen hadden gekregen en in die zin was het K. B. een belangrijke stap vooruit in de deculpabilisering van de syfilispatiënt (en de schurftlijder). Maar de soldaten die de schaamte voor hun ziekte niet onmiddellijk konden overwinnen, werden door de richtlijn verder in het nauw gedreven. Zij hadden geen enkel recht op een soldij62. Dezelfde tendens ging schuil achter een voorschrift van 25 april 1846 dat een officieel onderscheid invoerde tussen de zogenaamde 'déclarés' — de soldaten die hun ziekte na het verschijnen van de eerste symptomen onmiddellijk hadden gemeld — en de 'non-déclarés' — de soldaten die dat niet hadden gedaan. Op de 'toegangskaan' voor het hospitaal moest worden aangegeven tot welke categorie een soldaat behoorde. 60 'Instruction sur les moyens de prévenir la propagation de la maladie vénénenne', in: Meynne, Recueil des règlements, circulaires, arrêtés, lois et instructions concernant le service de santé de l'armée belge (Brussel, 1844) 318-319. Zie ook J.-R. Marinus, De la prostitution en Belgique (Parijs, 1857) 18. In Frankrijk werden in 1842 soortgelijke maatregelen genomen, maar volgens Quétel werden die van het Belgisch leger veel strikter toegepast. Zie Quétel, Le mal de Naples, 285. 61 In de civiele maatschappij werd heftig gediscussieerd over het al dan niet invoeren van de verplichte melding van geslachtsziekten. Velen achtten zo'n verplichte melding strijdig met het medisch geheim. Toen de Duitsers tijdens de Eerste Wereldoorlog in de door hen bezette delen van België de verplichte melding invoerden, zou dat op heel wat tegenwerking van de Belgische artsen zijn gestuit. Zie Craps, 'Règlements, législation et contributions médico-sociales belges', 485. 62 'Arrêté royal du 18 avril 1846, portant que les militaires atteints de syphilis ou de gale seront traités, dans les hôpitaux, sur le même pied que les autres malades, et qu'ils auront droit à la solde d'hôpital lorsqu'ils auront déclarés spontanément leur maladie des l'apparition des premiers symptômes', JMO (1846)90-91. 63 'Instruction sur l'exécution de l'arrêté du 18 avril 1846 modifiant le régime auquel sont soumis les 63 'Instruction sur l'exécution de l'arrêté du 18 avril 1846, modifiant le régime auquel sont soumis les vénériennes et les galeux traités dans les hôpitaux et les infirmeries', JMO (1846) 91-93. 62 'Arrêté royal du 18 avril 1846, portant que les militaires atteints de syphilis ou de gale seront traités, dans les hôpitaux, sur le même pied que les autres malades, et qu'ils auront droit à la solde d'hôpital lorsqu'ils auront déclarés spontanément leur maladie des l'apparition des premiers symptômes', JMO (1846)90-91. 64 Fromont, 'Des moyens de prévenir la propagation de la syphilis dans l'armée', 91-92. 65 'Seraient donc seuls vénériens, tout court, les non déclarés d'aujourd'hui. Le soidisant stigmate s'appliquerait non pas au mal vénérien mais à la mauvaise action de celui qui enfreindrait les dispositions réglementaires prises dans l'intérêt de tous', stelde Logie voor. Zie Logie, 'De la prostitution', 211. 66 'Circulaire prescrivant quelques dispositions pour arrêter les progrès de la maladie vénérienne dans les rangs de l'armée', JMO (1848) 167-168. 67 In januari 1899 werd echter beslist dat de informatie die soldaten vrijgaven over de publieke vrouw die hen had besmet, niet meer mocht worden doorgegeven aan de gemeentelijke overheden omdat het intieme informatie betrof waarvan de exactheid niet gegarandeerd was. Zie 'Circulaire faisant savoir que la circulaire du 7 mai 1848,6e direction, no. 464, a été abrogée par suite de la mise en vigueur du règlement du 1er août 1877',7AfO(1899) 160. Heel wat legerartsen hadden in de enquête van 1888 over het venerisch probleem in het leger al hun twijfel geuit over de efficiëntie van het 'verklikkingssysteem'. Zie bijvoorbeeld Logie, 'De la prostitution', 204 en Spruyt, e. a., 'De la prostitution', 271,284. Toch werd het systeem later opnieuw ingevoerd (zie bijvoorbeeld de circulaire van 7 december 1920), maar in 1921 drong legerarts Lakaye erop aan dat men er weer van zou afstappen omdat het nauwelijks succes oogstte en tot misbruiken leidde: R. Lakaye, 'Lutte antivénérienne à l'armée', in: Congrès international de médecine et de pharmacie militaires au palais mondial du 15 au 20 juillet 1921 (Brussel, 1921) 111. Meldingsplicht De 'déclarés' hadden recht op een behandeling die gelijkwaardig was aan die van de andere patiënten in het ziekenhuis, de 'non-déclarés' daarentegen konden geen aan- spraak maken op een soldij en zouden bij hun terugkeer naar het korps worden gestraft 'pour avoir dissimulé leur maladie et l'avoir, par là, volontairement aggravée'63. Legerartsen hadden vastgesteld dat veel soldaten hun geslachtsziekte onverzorgd lieten of de behandeling ervan aan kwakzalvers toevertrouwden om te ontsnappen aan het officieel ingestelde Strafregime dat hen in de militaire hospitalen te wachten stond. Een humanisering van de behandeling van de venerische patiënten moest de soldaten aansporen hun geslachtsziekte niet langer geheim te houden maar ze onmiddellijk aan de legerartsen te melden. De 'non-déclarés' werden uitdrukkelijk 412 Liesbet Nys uitgesloten van de humanere behandelingswijze. Zij leefden de bestaande richtlijnen niet na en vormden daardoor in de ogen van de legerartsen een enorm gevaar voor de overige soldaten. De legerartsen deden er alles aan om de 'non-déclarés' te stigmati- seren. In 1872 pleitte Fromont in de Archives voor een bikkelharde aanpak van de 'non-déclarés'. Hij wilde bijvoorbeeld dat ze door kwartierarrest van hun vrijheid konden worden beroofd64. In 1888 stelde Logie voor om de 'déclarés' verder te deculpa- biliseren door ze voortaan als 'blessés' en niet langer meer als 'vénériens' te bestem- pelen. Maar de 'non-déclarés' mochten van hem zeker niet van dit soort maatregelen profiteren. Zij moesten verder als 'vénériens' worden beschouwd65. Legerartsen zagen de 'non-déclarés' als een schandvlek op het leger en ze toonden dan ook geen enkele schijn van mededogen met deze soldaten. De gezondheidsdienst van het leger creëerde ook geregeld nieuwe redenen om sol- daten tot de groep van de 'non-déclarés' te rekenen. Deze categorie van soldaten werd herhaaldelijk opnieuw gedefinieerd. Een omzendbrief van 7 mei 1848 bepaalde bijvoorbeeld dat ook de soldaten die weigerden te onthullen van wie en waar ze hun ziekte hadden opgedaan, voortaan als 'non-déclarés' zouden worden ingeschreven en dus geen soldij meer zouden ontvangen. Wie foutieve of valse verklaringen aflegde over zijn infectiebron, moest door een maatregel van 5 april 1872 ook als 'non-déclaré' worden geregistreerd66. Blijkbaar zochten de legerartsen naarstig naar besmette prostituees om ze daarna met de hulp van de gemeentelijke overheden buiten het bereik van de garnizoenen te kunnen plaatsen67. Morele scheidsrechters Een snelle behandeling van de besmette soldaten en een efficiënte repressie van de geïnfecteerde prostituees moesten de gezonde soldaten voor geslachtsziekten behoe- den. Maar vanuit moreel standpunt was het interessanter om de rekruten te overtuigen van het gevaar van elke vorm van voor- of buitenhuwelijks seksueel contact. Daartoe besloot de minister van oorlog op 30 september 1885 tot de invoering van trimestriële conferenties. De artsen van het leger moesten de soldaten voortaan één keer per trimes- De grote school van de natie 413 ter wijzen op 'les déplorables conséquences de l'inconduite', 'les périls que l'immo- ralité fait courir à leur santé'68. Een primitieve vorm van seksuele voorlichting werd geboren. ter wijzen op 'les déplorables conséquences de l'inconduite', 'les périls que l'immo- ralité fait courir à leur santé'68. Een primitieve vorm van seksuele voorlichting werd geboren. Eigenlijk hield Petithan zich al vele jaren vóór de invoering van de verplichte confe- renties bezig met de voorlichting van soldaten. Al in 1863 zette hij in de Archives de methode uiteen die hij daarvoor gebruikte: Je leur racontai l'histoire de l'uréthrite, je leur exposai les agréments des rétrécissements, j l t ifi i l d i ti d P th l i bl h i j l d i i t Je leur racontai l'histoire de l'uréthrite, je leur exposai les agréments des rétrécissements, je les terrifiai par la description de Popthalmie blennorrhagique, je leur donnai une sainte et juste horreur pour le chancre, je les fis trembler à la pensée de la syphilis69. Angst voor de venerische ziekten moest de soldaten aansporen tot respect voor restrictieve seksuele waarden. Petithan vergeleek de rol van de legerarts met die van de goede huisvader70. De voorlichting die Petithan en de andere legerartsen aan de soldaten gaven, was duidelijk eerder van morele dan van medische aard71. Volgens Rulot en Sacré wezen de legerartsen de rekruten in hun verplichte conferenties vooral op 'les conséquences déplorables pour l'individu, pour la famille, et pour la société entière, de la débauche, de l'immoralité et des excès de toute manière'72. De legerartsen gedroegen zich als morele scheidsrechters. De voorlichtingssessies die zij voor de soldaten organiseerden, illustreren nogmaals hoe zij bij hun strijd tegen de venerische ziekten zeker niet louter door sanitaire, maar duidelijk ook door morele motieven werden gedreven, hoe ook zij de angst voor venerische ziekten hebben aangewend om de soldaten tot respect voor het burgerlijk fatsoen te dwingen. 73 Ook in de negentiende eeuw al waren zij niet helemaal onbetwist. Militair arts Célarier stelde in de commissie over de prostitutiekwestie de doelmatigheid van deze morele conferenties openlijk in vraag. Zie Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée, procès-verbaux des séances de la section d'hygiène, 113. 68 'Circulaire indiquant certaines mesures à prendre pour prémunir lesjeunes militaires contre la débauche et ses funestes conséquences', JMO (1885) 416-418. 69 Petithan, 'Note sur le traitement de l'uréthrite', 228. 71 Voor een uitvoerig fragment uit een voorlichtingsconferentie van Peüthan, dat dit bijzonder goed illustreert, zie Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée, procès-verbaux des séances plénières, 205-206. 70 Petithan, 'Prophylaxie des maladies vénériennes', 353. 68 'Circulaire indiquant certaines mesures à prendre pour prémunir lesjeunes militaires contre la débauche et ses funestes conséquences', JMO (1885) 416-418. 69 Petithan, 'Note sur le traitement de l'uréthrite', 228. 70 Petithan, 'Prophylaxie des maladies vénériennes', 353. 71 Voor een uitvoerig fragment uit een voorlichtingsconferentie van Peüthan, dat dit bijzonder goed illustreert, zie Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée, procès-verbaux des séances plénières, 205-206. 72 Rulot, Sacré, La vie du soldat belge, 8. 73 Ook in de negentiende eeuw al waren zij niet helemaal onbetwist. Militair arts Célarier stelde in de commissie over de prostitutiekwestie de doelmatigheid van deze morele conferenties openlijk in vraag. Zie Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée, procès-verbaux des séances de la section d'hygiène, 113. 74 Lakaye, 'Lutte antivénérienne à l'armée', 108. 75 Zie bijvoorbeeld Gliben, 'La lutte antivénérienne à l'armée belge', in: Congrès international de médecine et de pharmacie militaires au palais mondial du 15 au 20 juillet 1921 (Brussel, 1921) 51-54; L. Wilmaers, 'Lutte antivénérienne à l'armée', 82-86. Ook in andere landen bestond er veel discussie over het gebruik van de profylactische middelen. Vgl. B. A. Towers, 'Health education policy 1916-1926: ve- nereal disease and the prophylaxis dilemma', Medical history, XXIV (1980) 70-87; Mooij, Geslachts- ziekten en besmettingsangst, 107-116; S. M. Tomkins, 'Palmitate or permanganate. The venereal prophylaxis debate in Britain, 1916-1926*. Medical history, XXXVH (1993) 382-398. 76 Zie Derbraudrenghien, 'De la conspiration du silence', 71-74. Morele scheidsrechters Na de Eerste Wereldoorlog nam de kritiek op dit soort morele conferenties echter toe73. Steeds meer legerartsen stelden zich vragen bij de moraliserende aanpak van de strijd tegen de geslachtsziekten en wilden de klemtoon liever verleggen naar de thera- peutische bestrijding ervan. Legerarts Lakaye bijvoorbeeld had een erg beslist stand- punt over de efficiëntie van de morele conferenties: Il est hors de doute pour tous les médecins ayant une certaine pratique militaire, pour tous ceux qui ont été à la guerre, que les conférences morales ont fait faillite. Actuellement encore, certains luttent contre la syphilis en prêchant la chasteté. Allons donc! qu'on laisse 414 Liesbet Nys subsister, pour le bon renom de la morale, ces causeries somnifères, soit, mais disons bien qu'à notre point de vue de médecins de gardiens de la santé physique du soldat elles ne servent à rien et qu'on ne peut lutter contre un instinct aussi primordial, aussi impérieux avec de belles paroles et de sévères recommandations74. Op zieh was Lakaye de idee van seksuele voorlichting nochtans niet ongenegen, maar die voorlichting moest medisch en niet moraliserend van aard zijn. In korte conferenties met filmbeelden moesten de legerartsen de soldaten een beschrijving geven van de verschillende venerische ziekten. Daarnaast moesten ze de militairen wijzen op het bestaan van allerhande profylactische middelen die ter preventie van de geslachtsziekten konden worden aangewend. Hier raakte Lakaye een heikele kwestie. Het gebruik van het condoom en van chemische profylactica als protargol of calomelzalf was in de eerste helft van de twintigste eeuw bijzonder omstreden. Niet alleen werd de werkzaamheid van deze middelen vaak in twijfel getrokken, velen waren er tegen gekant omdat ze tot buitenechtelijke seksualiteit en bordeelbezoek zouden aansporen. Nogal wat legerartsen drongen er echter op aan dat deze middelen in het Belgisch leger ruimer bekend en verspreid zouden raken. Vooral het voorbeeld van het Amerikaans leger, dat tijdens de Eerste Wereldoorlog deze middelen op grote schaal had ingezet en daarmee volgens de legerartsen erg gunstige resultaten had geboekt, werkte inspirerend75. Vele van de vernieuwingen die legerartsen voorstelden, werden in de jaren twintig effectief gerealiseerd: de voorlichtingsconferenties voor soldaten kregen een mo- dernere aankleding, in elke kazerne werden profylactische kamertjes — ruimten waar soldaten na seksueel contact hun geslachtsdelen met allerlei zepen, zalven en andere middeltjes konden reinigen — ingericht en de opsporing van geslachtsziekten bij soldaten werd geoptimaliseerd76. De Belgische legerartsen hadden duidelijk lessen getrokken uit de oorlog. 77 Over Bayer. M.-T. Nisot, La question eugénique dans les divers pays. Il (Brussel, 1927-1929) 156- 163; Jacqué, Craps, 'La dermatologie et la syphiligraphie', 244-246; Derbraudrenghien, 'De la conspiration du silence', passim. Voor de lof van Bayet voor de aanpak van het venerisch probleem in het leger, zie bijvoorbeeld A. Bayet, La lutte contre la syphilis en Belgique. Son organisation et ses résultats (Brussel, 1926)12-13,24. Morele scheidsrechters Hun vernieuwde aanpak van het geslachtsziektenprobleem oogstte veel lof, niet in het minst van Adrien Bayet, die als hoogleraar van de Université de Bruxelles verbonden was aan de venerologische afdeling van het Brusselse Sint- Pietershospitaal en zich al voor de Eerste Wereldoorlog had ontpopt als één van de heftigste voorstanders van een medische (in de plaats van een moraliserende) aanpak van het venerisch probleem. In de jaren twintig werd een actieplan dat Bayet voor de Hoge Gezondheidsraad had opgesteld de basis van het regeringsbeleid inzake de venerische ziekten. De nadruk lag niet langer op de reglementering van de prostitutie, wel op de oprichting van dispensaria, de gratis ter beschikking stelling van medicatie, een intensieve propaganda bij de bevolking enzovoort. België nam in het Interbellum 415 De grote school van de natie op het vlak van de geslachtsziektenbestrijding zo'n toonaangevende positie in dat in internationale medische kringen van 'la grande expérience belge' werd gesproken77. op het vlak van de geslachtsziektenbestrijding zo'n toonaangevende positie in dat in internationale medische kringen van 'la grande expérience belge' werd gesproken77. 78 'Circulaire interdisant le débit des boissons alcooliques dans les casernes', JMO (1885) 414. Tot de alcoholische dranken werden alleen de gedistilleerde dranken gerekend en niet de gegiste -— zoals bier — die in de negentiende eeuw als hygiënische dranken werden beschouwd. 79 Zie bijvoorbeeld A. Meynne, De l'alimentation du soldat (Brussel, 1849). Ook Jansen en Petithan waren overtuigde abolitionisten. 80 Zie bijvoorbeeld de kritiek van Seutin op het hygiënistisch handboek van Marinus: J.-R. Marinus, Essai suri 'hygiène du soldat, 34. Omdat dronken soldaten steeds vaker in ruzies met burgers hun wapens gebruikten, werd in 1862 gedecreteerd dat het voor alle soldaten die in dronken toestand werden gezien voor onbepaalde duur verboden zou worden hun wapens nog buiten de diensturen te dragen. 'Circulaire portant que tout militaire qui aura été vu en état d'ivresse sera privé du port de l'arme pendant un temps indéterminé', JMO ( 1862) 201. Deze maatregel werd nader gespecificeerd door een aantal andere circulaires, bijvoorbeeld die van 3 april 1871: 'Circulaire portant que la durée de la privation du port de l'arme à infliger aux sous-officiers, ne pourra dépasser trois mois', JMO (1871) 314. In 1874 werd de draagkracht van de maatregel afgezwakt. 'Nouvelles prescriptions relatives à l'interdiction du port de l'arme pour ivresse', JMO (1874) 588). Dit was niet naar de zin van Auguste Jansen. Zie Jansen, 'De l'usage et de l'abus des boissons alcooliques', 564-565. 78 'Circulaire interdisant le débit des boissons alcooliques dans les casernes', JMO (1885) 414. Tot de alcoholische dranken werden alleen de gedistilleerde dranken gerekend en niet de gegiste -— zoals bier — die in de negentiende eeuw als hygiënische dranken werden beschouwd. 79 Zie bijvoorbeeld A. Meynne, De l'alimentation du soldat (Brussel, 1849). Ook Jansen en Petithan waren overtuigde abolitionisten. 80 Zie bijvoorbeeld de kritiek van Seutin op het hygiënistisch handboek van Marinus: J.-R. Marinus, Essai suri 'hygiène du soldat, 34. Omdat dronken soldaten steeds vaker in ruzies met burgers hun wapens gebruikten werd in 1862 gedecreteerd dat het voor alle soldaten die in dronken toestand werden gezien 78 'Circulaire interdisant le débit des boissons alcooliques dans les casernes', JMO (1885) 414. Tot de alcoholische dranken werden alleen de gedistilleerde dranken gerekend en niet de gegiste -— zoals bier — die in de negentiende eeuw als hygiënische dranken werden beschouwd. 80 Zie bijvoorbeeld de kritiek van Seutin op het hygiënistisch handboek van Marinus: J.-R. Marinus, Essai suri 'hygiène du soldat, 34. Omdat dronken soldaten steeds vaker in ruzies met burgers hun wapens gebruikten, werd in 1862 gedecreteerd dat het voor alle soldaten die in dronken toestand werden gezien voor onbepaalde duur verboden zou worden hun wapens nog buiten de diensturen te dragen. 'Circulaire portant que tout militaire qui aura été vu en état d'ivresse sera privé du port de l'arme pendant un temps indéterminé', JMO ( 1862) 201. Deze maatregel werd nader gespecificeerd door een aantal andere circulaires, bijvoorbeeld die van 3 april 1871: 'Circulaire portant que la durée de la privation du port de l'arme à infliger aux sous-officiers, ne pourra dépasser trois mois', JMO (1871) 314. In 1874 werd de draagkracht van de maatregel afgezwakt. 'Nouvelles prescriptions relatives à l'interdiction du port de l'arme pour ivresse', JMO (1874) 588). Dit was niet naar de zin van Auguste Jansen. Zie Jansen, 'De l'usage et de l'abus des boissons alcooliques', 564-565. 78 'Circulaire interdisant le débit des boissons alcooliques dans les casernes', JMO (1885) 414. Tot de alcoholische dranken werden alleen de gedistilleerde dranken gerekend en niet de gegiste -— zoals bier — die in de negentiende eeuw als hygiënische dranken werden beschouwd. 79 Zie bijvoorbeeld A. Meynne, De l'alimentation du soldat (Brussel, 1849). Ook Jansen en Petithan waren overtuigde abolitionisten Alcoholverbod Niet alleen door een krachtdadige bestrijding van het venerisch probleem, maar ook door een radicaal offensief tegen drankmisbruik in de kazerne trachtten de artsen van de Belgische strijdmacht de gezondheidstoestand én de moraal in het leger te bevor- deren. Legerartsen plaatsten de maatregelen die tegen alcoholisme bij soldaten werden genomen vaak zelf op één lijn met de richtlijnen die voor de bestrijding van de ge- slachtsziekten van militairen werden uitgevaardigd. Vanuit de nauwe relatie die zij tussen drankmisbruik en venerische ziekten veronderstelden, namen zij aan dat de strijd tegen het alcoholisme ook de strijd tegen de geslachtsziekten ten goede zou komen. De meest radicale maatregel tegen drankmisbruik bij soldaten werd op 12 september 1885 afgekondigd. Toen werd een absoluut verbod op alcoholische dranken in de kazerne uitgevaardigd. De invoering van deze maatregel moest gepaard gaan met de installatie van zogenaamde 'mess', kantines waar onderofficieren en soldaten hun vrije uren konden spenderen maar waar uiteraard geen alcohol werd verkocht78. Heel lang al hadden bepaalde legerartsen op de invoering van deze maatregel aange- drongen79. De tegenstanders van drankgebruik in het leger beweerden dat alcohol erg schadelijk was voor de lichamelijke, morele en intellectuele ontwikkeling van de soldaten. Ook meenden ze dat alcoholmisbruik bij soldaten kon leiden tot ongedisci- plineerd, gewelddadig of crimineel gedrag80. Toch waren lang niet alle legerartsen in de tweede helft van de negentiende eeuw te vinden voor een algemeen verbod op alcohol in het leger. Er heerste in de medische kringen van het leger heel wat verdeeldheid over de vraag of geestrijke dranken scha- 416 Liesbet Nys delijk, nuttig of noodzakelijk waren voor militairen. Nogal wat legerartsen meenden dat soldaten in de alcohol de noodzakelijke kracht vonden voor hun zware inspan- ningen of schreven aan alcohol een geneeskrachtige werking toe81. De meeste artsen namen een gematigde tussenpositie in. Ze meenden dat beperkt gebruik van alcoho- lische dranken in het leger wel moest kunnen, maar bepleitten dat dronkenschap hard zou worden bestreden82. Na 1885 klonk er uit de medische kringen van het leger een veel eenstemmiger geluid. De radicale maatregelen die tegen het alcoholisme waren genomen, werden vooral met lofbetuigingen onthaald. Dissidente meningen kwamen bijvoorbeeld in de Archives nauwelijks nog voor. Er werd algemeen aangenomen dat het aantal gevallen van dronkenschap en alcoholisme in het leger door de getroffen maatregelen erg zeldzaam was geworden83. 82 Zie onder andere Ch. Détienne, Hygiène de l'armée ou préceptes d'hygiène militaire, à l'usage des officiers et des sous-officiers de l'armée (Luik, 1849). 83 Zie bijvoorbeeld V. Desguin, De l'abus des boissons alcooliques. Ses causes, ses résultats, ses remèdes (Antwerpen, Parijs, 1876) 16; E. Dupont, 'Coup d'oeil sur l'hygiène militaire en Belgique depuis 1830', in: H. Kubom, e. a., Aperçu historique sur l'hygiène publique en Belgique depuis 1830 (Brussel, 1897) 255-256; De Broeu, 'Alcoolisme et manie ébrieuse. Responsabilité dans l'ivresse normale et pathologique', Archives, LH (juli 1899)6. 81 Zie bijvoorbeeld Jeanty, 'Quelques considérations sur l'emploi de l'alcool dans les affections pulmonaires'. Archives, XXIII (maart 1870) 151-155; Dereine, 'Des usages bienfaisants des boissons alcooliques et des actions physiologiques et thérapeutiques de l'alcool', Archives, XXXV (januari 1882) 5-36. 84 'Circulaire prescrivant certaines mesures spéciales contre l'ivrognerie', JMO ( 1886) 493-495. 86 'Arrêté royal no. 2421bisD, du 23 novembre 1914, interdisant l'usage des boissons alcoolisées', JMO (1914) 219-220. 85 Zie onder andere Rulot, Sacré, La vie du soldat Belge, 111. 82 Zie onder andere Ch. Détienne, Hygiène de l'armée ou préceptes d'hygiène militaire, à l'usage des officiers et des sous-officiers de l'armée (Luik, 1849). 88 Voorbeelden van deze lofzangen: Binard, 'De la prostitution dans le ville de Berlin et des mesures à prendre pour la combattre ainsi que la syphilis', Archives, III (april 1850) 304, 317; Idem, 'Des maladies vénériennes en Egypte', Archives, VII (maart 1854) 279; A. Jansen, Conférences militaires; entretiens sur l'hygiène militaire (Luik, 1878) 416; L. Mélis, 'Lésions oculaires et cérébrales de cause syphilitique. Observation recueillie à l'hôpital militairede Bruxelles', Archives, XVI (oktober 1879) 299; Umé, 'Névro- rétinite syphilitique', Archives, XVII (mei 1880) 293-294; Dupont, 'Coup d'oeil sur l'hygiène militaire', 253-254. Alcoholverbod Met het oog op een verbetering van de moraal in het leger had de minister van oorlog in november 1886 trouwens nog een belangrijke richtlijn uitgevaardigd om het drankmisbruik bij soldaten terug te dringen. Die richtlijn viseerde — als aanvulling op de maatregel van september 1885 die tegen drankmis- bruik in de kazerne was gericht — vooral het onmatig drankgebruik van soldaten buiten de muren van het garnizoen en hield onder meer in dat de namen van de soldaten die voor dronkenschap of openbaar schandaal waren gestraft, op een zichtbare plaats in de kazerne zouden worden opgehangen. Ook werden de legerartsen in de richtlijn opgeroepen om in hun trimestriële conferenties de soldaten voortaan uitdrukkelijk te wijzen op de gevaren van alcoholmisbruik84. Het begin van de twintigste eeuw bracht geen echte wijziging in de houding van de legerartsen tegenover het drankverbod in het leger. De meeste legerartsen bleven het verbod als een goede en noodzakelijke maatregel beschouwen85. In november 1914 werd omwille van de oorlogsomstandigheden op het hele Belgische grondgebied dat in handen was van de Belgische en de geallieerde troepen, een verbod op alcoholische dranken ingevoerd86. Na de oorlog bestendigde de socialistische minister van justitie Emile Vandervelde deze repressieve aanpak van het alcoholprobleem met een besluit- wet (van 15 november 1918) die het produceren, invoeren, vervoeren, verkopen en schenken van sterke dranken, bieren en wijnen met een alcoholgehalte van meer dan 15% verbood. Onder druk van aanhoudend protest moest Vandervelde deze wet echter De grote school van de natie 417 afzwakken. Op 29 augustus 1919 werd een nieuwe wet uitgevaardigd, de zogenaamde Wet Vandervelde. Die verbood de consumptie van alcohol in drankgelegenheden, maar liet wel toe dat thuis alcoholische dranken werden geconsumeerd op voorwaarde dat men minstens twee liter ineens kocht. In de Archives gaven legerartsen hun mening over de Wet Vandervelde niet echt te kennen, maar het valt aan te nemen dat zij, zoals hun collega's in de civiele maatschappij, in overwegend aantal achter deze wet stonden87. 89 Zie bijvoorbeeld 'De la prostitution. (Extraits de rapports)', 349. 87 Over deze alcohol wetgeving: B. de Ruyver, 'De alcoholwet Vandervelde in historisch en ideologisch perspectief', Tijdschrift voor sociale wetenschappen, XXVII (1982) 342-369; Scholliers, "Een vijand dien men kennen moet", 149-150. 88 V b ld d l f Bi d 'D l i i d l ill d B li d à 87 Over deze alcohol wetgeving: B. de Ruyver, 'De alcoholwet Vandervelde in historisch en ideologisch perspectief', Tijdschrift voor sociale wetenschappen, XXVII (1982) 342-369; Scholliers, "Een vijand dien men kennen moet", 149-150. 88 Voorbeelden van deze lofzangen: Binard, 'De la prostitution dans le ville de Berlin et des mesures à prendre pour la combattre ainsi que la syphilis', Archives, III (april 1850) 304, 317; Idem, 'Des maladies vénériennes en Egypte', Archives, VII (maart 1854) 279; A. Jansen, Conférences militaires; entretiens sur l'hygiène militaire (Luik, 1878) 416; L. Mélis, 'Lésions oculaires et cérébrales de cause syphilitique. Observation recueillie à l'hôpital militairede Bruxelles', Archives, XVI (oktober 1879) 299; Umé, 'Névro- rétinite syphilitique', Archives, XVII (mei 1880) 293-294; Dupont, 'Coup d'oeil sur l'hygiène militaire', 253-254. IMAGOVORMING Niet alleen de radicale maatregelen die in het leger tegen het drankmisbruik werden genomen, maar ook die ter bestrijding van de geslachtsziekten konden in de medische kringen van het leger op veel bijval rekenen. Heel de tweede helft van de negentiende eeuw staken artsen van het Belgisch leger de loftrompet over de maatregelen die Vleminckx en zijn opvolgers tegen de geslachtsziekten bij soldaten hadden genomen88. Net zoals de anti-alcoholmaatregelen het drankmisbruik bij soldaten sterk hadden teruggedrongen, hadden volgens hen de maatregelen tegen de venerische ziekten het aantal geslachtsziekten in het leger drastisch verminderd en de secundaire en tertiaire verschijnselen van syfilis in de meeste garnizoenen zelfs helemaal doen verdwijnen. De legerartsen spraken herhaaldelijk hun trots uit over de manier waarop in het leger de strijd tegen geslachtsziekten en drankmisbruik werd gevoerd. Vooral het ve- nerisch offensief was het paradepaard van de gezondheidsdienst van het leger. De samenleving kon er niet genoeg van worden doordrongen dat het leger op de voorste linie stond in de strijd tegen de geslachtsziekten en aan zijn moreel herstel was begonnen. Het offensief tegen de venerische ziekten in het leger beoogde een ver- betering van de gezondheid en de moraal van de soldaten, maar moest ook, net als dat tegen het drankmisbruik, dienen om het imago van de strijdmacht op te poetsen. Legerartsen haalden in hun pleidooien voor de afschaffing van de plaatsvervanging, die zij als een belangrijke oorzaak van drankmisbruik en venerische besmetting in het leger beschouwden, expliciet het argument aan dat deze ingreep het prestige van de strijdmacht ten goede zou komen89. En de voorkeur van de legerartsen voor het reglementarisme was zeker ook ingegeven door de wens de (onvermijdbare) illegitieme seksuele contacten van soldaten aan het zicht van de maatschappij te onttrekken en 418 Liesbet Nys zo een belangrijke oorzaak van de slechte morele reputatie van het leger tot op zekere hoogte te elimineren90. In het leger raakten bij de strijd tegen het alcoholisme en de venerische ziekten sanitaire en morele overwegingen verweven met drijfveren als faam en prestige, werd het drank- en het venerisch offensief een instrument voor imagovorming. g g Wie niet aan deze imagovorming van het leger meewerkte, haalde zichzelf een hoop ellende op de hals. Meynne bijvoorbeeld raakte hierdoor in het begin van de jaren zestig in een fikse rel met Vleminckx verwikkeld. 91 Vleminckx liet zich bijvoorbeeld in 1849 en in 1862bijzondertriomfantelijkuitoverdespectaculaire resultaten waartoe volgens hem zijn maatregelen tegen de venerische ziekten in het leger hadden geleid. Zie J.-F. Vleminckx, 'De la maladie vénérienne dans l'armée', Archives, II (april 1849) 274-278; Idem, 'Du mal vénérien en Belgique', BARMB, V (1862) 264-290. 90 Misschien moeten verder ook de richtlijnen die werden uitgevaardigd om de bordelen zoveel mogelijk uit de omgeving van de kazernes te weren niet alleen vanuit sanitair en moreel oogpunt maar ook vanuit dit imagoperspectief worden bekeken. In een circulaire van 30 september 1885 drong de minister van oorlog aan op een betere controle van de 'maisons mal famées qui, véritables parasites, se groupent d'ordinaire autour des quartiers de la troupe', in: 'Circulaire indiquant certaines mesures à prendre pour prémunir les jeunes militaires contre la débauche et ses funestes conséquences', JMO (1885) 416-418. De 'zuiverheid' van de kazerne werd bedreigd door 'parasieten', die blijkbaar onuitroeibaar waren want op 10 maart 1888 volgde opnieuw een maatregel om de bordelen in de buurt van de kazernes te laten verwijderen. 'Circulaire déterminant les mesures à prendre pour prémunir les jeunes militaires contre la débauche et ses funestes conséquences', JMO (1888) 54-55. In maart 1887 was al bepaald dat aan de rekruten de toegang tot bepaalde bordelen en zelfs tot de straten waarin deze etablissementen van verdacht allooi zich bevonden, kon worden ontzegd. 'Circulaire indiquant la marche à suivre lorsqu'il y a lieu d'interdire aux troupes la fréquentation de certains établissements publics', JMO (1887) 40. 92 Misschien speelde in deze kwestie mee dat Meynne zich ook al in 1855 behoorlijk sceptisch had uitgelaten over de toestand van de venerische ziekten in het leger. Zie: A. Meynne, 'Mesures prophylactiques contre la syphilis', Archives, VIII (maart 1855) 184-188. Voor het dispuut tussen Vleminckx en Meynne: dossier Antiand Meynne, R. M. 4384, Koninklijk museum van het legeren van krijgsgeschiedenis, Brussel. Zie ook Vleminckx, 'Du mal vénérien en Belgique', 264-290; 'Correspondance', BARMB, V (1862) 298- 306 en 367-369. Vleminckx is er wel zelf niet in geslaagd zijn optimisme te bewaren. In 1872 liet hij zijn triomfantelijke uitspraken varen en stelde hij voor de Hoge gezondheidsraad een rapport op om bij de overheid aan te dringen op een onderzoek naar het venerisch probleem in België. In dit onderzoek moest er ook specifieke aandacht worden besteed aan de toestand in het leger. Zie: J.-F. Vleminckx, 'Proposition d'enquête sur les ravages des maladies vénériennes', Conseil supérieur d'hygiène publique. Rapports adressés à mm. les ministres de l'intérieur et de la justice, IV (1867-1873) 475-477. Misschien hield deze omslag in de houding van Vleminckx verband met een rapport dat legerarts Fromont hem had toegestuurd. Fromont dramatiseerde daarin de situatie op het vlak van de geslachtsziekten in het leger en zocht de oorzaak van deze ongunstige toestand vooral in de clandestiene prostitutie. Hij pleitte er uitdrukkelijk voor om voortaan alleen nog meisjes met een certificaat van goede zeden toe te laten in de kazerne. Zie Fromont, 'Des moyens de prévenir la propagation de la syphilis dans l'armée', 84. IMAGOVORMING Vleminckx, die bijzonder zelfvol- daan was over de maatregelen die hij in het leger tegen de geslachtsziekten had genomen91, beschuldigde Meynne ervan dat hij aan de Franse arts Jeannel veel te hoge cijfers over het aantal venerische patiënten in de Belgische militaire hospitalen had doorgegeven. Meynne daarentegen beweerde dat hij Jeannel wel correcte gegevens had bezorgd, maar dat die er zelf, in zijn studie over de prostitutie in Bordeaux, fou- tieve en onverantwoorde bewerkingen had op toegepast. Dit suste Vleminckx aller- minst. Vleminckx zag zichzelf als inspecteur-generaal van de gezondheidsdienst van het leger als de enige betrouwbare bron over het aantal venerische patiënten bij soldaten en voelde zich duidelijk voorbijgestoken door Meynne. 'Est-ce que je ne suis pas la seule source officielle en Belgique pour cette espèce de choses? Quelqu'un a-t-il la prétention d'être plus officiel que moi?', schreef hij in het Bulletin van de Koninklijke Academie voor geneeskunde waarvan hij voorzitter was. Vleminckx maakte zich vooral zorgen over het feit dat 'ces fausses appréciations [... ] feront le tour du monde, et la vérité vraie aura bien de la peine à se faire jour'. Toen Meynne met een boze brief reageerde op de verwijten van Vleminckx, liet deze laatste prompt in de Academie een commissie aanstellen om de brief van Meynne te beoordelen. De commissie schaarde zich zoals verwacht achter Vleminckx en bestempelde de brief van Meynne als beledigend en ongepast. Verder heeft Vleminckx naar aanleiding van deze kwestie ook herhaaldelijk bij de minister van oorlog aangedrongen op sancties tegen Meynne. Het kon niet worden geduld dat legerartsen de reputatie van het leger schade toe- 419 De grote school van de natie brachten. Er mocht in de samenleving geen twijfel rijzen over het beleid dat Vleminckx in het leger ten aanzien van de geslachtsziekten voerde92. 93 Wie toch cijfers wil over het aantal geslachtsziekten in het leger, zie bijvoorbeeld Vleminckx, 'Du mal vénérien en Belgique', 264-290; A. Moelier, 'Les maladies vénériennes dans l'armée belge de 1868 à 1886', Uittreksel uit BARMB (Brussel, 1887); J. Gaudy, 'Les maladies vénériennes à l'armée', Archives, LXX (juni 1917) 510; A. Bayet, La lutte contre la syphilis, 35-37. 93 Wie toch cijfers wil over het aantal geslachtsziekten in het leger, zie bijvoorbeeld Vleminckx, 'Du mal vénérien en Belgique', 264-290; A. Moelier, 'Les maladies vénériennes dans l'armée belge de 1868 à g q g 1886', Uittreksel uit BARMB (Brussel, 1887); J. Gaudy, 'Les maladies vénériennes à l'armée', Archives, LXX (juni 1917) 510; A. Bayet, La lutte contre la syphilis, 35-37. MILITAIRE EN NATIONALE EFFICIËNTIE In de tweede helft van de negentiende en het begin van de twintigste eeuw was de idee vrij algemeen aanvaard dat soldaten een risicogroep vormden voor drankmisbruik en geslachtsziekten. In de kazerne werden verregaande maatregelen tegen beide 'plagen' genomen. De vraag of het drankmisbruik en de geslachtsziekten in het leger daadwerkelijk veel dramatischer proporties aannamen dan daarbuiten, is bij gebrek aan betrouwbaar statistisch materiaal moeilijk te beantwoorden. Legerartsen publi- ceerden — in de geest van de toenmalige statisticomanie — wel regelmatig cijfer- materiaal over het aantal alcoholisten of venerische patiënten in het leger, maar dit materiaal was lang niet onbetwist. Dat kon ook moeilijk anders want bijvoorbeeld in de tweede helft van de negentiende eeuw stond de diagnose van de verschillende geslachtsziekten nog helemaal niet op punt93. Annet Mooij gelooft alvast niet dat in Nederland de prevalentie van venerische ziekten onder soldaten een voldoende verklaring biedt voor de grote ongerustheid die vooral in het begin van de twintigste eeuw over het geslachtsziektenprobleem in het leger bestond. Immers, ook in voorgaande periodes kwamen geslachtsziekten frequent voor in de strijdmacht en toen lag niemand daar volgens haar echt wakker van. Mooij meent dat die ongerustheid veeleer in verband moet worden gebracht met het toenmalige streven naar national efficiency, naar een weerbare en efficiënt georganiseerde natie. Daar hoorde een goed functionerend leger bij, waarvan de werking niet voortdurend door horden venerische patiënten zou worden verlamd. Terwijl niemand zich voorheen echt had gestoord aan Liesbet Nys 420 Liesbet Nys Liesbet Nys een hoog aantal venerische patiënten in de garnizoenen, werd dat nu wel als een levensgrote bedreiging ervaren94. Dit lijkt ook een aannemelijke verklaring voor de panieksfeer die in België in de tweede helft van de negentiende en de eerste helft van de twintigste eeuw bij wijlen, niet alleen over het geslachtsziektenprobleem, maar ook over het alcoholmisbruik in het leger rees. Legerartsen zagen een gedisciplineerd en efficiënt werkend leger als een must om de veiligheid en stabiliteit van het land te garanderen. Drankmisbruik hypothekeerde de noodzakelijke discipline en kon zo het succes van de troepen da- nig compromitteren. 'Un militaire stupéfié par l'alcool ne pourrait faire une action héroïque, ni rendre les services que la patrie est en droit d'attendre de lui', schreef Auguste Jansen in 187695, Daarop somde hij een hele reeks militaire nederlagen op die volgens hem vooral aan het drankmisbruik van soldaten te wijten waren. 99 Gaudy, 'Les maladies vénériennes à l'armée', 511. 98 De Vaucleroy, De l'alcoolisme dans l'armée, 3. 94 Mooij, Geslachtsziekten en besmettingsangst, 105-107. 95 Jansen, 'De l'usage et de l'abus des boissons alcooliques', 5II-5I2. 96 Ibidem, 512. In Frankrijk speelde de zware nederlaag tegen Duitsland in 1870 een beslissende rol in de doorbraak van het anti-alcoholisme. 'Il faut un bouc-émissaire à cette série inexplicable de désastres, un exutoire capable de rassembler une élite désemparée: on trouve l'alcool', schrijft D. Nourrisson in 'Aux origines de l'antialcoolisme'. Histoire, économie et société, VII (1988) 498. Zie ook Nourrisson, Alcoolisme et antialcoolisme en France, 299. 97 Jansen, 'De l'usage et de l'abus des boissons alcooliques', 513-514. 98 De Vaucleroy, De l'alcoolisme dans l'armée, 3. 99 Gaudy, 'Les maladies vénériennes à l'armée', 511. 97 Jansen, 'De l'usage et de l'abus des boissons alcooliques', 513-514. 94 Mooij, Geslachtsziekten en besmettingsangst, 105-107. 95 Jansen, 'De l'usage et de l'abus des boissons alcooliques', 5II-5I2. 96 Ibidem, 512. In Frankrijk speelde de zware nederlaag tegen Duitsland in 1870 een beslissende rol in de doorbraak van het anti-alcoholisme. 'Il faut un bouc-émissaire à cette série inexplicable de désastres, un exutoire capable de rassembler une élite désemparée: on trouve l'alcool', schrijft D. Nourrisson in 'Aux origines de l'antialcoolisme'. Histoire, économie et société, VII (1988) 498. Zie ook Nourrisson, Alcoolisme et antialcoolisme en France, 299. MILITAIRE EN NATIONALE EFFICIËNTIE De verpletterende nederlaag in 1870 van de Fransen tegen de Duitsers bijvoorbeeld schreef hij toe aan het overmatig drankgebruik van een deel van de Franse bevolking, maar vooral ook van bepaalde fracties van het leger: C'est alors que nous avons vu une partie de la population et quelques indignes débris de l'armée du Rhin en proie à une épidémie alcoolique qui s'est terminée, d'une façon horrible, par l'assassinat des otages et l'incendie de la capitale96. De successen van de troepen van Frederik de Grote in de achttiende eeuw hadden volgens Jansen dan weer alles te maken met de geheelonthouding van deze grote Pruisische vorst97. De meeste legerartsen vonden matigheid in het drankgebruik van soldaten cruciaal opdat het leger de veiligheid van het land optimaal zou kunnen ga- randeren. De Vaucleroy schreef in 1900: Les autorités militaires ont reconnu, depuis longtemps, que la sobriété est une des conditions fondamentales de la discipline et de la vigueur d'une armée et qu'elle s'impose comme une nécessité à ceux qui ont reçu pour mission d'assurer la sécurité de la patrie98. Over de venerische ziekten in het Belgisch leger bestond een soortgelijk vertoog. Legerarts Jules Gaudy stelde in 1917: 'Au point de vue militaire, le vénérien ne peut produire qu'un rendement médiocre. L'effet déprimant exercé par ce genre d'affection sur le physique comme sur le moral de ceux qui en sont atteints n'est plus à démon- trer'99. Hoe meer soldaten omwille van een geslachtsziekte in een militair hospitaal De grote school van de natie 421 moesten worden opgenomen, hoe kleiner het aantal effectief inzetbare soldaten werd en hoe meer de veiligheid van het land in gevaar kwam. Legerartsen berekenden voortdurend de kosten die venerische patiënten en dronkaards voor het leger met zich brachten. Ze telden het aantal rustdagen van deze soldaten en trokken daaruit onrustbarende conclusies over de verspilling van mankracht ten gevolge van alcohol- en seksuele uitspattingen. Het zedeloos gedrag van soldaten werd als een ernstige bedreiging voor de military efficiency gezien, het bevorderen van de moraal in het leger als de beste manier om de discipline te garanderen. Al waren fysiek krachtige soldaten zonder meer een vereiste voor een goed functionerend leger, daarnaast was militaire efficiëntie voor de legerartsen ook sterk gebonden aan een hoogstaande moraal. MILITAIRE EN NATIONALE EFFICIËNTIE Soldaten die zich te buiten gingen aan alcohol- en venerische excessen haalden niet alleen de morele reputatie van het leger onderuit, maar ondermijnden ook de slagkracht van de troepen. Moraal, eer en militaire efficiëntie gingen voor de legerartsen hand in hand. Vaak namen zij in hun bijdragen voor de Archives over het alcoholmisbruik of de venerische ziekten van soldaten de gelegenheid te baat om het belang van een gezonde moraal voor het behoud van de discipline in de kazerne te benadrukken. Vooral Petithan liet op dit vlak geen enkele beurt aan zich voorbij gaan. Hij verkondigde herhaaldelijk de idee dat het leger een morele leerschool van de natie moest zijn: L'armée nationale moderne est essentiellement composée de la partie de la plus virile et la meilleure du peuple. Elle n'est pas seulement l'agent de la force, le soutien de l'autorité, mais encore une véritable école d'éducation. Bonne ou mauvaise, que nous le voulions ou non, elle sera ce que nous la ferons. C'est l'avenir du pays qui est confié à nos mains. Dans notre temps on ne fait plus de la discipline avec la force seulement, la moralité est indis- pensable100. 100 Petithan, 'Prophylaxie des maladies vénériennes', 357. 101 Documents parlementaires. Recueil des pièces, nr. 136, séance du 5 avril 1892. Commission chargée, procès-verbaux des séances plénières, 207. 102 Petithan, 'Note sur le traitement de l'uréthrite', 229. 103 Petithan, 'Prophylaxie des maladies vénériennes', 352-353. 104 Ch. Petithan, 'La dégénérescence de la race belge, ses causes et ses remèdes', Bulletin de la société royale de médecine publique du royaume de Belgique, VII (september 1889) 66. 105 Over alcoholisme en degeneratie: J. Borel, Du concept de dégénérescence à la notion d'alcoolisme dans la médecine contemporaine. Les campagnes antialcooliques de 1865 à 1965 (Montpellier, 1968); C. Finzen, Der Alkoholismus als Problem der Degeneration um die Jahrhundertwende (Kiel, 1977); F. Bynum, DE REGENERATIE VAN HET RAS Vooral Petithan werd in zijn strijd tegen het alcoholisme en de geslachtsziekten bij soldaten sterk door morele overwegingen gedreven en hij kwam daar ook openlijk voor uit. In de parlementaire commissie over de prostitutiekwestie maakte hij zich zelfs een enkele keer behoorlijk boos op de abolitionisten die hem verweten dat hij in zijn pleidooien voor het reglementarisme geen rekening hield met de moraal. Hij weerlegde dit verwijt door aan de term 'moraal' een ruimere betekenis toe te kennen: Je proteste contre cette allégation. Seulement, il y a plusieurs interprétations de cette loi morale dans l'ordre relatif où nous vivons. Nous ne méconnaissons pas la morale indivi- duelle, mais nous affirmons l'existence d'une morale nationale. Nous croyons que la visite d'une prostituée est une immoralité infiniment moindre que l'empoissonement de la race101. etithan toonde zich in zijn bijdragen voor de Archives over alcoholisme en geslachts- 422 Liesbet Nys ziekten meermaals erg bezorgd om de toekomst van het ras. In 1863 al achtte hij voorlichtingsconferenties over de geslachtsziekten in het leger gewenst 'pour la beauté et la prospérité de la race humaine'102 en in november 1882 beweerde hij dat het (on- danks de genomen maatregelen) nog altijd hoge aantal geslachtsziekten in het leger ervoor verantwoordelijk was dat 'chaque année l'armée verse dans la nation une cause puissante d'affaiblissement et de dégénérescence de la race'103. Als er niet dringend bijkomende maatregelen tegen alcoholisme en venerische ziekten in het leger zouden worden genomen, dan was volgens Petithan het 'Belgische ras' gedoemd te verdwijnen. In 1889 sloeg hij in de Société royale de médecine publique du royaume de Belgique een luide alarmkreet: 150,000 tuberculeux, 100,000 al-coolisés, 50,000 syphilitiques, 50,000 autres affections constitutionnelles sur une popu-lation de 2 millions de citoyens propres à la procréation, voilà l'état de la race à ce point de vue! Et notons que tous ces vices constitutionnels marchent en progression géométrique, car il est peu de ceux qui en sont atteints qui aient l'honnêteté de reculer devant la transmission de leurs maux à leurs descendants. N'ai je pas raison de dire que notre race est profondément malade et qu'il est temps de songer à la guérir104! Petithan rekende naast het alcoholisme en de geslachtsziekten nog vele andere aandoeningen tot de oorzaken van degeneratie, maar in het leger was zijn aandacht in de eerste plaats op het drank- en het venerisch probleem gericht. 105 Over alcoholisme en degeneratie: J. Borel, Du concept de dégénérescence à la notion d'alcoolisme dans la médecine contemporaine. Les campagnes antialcooliques de 1865 à 1965 (Montpellier, 1968); C. Finzen, Der Alkoholismus als Problem der Degeneration um die Jahrhundertwende (Kiel, 1977); F. Bynum, 04 Ch. Petithan, 'La dégénérescence de la race belge, ses causes et ses remèdes', Bulletin de la sociét oyale de médecine publique du royaume de Belgique, VII (september 1889) 66. 102 Petithan, 'Note sur le traitement de l'uréthrite', 229. 03 Petithan, 'Prophylaxie des maladies vénériennes', 352-353. 'Alcoholism and degeneration in nineteenth-century European medicine and psychiatry', British journal of addiction, LXX1X (1984) 59-70; Nourrisson, Alcoolisme et antialcoolisme en France, 326-332; J. C. van der Stel, Drinken, drank en dronkenschap. Vijf eeuwen drankbestrijding en alcoholhulpverlening in Nederland (Hilversum, 1995) 199-201. Voorde geschiedenis van het degeneratiebegrip in het algemeen: R. Nye, 'Degeneration and the medical model of cultural crisis in the French Belle Epoque', in: S. Drescher, D. Sabean, A. Sharlin, ed., Political symbolism in modern Europe. Essays in honor of George L. Mosse (New Brunswick, Londen, 1982) 19-41 ; J. E. Chamberlin, S. L. Gilman, ed., Degeneration. The dark side of progress (New York, 1985); D. Pick, Faces of degeneration. A European disorder, c. 1848-1918 (Cambridge, 1989); A. Carol, Histoire de l'eugénisme en France. Les médecins et la procréation XlXe- XXe siècle (Parijs, 1995) 87-114. Over het degeneratiedenken in België: G. Bustraen, 'Degeneratiedenken in de Belgische criminele antropologie' (Onuitgegeven licentiaatsverhandeling KU Leuven; Leuven, 1997); L. Beyers, 'Rasdenken tussen geneeskunde en natuurwetenschap. Emile Houzé en de Société d'anthro- pologie de Bruxelles (1882-1921)', BMGN, CXIV (1999) 481-505. DE REGENERATIE VAN HET RAS Petithan rekende naast het alcoholisme en de geslachtsziekten nog vele andere aandoeningen tot de oorzaken van degeneratie, maar in het leger was zijn aandacht in de eerste plaats op het drank- en het venerisch probleem gericht. Petithan stond met zijn ideeën over een mogelijke degeneratie van het ras ten gevolge van alcoholisme en geslachtsziekten niet alleen. Vele artsen beschouwden drankmis- bruik en venerische ziekten in de tweede helft van de negentiende en in het begin van de twintigste eeuw als belangrijke oorzaken van degeneratie. Hoewel het degene- ratiebegrip eigenlijk zijn oorsprong had in de achttiende-eeuwse natuurgeschiedenis, kende het in de tweede helft van de negentiende eeuw door het werk van de Franse psychiater Benedict Augustin Morel een revival in de psychiatrie en de geneeskunde, en later ook in de populaire literatuur. Morel had in 1857 in zijn Traité des dégéné- rescences physiques, intellectuelles et morales de l'espèce humaine beschreven hoe milieufactoren als voeding en klimaat bij de mens allerhande afwijkingen konden veroorzaken. Die afwijkingen werden erfelijk overgedragen en konden in vier ge- neraties tot steriliteit en uitsterving leiden. Morel zelf had aan het alcoholisme al een gepriviligeerde plaats onder de oorzaken van degeneratie toegewezen. Valentin Magnan en Paul-Maurice Legrain, twee andere toonaangevende Franse psychiaters, versterkten deze tendens nog. Vooral Legrain, die in 1897 de Union française antialcoolique oprichtte, oriënteerde Morels dege- neratietheorie sterk in anti-alcoholische richting105. Naast Petithan lieten nog vele De grote school van de natie 423 andere Belgische militaire geneeskundigen zich in het spoor van deze Franse zenuw- artsen in verband met alcoholisme tot allerhande minder of meer uitgewerkte doem- scenario's verleiden. Zij wezen op de mogelijke fysieke, intellectuele en morele gebre- ken die de nakomelingen van alcoholici konden vertonen en waarschuwden in het bijzonder voor de dramatische gevolgen van conceptie in dronkenschap voor het nageslacht106. Ook probeerden ze de soldaten tot matig drankgebruik of zelfs tot ge- heelonthouding aan te sporen door hen te wijzen op de relatie die tussen drankmis- bruik en waanzin bestond107. Verder waren, zoals gezegd, vele legerartsen ook gefas- cineerd door het verband tussen alcoholisme en crimineel gedrag. De vraag naar de strafrechtelijke verantwoordelijkheid van normale en pathologische dronkaards die in het leger criminele daden hadden gesteld, werd in de Archives meermaals opge- worpen108. Vooral in de periode 1880-1914 dreven legerartsen op alle mogelijke ma- nieren de angst voor de degeneratieve gevolgen van drankmisbruik ten top. 106 Zie bijvoorbeeld V. de Vaucleroy, L'hérédité alcoolique. Conférence faite à l'assemblée générale de la Ligue patriotique contre l'alcoolisme (Brussel, 1893) 11; Lemaire, 'L'Hérédité physiologique et pathologique', Archives, LVII (augustus 1904) 113-114. In enkele studentencursussen die aan de militaire school werden gebruikt, is met betrekking tot het alcoholisme een rudimentaire vorm van de dege- neratietheorie terug te vinden. Zie bijvoorbeeld A. de Marneffe, Cours de physiologie et d'hygiène appliquées à l'éducation physique (Brussel, 1904) 174-175, aanwezig in: Fonds Gaston Dothey, 22.5, Koninklijk museum van het leger en van krijgsgeschiedenis, Brussel. 107 Legerartsen stelden niet alleen een correlatie vast tussen een toegenomen alcoholconsumptie en een stijgend aantal krankzinnigen, maar rekenden ook bepaalde vormen van dronkenschap tot de mentale aandoeningen. In de Archives verschenen vaak gedetailleerde observatieverslagen van soldaten die aan delirium tremens, dipsomanie of andere pathologische vormen van dronkenschap leden. Legerartsen testten diverse behandelingswijzen voor deze psychische kwalen uit. Zie bijvoorbeeld Déchange, 'Observation d'alcoolisme sur aigu; mort par hemorrhagic méningée', Archives, XIII (oktober 1860) 234-236; Pirotte, 'Observation de delirium tremens guéri par la teinture de digitale à haute dose', Archives, XVIII (mei 1865) 330-334; Wilmaers, 'Ivresse anormale', Archives, LV (mei 1902) 301-311. Vgl. Nourrisson, Al- coolisme et antialcoolisme en France, 527-544; C. Quétel, J.-Y. Simon, 'L'aliénation alcoolique en France (XIXe et première moitié du XXe siècle)', Histoire, économie et société, VII (1988) 507-533. 108 Zie bijvoorbeeld De Broeu, 'Alcoolisme et manie ébrieuse', 5-22. Zie ook De Vaucleroy, De l'al- coolisme dans l'armée, 4-5. 110 De erfelijkheid van syfilis werd, naast de besmettelijkheid van de ziekte, in de tweede helft van de negentiende en de eerste helft van de twintigste eeuw vrij algemeen aangenomen. Er bestond wel onenigheid over de vraag of alleen de vader, alleen de moeder of beiden hierin een aandeel konden hebben. Ook bleef lange tijd onduidelijk op welke manier die overdracht precies verliep. Legerarts Delforge beschreef in de Archives bijvoorbeeld een geval van erfelijke syfilis dat volgens hem louter aan 'l'hérédité paternelle' te wijten was. De moeder van het kind met erfelijke syfilis vertoonde — aldus Delforge — nooit enig symptoom van de ziekte. Delforge, 'A propos d'un cas de syphilis héréditaire', Archives, XLI (mei 1888) 319-325. Over de negentiende- en vroeg-twintigste-eeuwse opvattingen over de erfelijkheid van syfilis: E. Lomax, 'Infantile Syphilis as an exemple of nineteenth century belief in the inheritance of acquired characteristics', Journal of the history of medicine and allied sciences, XXXIV (januari 1979) 23-39; A. Corbin, 'L'hérédosyphilis ou l'impossible rédemption. Contribution à l'histoire de l'hérédité morbide', Romantisme. Revue du dix-neuvième siècle, XI (1981) 131-149. 109 Over syfilis en de degeneratie-angst: A. Corbin, 'Le péril vénérien au début du siècle. Prophylaxie sanitaire et prophylaxie morale', in: L. Murard, P. Zylberman, ed., Recherches. L'haleine des faubourgs. Ville, habitat et santé au XIXe siècle (Fontenay-sous-Bois, 1978) 283. Zie ook de al genoemde algemene werken over het degeneratiebegrip. 111 A. Fournier, Syphilis et mariage (Parijs, 1880). Zie ook Quétel, La mal de Naples, 186-198; Carol, Histoire de l'eugénisme, 51-61. 111 A. Fournier, Syphilis et mariage (Parijs, 1880). Zie ook Quétel, La mal de Naples, 186-198; Carol, Histoire de l'eugénisme, 51-61. 112 Verhagen, 'Du traitement de la syphilis', Archives, LX (maart 1907) 168. 112 Verhagen, 'Du traitement de la syphilis', Archives, LX (maart 1907) 168. 111 A. Fournier, Syphilis et mariage (Parijs, 1880). Zie ook Quétel, La mal de Naples, 186 198; Carol, Histoire de l'eugénisme, 51-61. 112 Verhagen, 'Du traitement de la syphilis', Archives, LX (maart 1907) 168. DE REGENERATIE VAN HET RAS De Belgische eugenetische beweging kan het best als een sociaal-hygiënis- tische beweging worden getypeerd, die in opvoeding en moralisering veeleer dan De grote school van de natie 425 in dwang een oplossing zocht voor onder meer het drank- en het venerisch probleem in dwang een oplossing zocht voor onder meer het drank- en het venerisch probleem. Legerarts Albert Govaerts, als secretaris-generaal van de Société belge d'eugénique één van de actiefste propagandisten van de eugenetica in België, maakte het euge- netisch gedachtegoed via de Archives bij zijn collega's legerartsen bekend. Hij trachtte hen ervan te overtuigen dat de eugenetica de militaire geneeskunde erg tot nut kon zijn, bijvoorbeeld bij de rekrutering voor het leger. Govaerts kon alvast rekenen op de steun van de toenmalige inspecteur-generaal van de gezondheidsdienst van het leger Léon Wilmaers. Die gaf in zijn voordrachten en geschriften over de strijd tegen de venerische ziekten in het leger blijk van een grote bekommernis om de kwaliteit van het ras en wees in de Journées internationales d'eugénique van oktober 1922 op de bijdrage die de gezondheidsdienst van het leger, bijvoorbeeld met haar antropo- metrisch onderzoek van soldaten, aan de eugenetica kon leveren113. in dwang een oplossing zocht voor onder meer het drank- en het venerisch probleem. Legerarts Albert Govaerts, als secretaris-generaal van de Société belge d'eugénique één van de actiefste propagandisten van de eugenetica in België, maakte het euge- netisch gedachtegoed via de Archives bij zijn collega's legerartsen bekend. Hij trachtte hen ervan te overtuigen dat de eugenetica de militaire geneeskunde erg tot nut kon zijn, bijvoorbeeld bij de rekrutering voor het leger. Govaerts kon alvast rekenen op de steun van de toenmalige inspecteur-generaal van de gezondheidsdienst van het leger Léon Wilmaers. Die gaf in zijn voordrachten en geschriften over de strijd tegen de venerische ziekten in het leger blijk van een grote bekommernis om de kwaliteit van het ras en wees in de Journées internationales d'eugénique van oktober 1922 op de bijdrage die de gezondheidsdienst van het leger, bijvoorbeeld met haar antropo- metrisch onderzoek van soldaten, aan de eugenetica kon leveren113. In de tweede helft van de negentiende en het begin van de twintigste eeuw toonden de legerartsen zich in hun vertoog over drankmisbruik en venerische ziekten bij soldaten in toenemende mate bezorgd om de toekomst van het ras. Een fysiek verzwakt en moreel verdorven leger zagen zij als symptomatisch voor een nakende degeneratie van de hele maatschappij. 113 Zie A. Govaerts: 'L'Eugénique', Archives, LXXIV (december 1922) 1201-1221. Voor Wilmaers: Wilmaers, 'Lutte antivénérienne à l'armée, 77-95; A. Govaerts, 'Journées internationales d'eugénique', Archives, LXXIV (december 1922) 1271-1274. Voor de eugenetica: D. J. Kevies, In the name of eugenics. Genetics and the uses of human heredity (Harmondswoith, 1986); W. Schneider, Quality and quantity. Eugenics and the biological regeneration of twentieth century France (Cambridge, 1987); J. Noordman, Om de kwaliteit van het nageslacht. Eugenelika in Nederland (1900-1950) (Nijmegen, 1989); W. de Raes, 'Eugenetika in de Belgische medische wereld tijdens het Interbellum', BTNG, XX (1989) 399-464; P. Weindling, Health, race and german politics between national unification and nazism, 1870-1945 (Cambridge, 1989); R. A. Soloway, Demography and degeneration. Eugenics and the declining birthrate in twentieth-century Britain (Chapel Hill, Londen, 1990); Carol, Histoire de l'eugénisme; J.-F. Crombois, 'De eugenetica in België vóór 1914. Het Institut de sociologie Solvay (1902-1914)', in: M. Beyen, G. Vanpaemel, ed., Rasechte wetenschap? Het rasbegrip tussen wetenschap en politiek vóór de Tweede Wereldoorlog (Leuven, Amersfoort, 1998) 31-41. 114 'Circulaire interdisant le débit des boissons alcooliques dans les casernes', JMO (1885) 416. 115 Petithan, 'La dégénérescence de la race belge', 75 DE REGENERATIE VAN HET RAS g g g g p In zijn bijbel van de degeneratie-idee van 1857 rekende Morel de venerische ziekten in tegenstelling tot het alcoholisme duidelijk niet tot de hoofdoorzaken van degeneratie. Het was de Franse veneroloog Alfred Fournier die op het einde van de negentiende eeuw voor het eerst uitdrukkelijk over mogelijke degeneratieprocessen ten gevolge 424 Liesbet Nys van syfilis (en in mindere mate ook gonorroe) sprak. Fournier streefde er vooral naar om met uitvoerige beschrijvingen van de zogenaamde 'hérédo's' — het lichamelijk misvormde en mentaal onderontwikkelde nageslacht van syfilislijders — de angst voor de venerische ziekten op te drijven109. Zijn voorbeeld werd door artsen in verschil- lende landen nagevolgd. In de tweede helft van de negentiende en de eerste helft van de twintigste eeuw verschenen in de medische tijdschriften, ook in de Archives, gere- geld gedetailleerde beschrijvingen van de verschrikkelijke ravages die syfilis bij het nageslacht kon aanrichten110. De vrees voor degeneratie ten gevolge van syfilis bracht Fournier er zelfs toe nauw- keurige richtlijnen op te stellen om te bepalen of een huwelijk van een (ex-)syfilis- patiënt al dan niet aanvaardbaar was: de patiënt mocht minstens drie à vier jaar geen symptomen van de ziekte meer hebben vertoond en moest een volledige behandeling hebben gevolgd111. 'La question du mariage des syphilitiques' was op het einde van de negentiende en in het begin van de twintigste eeuw ook een veelbesproken onder- werp in Belgische medische kringen. Legerarts Verhagen bijvoorbeeld stelde in 1907 in de Archives zijn eigen huwelijksrichtlijnen op. Die moesten in radicaliteit zeker niet onderdoen voor die van Fournier112. De stap van dit soort huwelijksvoorschriften naar de eugenetica was niet groot. De angst voor degeneratie ten gevolge van alcoholisme en venerische ziekten speelde een cruciale rol in de eugenetische beweging die op het einde van de negentiende en in het begin van de twintigste eeuw in het spoor van Francis Galton in verschillende landen tot ontwikkeling kwam. De eugenetica bestudeerde de menselijke erfelijkheid met het oog op maatregelen ter verbetering van de kwaliteit van het nageslacht. De kleinschalige eugenetische beweging die België in het Interbellum kende, hield zich ver van de radicale maatregelen (sterilisatie en dies meer) die bijvoorbeeld door Ameri- kaanse eugenetici voor de preventie van morbide erfelijkheid naar voren werden geschoven. 115 Petithan, 'La dégénérescence de la race belge', 75. 114 'Circulaire interdisant le débit des boissons alcooliques dans les casernes', JMO (1885) 416. 115 Petithan, 'La dégénérescence de la race belge', 75. DE REGENERATIE VAN HET RAS Het leger was voor hen een index, een maatstaf van de nationale vitaliteit. Een regeneratie van het ras kon volgens hen alleen maar in het leger beginnen. Een offensief tegen drankmisbruik en venerische ziekten bij soldaten moest het leger moreel verheffen en omvormen tot 'la grande école nationale'. Voortaan moesten soldaten het leger als herboren verlaten: 'Il faut qu'à l'expiration de leur service, les miliciens soient rendus en leurs foyers moralement et physiquement améliorés114. Zo zouden de in het leger genomen maatregelen tegen alcoholisme en geslachtsziekten ook op de civiele maatschappij een heilzame uitwerking kennen. Legerartsen beschouwden het leger als de voornaamste waarborg voor de fysieke en morele gezondheid van de natie: Officiers et médecins militaires doivent comprendre que le pays favorisera d'autant plus l'armée qu'elle donnera plus de gages de moralité. Telle sera l'armée, telle sera la nation! Tâchons donc, par tous les moyens, d'avoir une armée saine, morale, énergique, pour avoir une Race semblable115.
https://openalex.org/W2123901837	http://uu.diva-portal.org/smash/get/diva2:812307/FULLTEXT01	English	null	Contribution of indoor-generated particles to residential exposure	Atmospheric environment	2,015	cc-by	8,267	a b s t r a c t Article history: Received 4 April 2014 Received in revised form 18 July 2014 Accepted 30 July 2014 Available online 1 August 2014 Keywords: Ultraﬁne particles Indoor sources Exposure Indoor measurements Soot Source strength The majority of airborne particles in residences, when expressed as number concentrations, are gener- ated by the residents themselves, through combustion/thermal related activities. These particles have a considerably smaller diameter than 2.5 mm and, due to the combination of their small size, chemical composition (e.g. soot) and intermittently very high concentrations, should be regarded as having po- tential to cause adverse health effects. In this study, time resolved airborne particle measurements were conducted for seven consecutive days in 22 randomly selected homes in the urban area of Lund in southern Sweden. The main purpose of the study was to analyze the inﬂuence of human activities on the concentration of particles in indoor air. Focus was on number concentrations of particles with diameters <300 nm generated by indoor activities, and how these contribute to the integrated daily residential exposure. Correlations between these par- ticles and soot mass concentration in total dust were also investigated. g It was found that candle burning and activities related to cooking (using a frying pan, oven, toaster, and their combinations) were the major particle sources. The frequency of occurrence of a given concentration indoors and outdoors was compared for ultraﬁne particles. Indoor data was sorted into non-occupancy and occupancy time, and the occupancy time was further divided into non-activity and activity inﬂuenced time. It was found that high levels (above 104 cm3) indoors mainly occur during active periods of occupancy, while the concentration during non- activity inﬂuenced time differs very little from non-occupancy time. Total integrated daily residential exposure of ultraﬁne particles was calculated for 22 homes, the contri- bution from known activities was 66%, from unknown activities 20%, and from background/non-activity 14%. The collected data also allowed for estimates of particle source strengths for speciﬁc activities, and for some activities it was possible to estimate correlations between the number concentration of ultraﬁne particles and the mass concentration of soot in total dust in 10 homes. Particle source strengths (for 7 speciﬁc activities) ranged from 1.6$1012 to 4.5$1012 min1. The correlation between ultraﬁne particles and mass concentration of soot in total dust varied be- tween 0.37 and 0.85, with an average of 0.56 (Pearson correlation coefﬁcient). Contribution of indoor generated particles to residential exposure C. Isaxon a, , A. Gudmundsson a, E.Z. Nordin a, L. L€onnblad b, A. Dahl a, G. Wieslander c, M. Bohgard a, A. Wierzbicka a a Ergonomics and Aerosol Technology, Lund University, 22100 Lund, Sweden b Astronomy and Theoretical Physics, Lund University, Sweden a Ergonomics and Aerosol Technology, Lund University, 22100 Lund, Sweden b Astronomy and Theoretical Physics, Lund University, Sweden c Occupational and Environmental Medicine, Uppsala University, Uppsala, Sweden y y y c Occupational and Environmental Medicine, Uppsala University, Uppsala, Sweden h i g h l i g h t s Several high time resolution instruments were operated for seven days in 22 homes. Concentrations above 104 cm3 almost only occur during active periods of occupancy. Several high time resolution instruments were operated for seven days in 22 homes. Concentrations above 104 cm3 almost only occur during active periods of occupancy. Known and unknown indoor activities were 86% of the total integrated daily residential Several high time resolution instruments were operated for seven days in 22 homes. Concentrations above 104 cm3 almost only occur during active periods of occupancy. Known and unknown indoor activities were 86% of the total integrated daily residential exposure. Source strengths of speciﬁc activities ranged from 1.6$1012 to 4.5$1012 min1. Correlation between UFP and mass conc of soot in total dust was on average 56%. Concentrations above 104 cm 3 almost only occur during active periods of occupancy. Known and unknown indoor activities were 86% of the total integrated daily residential exposure. Source strengths of speciﬁc activities ranged from 1.6$1012 to 4.5$1012 min1. Correlation between UFP and mass conc of soot in total dust was on average 56% Known and unknown indoor activities were 86% of the total integrated daily residential exposure. Source strengths of speciﬁc activities ranged from 1 6$1012 to 4 5$1012 min1 Source strengths of speciﬁc activities ranged from 1.6 10 to 4.5 10 min . Correlation between UFP and mass conc of soot in total dust was on average 56%. Corresponding author. E-mail address: christina.isaxon@design.lth.se (C. Isaxon). Atmospheric Environment 106 (2015) 458e466 Atmospheric Environment 106 (2015) 458e466 * Corresponding author. E-mail address: christina.isaxon@design.lth.se (C. Isaxon). http://dx.doi.org/10.1016/j.atmosenv.2014.07.053 1352-2310/© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 2.1. Measurements and instrumentation Number concentrations, and mean diameter, of ultraﬁne parti- cles were monitored with Mini Diffusion Size Classiﬁer (DiSC) (University of Applied Sciences, Windisch, CH) or Nanotracer (Philips Aerasense). These instruments are based on an electrical measurement technique, with a diffusion charger, by which the average acquired charge of a particle depends on its size, followed by a diffusion stage. This stage consists of a stainless steel grid, where the particles will deposit due to diffusion, connected to electrometers that detect the current carried by the deposited particles. The current is dependent on ﬂow rate, number concen- tration and particle size. Both instruments have an upper size limit of 300 nm. Since over 80% of particles measured by these two in- struments were below 120 nm, and the majority were below 100 nm, with a minimal contribution of particles between 100 and 300 nm, the measurements of the Nanotracer and DiSC can be considered as good approximations of ultraﬁne particles (number concentrations measured with Nanotracer and DiSC will in this article be entitled ultraﬁne particles). The accuracy of both the Nanotracer and DiSC are ±30% (Asbach et al., 2012). It has been shown (Hussein et al., 2006; Wallace, 2006; Turpin et al., 2007 Wierzbicka, 2008) that the major sources contributing to indoor air concentrations are combustion related or related to thermal processes (e.g. cooking, smoking and candle burning) and electric appliances. Peak number concentrations from cooking have been found to be higher than reported outdoor peak concentrations (Dennekamp et al., 2001; He et al., 2004; Wan et al., 2011), by at least an order of magnitude. It is well known that combustion generated particles generally are considerably smaller than 2.5 mm, often smaller than 300 nm (Gehin et al., 2008), which justiﬁes that number concentration of ultraﬁne particles would be a more rele- vant metric than mass concentration to determine residential exposure to combustion-related particles. Ultraﬁne (<100 nm) particles are of special interest from a health perspective, since they, due to their small size, can penetrate deep into the respiratory system and cause inﬂammatory effects (Long et al., 2001). Particles <100 nm also have a higher deposition rate in both the upper air- ways and the alveolar tract, which in itself can cause adverse effects (Hirano, 2009; Araujo et al., 2008; Oberd€orster et al., 2005). 1. Introduction The main aims of this study were to analyze the inﬂuence of human activities on particles in indoor air, with focus on deter- mining the differences between residential and outdoor number concentrations of ultraﬁne particles; to estimate contribution of indoor sources to integrated daily residential exposure; and to check correlation between mass concentration of soot in total dust and number concentration of ultraﬁne particles in residences in general as well as during different activities. In the industrialized part of the world, we spend approximately 65% of our lives in our homes (Leech et al., 2002; Brasche and Bischof, 2005). Hereby we are subjected to various indoor- generated airborne particles as well as to background particles originating from outdoors. It is widely known that outdoor parti- cles contribute to the indoor aerosol concentration levels, and in epidemiology, exposure is often determined based on the outdoor particle concentration. However the outdoor contribution to the indoor aerosol size distribution is modiﬁed compared to the dis- tribution outdoors, due to size-speciﬁc differences in penetration efﬁciency (Thatcher et al., 2003; Liu and Nazaroff, 2003; Nazaroff, 2004). Accumulation mode particles have the highest penetration (Nazaroff, 2004) since these particles have a rather low diffusivity and are too small to be signiﬁcantly affected by sedimentation or impaction. To a far greater extent than outdoor generated particles, the particle concentration and size distribution indoors are domi- nated by particles generated by the residents' activities. For natu- rally ventilated buildings, Morawska and Salthammer (2003) summarized indoor/outdoor ratios (I/O) for PM10 and PM2.5 (par- ticulate matter smaller than 10 mm and 2.5 mm, respectively) from 0.5 to 0.98 and 0.54 to 1.08, respectively, in the absence of indoor sources. However, when indoor sources were present, I/O ratios for PM10 and PM2.5 ranged from 1.14 to 3.91 and 1 to 2.4, respectively, which stress the signiﬁcance of indoor source contributions (Morawska et al., 2013). a b s t r a c t This study clearly shows that due to the importance of indoor sources, residential exposure to ultraﬁne particles cannot be characterized by ambient measurements alone. © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 459 polycyclic aromatic hydrocarbons (PAH) (Pagels et al., 2009), but are of health interest also due to their small size and because of the large surface area available for adsorption of gases and other par- ticle phase toxins. Since the major sources of ultraﬁne particles indoors have been shown to be combustion/thermal related, it is likely that soot is a major component of this aerosol and it is therefore of interest to monitor the soot concentration indoors. The importance of the potential health effects of indoor generated soot has been pointed out by e.g. Buonanno et al. (2013). Monitoring soot concentrations in parallel to ultraﬁne particle number con- centrations allows determination of their correlation, and, hypo- thetically, as a consequence gives a possibility of predicting the soot component on the basis of monitoring ultraﬁne particles. 2. Methods For seven consecutive days, time resolved stationary air mea- surements were conducted in 22 randomly selected homes in the urban area of Lund in southern Sweden. The measurements were conducted during wintertime. Home characteristics, including ventilation type, air exchange rate, volume, indoor temperature and RH, and instruments used are summarized in Table 1. Initially measurements were conducted in 39 homes; however mainly due to technical reasons (see data processing for details) results from only 22 homes were included in this article. The habitants ﬁlled in detailed activity log books specifying conducted activities and made notes of when they were present or absent in the dwelling. Table 1 Table 1 Homes and measurement characteristics including measurement duration, temperature, RH and used instruments. Type of home Measurement duration [days] AER [h1] Vent. Volume [m3] Temp (C) mean (std) RH (%) mean (std) Use of Nanotracer/DiSCa Use of Aethb Use of IAQc 01 House 6.12 0.4 A 293 22.0 (0.4) 47 (3) Yes Yes No 02 House 6.10 0.7 C 350 22.0 (0.4) 47 (3) Yes No No 03 Apartm. 7.26 0.4 D 250 23.6 (0.6) 35 (3) Yes Yes No 04 House 6.95 N/A A N/A 22.4 (0.4) 47 (3) Yes No No 05 Apartm. 2.42 1.6 B 168 21.9 (1.5) 37 (3) Yes Yes No 06 Apartm. 7.14 1.5 A 96 21.8 (0.9) 38 (4) Yes Yes No 07 Apartm. 6.59 1.4 C 227 23.4 (0.6) 36 (4) Yes No No 08 House 6.50 2.3 C 327 23.5 (0.5) 37 (2) Yes No No 09 Apartm. 6.30 1.0 B 202 22.9 (0.3) 34 (4) Yes No Yes 10 Apartm. 6.64 2.9 A N/A N/A N/A Yes No No 11 Apartm. 6.82 1.1 D 118 20.7 (0.4) 43 (4) Yes No Yes 12 House 6.61 1.0 C 288 22.8 (0.7) 35 (2) Yes Yes No 13 House 6.55 1.0 N/A N/A 22.3 (0.6) 43 (2) Yes Yes No 14 Apartm. 7.10 0.3 A 195 22.3 (0.4) 38 (3) Yes No Yes 15 Apartm. 5.69 0.4 B 132 N/A N/A Yes No No 16 Apartm. 8.95 1.4 A 208 23.4 (1.4) 25 (2) Yes Yes Yes 17 House 6.33 N/A A 442 23.3 (0.9) 26 (2) Yes Yes Yes 18 House 6.25 N/A C N/A 22.3 (1.0) 26 (2) Yes No Yes 19 Apartm. 6.92 N/A A N/A 25.2 (1.6) 28 (5) Yes Yes No 20 Apartm. 7.01 N/A C N/A 23.0 (1.3) 30 (5) Yes No Yes 21 House 7.00 N/A A 328 21.8 (1.3) 35 (4) Yes No No 22 House 6.62 N/A N/A N/A N/A N/A Yes Yes No A) Additional exhaust mechanical ventilation in kitchen only (kitchen exhaust hood). B) Additional exhaust mechanical ventilation in kitchen and bathroom(s). C) Mechanical exhaust air in kitchen and bathroom(s) and fresh air supply valve ventilation in other rooms. D) Natural ventilation. a Instruments measuring number concentration of particles <300 nm. b Instrument measuring mass concentration of black carbon. c Instrument measuring number (and mass) concentration of particles 0.3e10 mm. A thorough examination of each home e e.g. Table 1 construction year, ﬂoor and wall materials, and ventilation system e was conducted according to a structured protocol. In addition, the participants ﬁlled in a translated and modiﬁed version of the IUATLD (Interna- tional Union Against Tuberculosis and Lung Disease) questionnaire (Burney and Chinn, 1987; Burney et al., 1989) covering both the characteristics of the dwelling and the residents and their everyday habits. The results of these examinations will be presented elsewhere. related) activities. The soot monitor measures the transmission of infrared (IR) light (880 nm) through a ﬁlter, which is continuously loaded with airborne particles by the suction of an internal pump. The accumulation of particles on the ﬁlter over time increases the absorbance, which is calculated relative to a reference cell. The attenuation IR is then transferred to mass concentration of black carbon (described in detail by Cheng and Lin (2013)). related) activities. The soot monitor measures the transmission of infrared (IR) light (880 nm) through a ﬁlter, which is continuously loaded with airborne particles by the suction of an internal pump. The accumulation of particles on the ﬁlter over time increases the absorbance, which is calculated relative to a reference cell. The attenuation IR is then transferred to mass concentration of black carbon (described in detail by Cheng and Lin (2013)). Temperature and relative humidity were logged (USB-500, Measurement Computing). All measurements were made during off-pollen season (OctobereApril). The instruments were zero- calibrated before each home, and placed between 1 and 1.5 m above the ﬂoor centrally in the dwelling, but not in, or in direct connection to, the kitchen, on a rack of shelves. The rack was constructed for occupying a minimum of space so that the in- struments could be placed at a central spot without being in the way of daily living. 2.1. Measurements and instrumentation Studies (Wang et al., 2009; Kennedy, 2007) have shown that the number concentration of ultraﬁne particles induce more adverse health effects than the mass concentrations of PM10 and PM2.5 (to which the ultraﬁne particles are a poor contributor). The measurements of particles larger than 0.3 mm were per- formed by an optical particle counter, Indoor Air Quality monitor (IAQ3016, Lighthouse). Particles passing a laser beam inside the instrument scatter light. A photo detector converts the scattered light to an electrical pulse. For a single particle, the light intensity of the generated pulse depends on the size, refractive index and color of the particle. According to a reference aerosol of monodisperse PSL particles, the pulse height is transferred to six particle size intervals. The pulse intensity thresholds in the IAQ3016 correspond to particle sizes of 0.3, 0.5, 1.0, 2.5, 5.0 and 10 mm. Soot (black or elemental carbon), is a primary component in several emissions known to affect human health indoors, such as wood burning, cigarette smoke, cooking with poor ventilation but also e.g. diesel exhaust, which can inﬁltrate the building from outdoors. Soot particles are often carriers of elements such as Black carbon (soot) levels were monitored by microAeth (AE51, Magee Scientiﬁc) in 10 of the homes, to investigate the correlation between soot measurements and indoor (particularly combustion C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 460 Table 2 The total daily integrated residential exposure was calculated for pe- riods of activities and periods of non-activities. Candle burning and activities related to cooking (using a frying pan, oven, toaster, and their combinations) were the major particle sources. The elevated concentrations of ultraﬁne particles when cleaning (i.e. dusting, vacuum cleaning and mopping) are likely to be originating from the motor of the vacuum cleaner (Lioy et al., 1999; Afshari et al., 2005). Increased indoor concentration due to opening a window cannot be considered as an indoor source, but is in this case given for comparative purposes. Due to that the Indoor Air Quality monitor reported ﬂow problems in several cases, data from only 9 homes (of which 7 matched the abovementioned 22 homes), where the instrument ran without complications for the whole measurement duration, could be evaluated. This data was sorted into occupancy time and non-occupancy time. The six channels were then compressed to PM1e0.3 and PM10e1, since these fractions match the accumulation mode and the coarse mode fairly well (Lundgren and Burton, 1995; Morawska et al., 2008). The contribution of ultraﬁne particles due to activities was further studied by comparing the activity inﬂuenced time data to the outdoor (ambient) concentrations of ultraﬁne particles as extracted from the SMPS measurements. Due to that the SMPS and the Nanotracer/DiSC have very different time resolution, it is difﬁcult to visualize them in the same ﬁgure (the number of mea- surement points from the SMPS is much lower from the Nano- tracer/DiSC). This was taken care of by normalizing the data, i.e. the concentrations were divided by the number of concentration measurements done by the SMPS and the Nanotracers, respectively. Data was sorted into concentration bins and the frequency of a given concentration was studied. The resulting ﬁgure shows the normalized frequency of a given concentration to occur (y-axis) as a function of concentrations (x-axis). From Fig. 2a and b it is obvious that levels of 104 cm3 indoors almost only occur during human presence, which illustrates the huge inﬂuence residents have on the air at home. When number concentration data is plotted separately for non-activity inﬂuenced periods and activity inﬂu- enced periods (Fig. 3a and b) the impact of the residents' activities on number concentration of ultraﬁne particles is even more apparent. The indoor air particle concentration during non-activity inﬂuenced periods differs very little from non-occupancy time. Table 2 Table 2 Source strengths, S, estimated for 7 activities. Activity Number of peaks Peak concentration [#/cm3] (std) Mean S, [#/min] (std) Boiling 11 5.6$104 (3.5$104) 1.7$1012 (1.4$1012) Frying 8 1.4$104 (1.3$104) 1.6$1012 (1.7$1012) Oven 10 2.3$105 (1.2$105) 2.4$1012 (2.6$1012) Hairspray 3 2.4$104 (1.3$104) 3.1$1012 (1.4$1012) Cleaning 3 2.8$105 (9.3$104) 2.4$1012 (1.7$1012) Laundry 1 2.5$104 4.5$1012 Toaster 3 1.6$105 (1.2$105) 1.9$1012 (1.3$1011) S ¼ Cmax V t where S is the source strength or emission rate (particles/min), Cmax is the maximum concentration (cm3), V is the mixing volume (cm3) and t is the time (min) during which a source is on. Activity logs did not provide information of when a source was turned off; instead t was approximated to the time from a start in concentra- tion elevation to the corresponding peak concentration was reached. Everything which was elevated above the baseline, was possible to identify from the activity logs and did not overlap with other peaks, was counted as a peak. It was assumed that the entire volume of the house/apartment was a single well mixed volume. where S is the source strength or emission rate (particles/min), Cmax is the maximum concentration (cm3), V is the mixing volume (cm3) and t is the time (min) during which a source is on. Activity logs did not provide information of when a source was turned off; instead t was approximated to the time from a start in concentra- tion elevation to the corresponding peak concentration was reached. Everything which was elevated above the baseline, was possible to identify from the activity logs and did not overlap with other peaks, was counted as a peak. It was assumed that the entire volume of the house/apartment was a single well mixed volume. Several of the periods with elevated concentration were results of a combination of two or more particle generating activities. 13 different single activity types, shown in Fig. 1, could however be identiﬁed as contributing signiﬁcantly to the indoor air particle concentration. From the particle number concentration data, the integrated resi- dential exposure was calculated by integrating the concentration over time (particles/cm3 h). The total integrated daily residential exposure was calculated by dividing the integrated residential exposure by the measurement period (in days)resulting in units of particles percm3$h/ day (Bhangar et al., 2011; Mullen et al., 2011; Wallace and Ott, 2011). Table 2 ) In 10 of the 22 houses mentioned above, soot data from microAeth was analyzed. When the sampling time is short and/or the soot concentration is low, noise from the instrument can cause attenuation values to be unchanged or even decline, from one period to the next. This may result in an erroneously low value at one time followed by an erroneously high value at the next (Cheng and Lin, 2013). Noise obstructs the actual variations in soot con- centrations and makes comparison of the microAeth data and the data from Nanotracer and DiSC difﬁcult. A noise-reduction aver- aging (ONA) algorithm based on a user-speciﬁed minimum change in attenuation over a time interval, developed and described in detail by Hagler et al. (2011) and evaluated by Cheng and Lin (2013) was used to eliminate the negative values and reduce the noise, while maintaining high time resolution. The particle source strengths ranged from 1.6$1012 to 4.5$1012 min1, which is in accordance with the higher range of emission rates reported by e.g. Wallace and Ott (2011). In some studies (e.g. He et al., 2004) lower source strengths were reported. 2.2. Data processing In total, data measured in 24 homes is presented here. Initially measurements were conducted in 39 homes, however three of these residences were student dormitories, which were not considered representative of common home settings and will be presented elsewhere. Further reduction of presented results to 24 homes is due to technical problems with the instruments. Ambient (outdoor) number concentrations were monitored constantly by a scanning mobility particle sizer (SMPS, consisting of a TSI 3071 Differential Mobility Analyzer, DMA and a TSI 3010 Condensation Particle Counter, CPC) from a station in northern Lund. All homes were located within 5 km radius from the ambient monitoring station. Nanotracer or DiSC data, without reporting any problems, were collected in 22 homes and analyzed in this article (see details in Table 1). A classiﬁcation of the ultraﬁne particle number concentration measurements was carried out by sorting the data into two cate- gories: occupancy time (at least one person present in the home) and non-occupancy time. The known and unknown activity inﬂu- enced concentrations are identiﬁed as apparent concentration ele- vations from baseline concentration, which could, respectively could not, be identiﬁed by the residents' notes in the activity logs. The activity inﬂuenced period was deﬁned as starting at the time when concentrations started to increase and ending at the time when concentrations had returned to baseline, thus it does not represent the duration of given activity but rather the periods of elevated concentrations being the effect of it. During non-activity inﬂuenced periods, it was assumed that the particles indoors origi- nate mainly from outdoor sources. g CO2 data was collected in one bedroom in each residence. CO2 concentration was measured with CARBOCAP CO2 monitors (GMW22, Vaisala, Finland) connected to a HOBO12-012 data log- gers (Onset Computer Corp., USA) to record the measured values. CO2 levels were used for estimation of air exchange rates on the basis of occupant-generated CO2 mass balance method as described by Bek€o et al. (2010). The method is based on analyzing steady state concentrations or buildups and decays of CO2 in bed rooms. In some cases the bedroom design was such that no obvious build-up occurred (e.g. no door, open window, bed placed in living room), and in a few cases it was not possible to estimate the bedroom volume (open space apartment). In total, it was possible to estimate air exchange rates in 15 homes. C. Isaxon et al. 2.2. Data processing / Atmospheric Environment 106 (2015) 458e466 461 Table 2 Source strengths, S, estimated for 7 activities. Approximate source strengths, S, of ultraﬁne particles were estimated (Table 2) for 7 of the 13 activity types in Fig. 1, using a method described by Wallace and Ott (2011): 3.2. Comparison with number concentration of accumulation and coarse mode particles The data analysis was focused on the results from the ultraﬁne particle measurements by the Nanotracer and DiSC. Occupancy time occurred during an average of 77% of the measurement period. The geometric mean of the concentration of ultraﬁne particles during occupancy time was 4500 cm3, with a lower quartile of 1200 and an upper quartile of 17 000 cm3 (average ± standard deviation: 20 500 ± 99 000 cm3), and during non-occupancy time 2400 [1200, 4800] cm3 (average ± standard deviation: 3200 ± 5100 cm3). The origins of elevated concentra- tions were identiﬁed using the activity logs from the 22 homes in Table 1. Activity inﬂuenced periods comprised on average 33% of the occupancy time. The results of Indoor Air Quality monitor measurements of mass concentration of particles larger than 300 nm, together with the particle number concentrations measured with Nanotracer or DiSC devices are shown in Fig. 4. Mass concentrations were obtained with assumption of a particle density of 2500 g/cm3 (proxy for ambient particles). Median PM10e1 (corresponding to coarse mode) mass concentration was 17.9 [4.8, 49.9] mg/m3 during occupancy time and was about 6 times higher than during non-occupancy time 3.3 [2.0, 9.2] mg/m3. The median PM1e0.3 (accumulation C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 462 Fig. 1. Summary of ultraﬁne particle number concentrations from various activity inﬂuenced periods. The boxes denote the quartiles, the horizontal line the medians and the whiskers maximum and minimum concentrations. N is the number of identiﬁed periods the calculations are based on. Fig. 1. Summary of ultraﬁne particle number concentrations from various activity inﬂuenced periods. The boxes denote the quartil whiskers maximum and minimum concentrations. N is the number of identiﬁed periods the calculations are based on. ber concentrations from various activity inﬂuenced periods. The boxes denote the quartiles, the horizontal line the medians and the trations. N is the number of identiﬁed periods the calculations are based on. Fig. 1. Summary of ultraﬁne particle number concentrations from various activity inﬂuenced periods. The boxes denote the quartiles, the horizontal line the medians and the whiskers maximum and minimum concentrations. N is the number of identiﬁed periods the calculations are based on. particle mass (due to their size) and this is the reason why authors decided to present the values as mass concentration in Fig. 4. 3.3. Total integrated daily residential exposure where Xi and Yi are the logarithms of the values from the Nano- tracer/DiSC and microAeth respectively, n is the number of data points in one set, and X and Y (sX and sY) are the corresponding means (standard deviations). Fig. 5 shows contribution of known and unknown activities and background/non-activities to the average total integrated daily exposure for 22 residences. The average total integrated daily exposure was 4.0$105 ± 3.5$105 cm3 h/d, median total integrated daily exposure was 2.7$105 cm3 h/d. Of this, the contribution from known activities was 2.7$105 ± 2.6$105 cm3 h/d (66%) (median 1.8$105 cm3 h/d), from unknown activities 1.1$105 ± 1.9$105 cm3 h/d (20%) (median 2.9$104 cm3 h/d), and from background/non-activity 3.6$104 ± 1.9$104 cm3 h/d (14%) (median 3.2$104 cm3 h/d). The correlation between ultraﬁne particles and mass concen- tration of soot in total dust in the 10 homes varied between 0.37 and 0.85, with an average of 0.56. In 3 homes, the correlations were higher than 0.60, in these homes a majority of the activities con- sisted of cooking, candle burning (in two of them) and cigarette smoking (in one). The correlations of the instruments for identiﬁed activities were checked and are summarized in Table 3. The activities for which correlation is the highest coincide with activities which generate the highest number concentrations of ultraﬁne particles (Fig. 1). Soot mass concentration measurements correlate to measurements of ultraﬁne particles above 0.60 in the case of smoking cigarettes, burning candles, and cleaning. The relatively high correlation in the case of cleaning (0.64) is most likely due to the particles emitted from the engine of the vacuum cleaner. Correlation was also found for using microwave (0.61 ± 0.11) based on two events occurring in the same home. Due to that this may have resulted from a mal- functioning microwave it is not included in Table 3. Three other events (toaster, laundry and printer, with correlations of 0.56, 0.46 and 0.38, respectively) only occurred once, and are hence left out from Table 3. Total integrated daily residential exposure was calculated for the different known activities, to determine their speciﬁc contribution in each home. In the 22 homes overall, cooking related activities (using a frying pan, boiling oven, micro, toaster and their combi- nations) contributed to the total daily integrated exposure with 31% and candles with 26%. 3.3. Total integrated daily residential exposure In 6 of the homes, the contribution from candles were more than 60%, and in two cases candles contribution reached 80%. Candles have been reported earlier as signiﬁcant contributors to high particle levels indoors (Bek€o et al., 2013), especially during winter time in northern Europe. 3.2. Comparison with number concentration of accumulation and coarse mode particles mode) mass concentration showed less differences: 8.0 [4.3, 14.3] mg/m3 during occupancy time and 5.2 [2.9, 9.0] mg/m3 during non- occupancy time. The number concentrations of PM10e0.3 were very low with median 22 [12, 61] cm3 during occupancy time and 14 [10, 33] cm3 during non-occupancy time. However, despite their low number concentration they contribute considerably to the Generally, concentrations during occupancy time for accumu- lation mode, coarse mode and ultraﬁne were higher in comparison to non-occupancy time. Higher concentrations of coarse mode during occupancy time may reﬂect resuspension of settled dust Fig. 2. Occurrence of ultraﬁne particle number concentrations outdoor (transparent) and indoors (orange) when no one is present in the residences (a) and when at least one person is present (b). Measurement data are unit normalized. Fig. 3. Occurrence of ultraﬁne particle number concentrations outdoor (transparent) and indoors (orange) during non-activity inﬂuenced periods and activity inﬂuenced periods (i.e. times with elevated concentrations including decay of peaks after the activity ceased). Measurement data are unit normalized. Fig. 3. Occurrence of ultraﬁne particle number concentrations outdoor (transparent) and indoors (orange) during non-activity inﬂuenced periods and activity inﬂuenced periods (i.e. times with elevated concentrations including decay of peaks after the activity ceased). Measurement data are unit normalized. Fig. 2. Occurrence of ultraﬁne particle number concentrations outdoor (transparent) and indoors (orange) when no one is present in the residences (a) and when at least one person is present (b). Measurement data are unit normalized. C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 463 Fig. 4. Boxplots of particle mass concentrations (PM1e0.3 and PM10e1) (left) and particle <300 nm number concentration (right) during occupancy and non-occupancy time. The boxes denote the quartiles, the horizontal line the medians and the whiskers maximum and minimum concentrations. Fig. 4. Boxplots of particle mass concentrations (PM1e0.3 and PM10e1) (left) and particle <300 nm number concentration (right) during occupancy and non-occupancy time. The boxes denote the quartiles, the horizontal line the medians and the whiskers maximum and minimum concentrations. indoors, while higher number concentrations of ultraﬁne particles reﬂect the (mainly combustion related) activities of the residents. r ¼ 1 n 1 X n i¼1 Xi X sX ! Yi Y sY ! 3.4. Correlation between soot concentration and number concentration of ultraﬁne particles The calculated correlations suggest that a signiﬁcant part of the soot indoors originate from outdoors, however, a limitation that should be considered is the different size ranges covered by the two instruments. Soot indoors may originate from outdoors and/or from com- bustion related activities indoors. Contribution of indoor sources to soot levels indoors remains not fully understood. By correlating data from microAeth (measures mass concentration of soot in total dust) and ultraﬁne particle number concentration data from Nanotracer and DiSC, this was investigated. 3.5. Limitations Using the ONA algorithm (Hagler et al., 2011) to reduce the noise in the soot data, shown in Fig. 6a, enabled comparison between the two instruments, as seen in Fig. 6b. It is crucial to be able to identify and characterize the sources. In this study, this has been done by asking the residents to ﬁll in detailed activity log books. These documents are not fully reliable (difﬁcult to interpret, lack of consistency, parts not ﬁlled in etc.), thus they are rather a blunt means of gathering important infor- mation, and probably the major limitation of this study. For future studies, use of some other way of source identiﬁcation, such as sensors giving information about operation of stove, oven, toaster, window, door opening, maybe in combination with GPS equipment worn by occupants allowing accurate location determination As shown in Fig. 6b, a majority of the peaks identiﬁed in the Nanotracer data could be identiﬁed also in the microAeth data, suggesting that the major part of the soot in this home was actually generated indoors. Correlation factors were calculated for 10 homes by ﬁrst calculating averages of the Nanotracer/DiSC data (data sampled every 16 s) to match the data of the microAeth (data sampled every 60 s). Correlation coefﬁcients, r, between the two data sets were then calculated as: Fig. 6. Non-post-processed microAeth data (red) and Nanotracer data (blue) from one of the studied residences (a). Post-processed microAeth data (red) and Nanotracer data (blue) from the same residence (b). vironment 106 (2015) 458e466 C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 464 (occupancy/non-occupancy time), could be recommended. If all activities (or at least the major part) could be known (see Fig. 5) the concentrations inside the residences could be modeled based on information of the resident's living habits, considering that the major contribution to observed concentrations in residences is inﬂuenced by the habitant's activities. (occupancy/non-occupancy time), could be recommended. If all activities (or at least the major part) could be known (see Fig. 5) the concentrations inside the residences could be modeled based on information of the resident's living habits, considering that the major contribution to observed concentrations in residences is inﬂuenced by the habitant's activities. y Another limitation of the study is the accuracy of used in- struments. Asbach et al. 3.5. Limitations reported that the Nanotracer over- estimated particle number concentrations by a factor of ~7 for di(2- ethylhexyl) sebacate particles with a modal diameter of 180 nm, which is above the instrument's upper range for the mean diameter (120 nm). However, chamber experiments with candle smoke (resulting in the highest concentrations measured in this study) proved the opposite, i.e. underestimation of measured concentra- tion by Nanotracer for the sizes between 20 and 120 nm (Bek€o et al., 2013). Only 20% of measurement points in this study showed measured average diameter above 120 nm (mainly occurring dur- ing non-activity time), thus it is believed that presented results are not overestimated more than the assessed instrumental accuracy i.e. 30%. One has to bear in mind that the comparison between the aethalometer and Nanotracer measures two different size ranges and two different particle properties. We know from outdoor studies that a majority of soot is in the ultraﬁne particle range, which was the basis of the assumption that the two instruments could be worthwhile comparing. The correlation showed that soot also, but not exclusively, originate from indoor sources. 4. Conclusions Due to the importance of indoor sources, residential exposure to ultraﬁne particles cannot be characterized by ambient measure- ments alone, which has been clearly shown in this study. Indoor sources are of a transient nature. They quickly generate high peaks in concentration which decay at a rate mainly determined by the air Fig. 6. Non-post-processed microAeth data (red) and Nanotracer data (blue) from one of the studied residences (a). Post-processed microAeth data (red) and Nanotracer data (blue) from the same residence (b). Fig. 5. Total integrated daily residential exposure for 22 residences and the contri- bution of time inﬂuenced by known and unknown activities and background/non- activity. exchange rate and deposition on interior surfaces (and, in cases of number concentrations higher than 10 000e20 000 #/cm3, by coagulation). It is not fully understood whether it is the long term mean exposure or the events with elevated concentrations that has the greatest impact on human health. Until this has been further investigated and determined, it is of importance to gather exposure data based on high time resolution instruments. Comparison of ultraﬁne particle number concentration frequency distributions for 22 studied homes showed that outdoor concentrations can be used to describe concentrations in residences only during the time when occupants are not at home (which is not interesting from the exposure assessment perspective) or when they are not involved in any activities. Concentrations indoors during times inﬂuenced by activities, which comprise 33% of time spent at home, are Table 3 Correlation between number concentrations of ultraﬁne particles and mass con- centration of soot in total dust during speciﬁc activities. Activity Number of events Mean correlation ± standard deviation (%) Cigarettes 2 0.74 ± 0.0 9 Candles 13 0.64 ± 0.26 Cleaning 6 0.64 ± 0.27 Frying 7 0.49 ± 0.49 Oven 6 0.46 ± 0.36 Open window 4 0.44 ± 0.38 Boiling 14 0.36 ± 0.35 Table 3 l Table 3 Correlation between number concentrations of ultraﬁne particles and mass con- centration of soot in total dust during speciﬁc activities. Fig. 5. Total integrated daily residential exposure for 22 residences and the contri- bution of time inﬂuenced by known and unknown activities and background/non- activity. C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 465 Bek€o, G., Lund, T., Nors, F., Toftum, J., Clausen, G., 2010. Ventilation rates in the bedrooms of 500 Danish children. Build. Environ. 45, 2289e2295. signiﬁcantly different (p < 0.01). In epidemiological studies, it is not uncommon to estimate the exposure of a population based on measurements from one outdoor station. If this approach would be used in the case of the homes studied here, the exposure estimation would only be approximately relevant when no activities at all are conducted at home (see Fig. 3a), i.e. mainly when people are asleep. Bek€o, G., Weschler, C.J., Wierzbicka, A., Karottki, D.G., Toftum, J., Loft, S., Clausen, G., 2013. Ultraﬁne particles: exposure and source apportionment in 56 Danish homes. Environ. Sci. Technol. 47 (18), 10240e10248. 2013. Ultraﬁne particles: exposure and source apportionment in 56 Danish homes. Environ. Sci. Technol. 47 (18), 10240e10248. Bhangar, S., Mullen, N.A., Hering, S.V., Kreisberg, N.M., Nazaroff, W.W., 2011. Ul- traﬁne particle concentrations and exposures in seven residences in northern California. Indoor Air 21, 132e144. Integrated daily residential exposure of particles <300 nm was on average 4.0$105 ± 3.5$105 cm3 h/d, where known indoor ac- tivities contributed with 66% to the integrated daily residential exposure, unknown activities 20% and outdoor particles 14%. These numbers are in agreement with results published by Bek€o et al. (2013), Bhangar et al. (2011), and Wallace and Ott (2011). If un- known activities were not included as indoor sources, Bek€o et al. (2013) found a 51% contribution from known activities, and a 65% contribution from known þ unknown activities. The difference between known indoor contribution and unknown indoor contri- bution will depend on how unknown activities are classiﬁed in the different studies. We only included an indoor activity among the known if it was speciﬁcally stated in the activity log. Several of the peaks classiﬁed as “unknown” are however most likely originating from indoor activities. A typical example is a peak occurring at the same time every morning, which is only the ﬁrst couple of days stated as “cooking breakfast”. Table 3 l It should be noted that this study was performed in Northern Europe where outdoor air pollution is relatively low, and during winter time, when it is common to burn candles (which contribute signiﬁcantly to the particle number concentration). Brasche, S., Bischof, W., 2005. Daily time spent indoors in German homes e baseline data for the assessment of indoor exposure of German occupants. Int. J. Hyg. Environ. Health 208, 247e253. Buonanno, G., Stabile, L., Morawska, L., Russi, A., 2013. Children exposure assess- ment to ultraﬁne particles and black carbon: the role of transport and cooking activities. Atmos. Environ. 79, 53e58. Burney, P.G., Chinn, S., 1987. Developing a new questionnaire for measuring the prevalence and distribution of asthma. Chest J. 91 (6), 79Se83S. Burney, P.G., Laitinen, L.A., Perdrizet, S., Huckauf, H., Tattersﬁeld, A.E., Chinn, S., Poisson, N., Heeren, A., Britton, J.R., Jones, T., 1989. Validity and repeatability of the IUATLD (1984) Bronchial Symptoms Questionnaire: an international com- parison. Eur. Respir. J. 2 (10), 940e945. Cheng, Y.-H., Lin, M.-H., 2013. Real-time performance of the microAeth AE51 and the aerosol loading effects on its measurement results at a trafﬁc site. Aerosol Air Qual. Res. 13, 1853e1863. Dennekamp, M., Howarth, S., Dick, C.A.J., Cherrie, J.W., Donaldson, K., Seaton, A., 2001. Ultraﬁne particles and nitrogen oxides generated by gas and electric cooking. Occup. Environ. Med. 58, 511e516. Gehin, E., Ramalho, O., Kirchner, S., 2008. Size distribution and emission rate measurement of ﬁne and ultraﬁne particle from indoor human activities. Atmos. Environ. 42, 8341e8352. Hagler, G.S.W., Yelverton, T.L.B., Vedantham, R., Hansen, A.D.A., Turner, J.R., 2011. Post-processing method to reduce noise while preserving high time resolution in aethalometer real-time black carbon data. Aerosol Air Qual. Res. 11, 539e546. H C M k L Hi hi J Gilb D 2004 C ib i f i d He, C., Morawska, L., Hitchins, J., Gilbert, D., 2004. Contribution from indoor sources to particle number and mass concentrations in residential houses. Atmos. En- viron. 38 (21), 3405e3415. Hirano, S., 2009. A current overview of health effect research on nanoparticles. Environ. Health Prev. Med. 14, 223e225. Based on the correlation calculations of soot and ultraﬁne par- ticles, one cannot draw conclusions that measurements of ultraﬁne particles could be used for predicting soot levels indoors. If there would be only one source present, which generates a known frac- tion of soot, this approach can be relevant. Table 3 l However, in indoor settings, there is a variety of sources present, which can be operated under different conditions from time to time, and from household to household, resulting in very different ultraﬁne particles-to-soot ratios. Often elemental carbon is used as proxy of exposure to diesel exhaust particles. Interestingly enough, this study has shown that indoors a large part of the soot has indoor sources, and clearly elemental carbon should not be treated as a tracer for particles originating from outdoors. Hussein, T., Glytsos, T., Ondracek, J., Dohanyosova, P., Zdimal, V., H€ameri, K., Lazaridis, M., Smolik, J., Kulmala, M., 2006. Particle size characterization and emission rates during indoor activities in a house. Atmos. Environ. 40, 4285e4307. Kennedy, I.M., 2007. The health effects of combustion-generated aerosols. Proc. Combust. Inst. 31, 2757e2770. Leech, J., Nelson, W., Burnett, R., Aaron, S., Raizenne, M., 2002. It's about time: a comparison of Canadian and American time-activity patterns. J. Expo. Anal. Environ. Epidemiol. 12, 427e432. Lioy, P.J., Wainman, T., Zhang, J.J., 1999. Typical household vacuum cleaners: the collection efﬁciency and emissions characteristics for ﬁne particles. J. Air Waste Manag. Assoc. 49, 200e206. Liu, D.L., Nazaroff, W.W., 2003. Particle penetration through building cracks. Aerosol Sci. Technol. 37 (7), 565e573. Long, C., Suh, H., Catalano, P., Koutrakis, P., 2001. Using time- and size-resolved particulate data to quantify indoor penetration and deposition behavior. Envi- ron. Sci. Technol. 35, 4584. Strictly speaking, the results of this study can only represent the 22 households studied during the time period in the winter when the measurements were conducted. The households were chosen from randomly selected addresses in and in the vicinity of Lund. However, only households where inhabitants accepted to be included in the studies became objects of measurements, and it may be possible that people willing to participate in scientiﬁc studies not necessarily represent an average of households in the community. There are, however, other studies that support the general conclusion that periods of various activities in dwellings dominate the sources which cause the major exposures of the in- habitants. More studies are needed in order to be able to generalize on a national, continental or global scale. Lundgren, D.A., Burton, R.M., 1995. Effect of particle size distribution on the cut point between ﬁne and coarse ambient mass fractions. Inhal. Toxicol. 7, 131e148. Morawska, L., Salthammer, T., 2003. Indoor Environment. Airborne Particles and Settled Dust. WILEY-VCH, Weinhem. Table 3 l Morawska, L., Keogh, D.U., Thomas, S.B., Mengersen, K., 2008. Modality in ambient particle size distributions and its potential as a basis for developing air quality regulation. Atmos. Environ. 42, 1617e1628. Morawska, L., Afshari, A., Bae, G.N., Buonanno, G., Chao, C.Y.H., H€anninen, O., Hofmann, W., Isaxon, C., Jayaratne, E.R., Pasanen, P., Salthammer, T., Waring, M., Wierzbicka, A., 2013. Indoor aerosols: from personal exposure to risk assess- ment. Indoor Air 23 (6), 467e487. Mullen, N.A., Liu, C., Zhang, Y., Wang, S., Nazaroff, W.W., 2011. Ultraﬁne particle concentrations and exposures in four high-rise Beijing apartments. Atmos. Environ. 45, 7574e7582. Nazaroff, W.W., 2004. Indoor particle dynamics. Indoor Air 14 (s7), 175e183. Ob d€ t G Ob d€ t E Ob d€ t J 2005 N t i l i Oberd€orster, G., Oberd€orster, E., Oberd€orster, J., 2005. Nanotoxicology: an emerging discipline evolving from studies of ultraﬁne particles. Environ. Health Perspect. 113, 823e839. Afshari, A., Matson, U., Ekberg, L.E., 2005. Characterization of indoor sources of ﬁne and ultraﬁne particles: a study conducted in a full-scale chamber. Indoor Air 15, 141e150. Asbach, C., Kaminski, H., Von Barany, D., Kuhlbusch, T.A.J., Monz, C., Dziurowitz, N., Pelzer, J., Vossen, K., Berlin, K., Dietrich, S., Gotz, U., Kiesling, H.J., Schierl, R., Dahmann, D., 2012. Comparability of portable nanoparticle exposure monitors. Ann. Occup. Hyg. 56, 606e621. Araujo, J.A., Baraja, B., Kleinman, M., Wang, X.P., Bennett, B.J., Gong, K.W., Navab, M., Harkema, J., Sioutas, C., Lusis, A.J., Nel, A., 2008. Small ambient particulate pollutants in the ultraﬁne range promote atherosclerosis and systemic oxida- tive stress. Circulation 102, 589e596. Wang, S.M., Inthavog, K., Wen, J., Tu, J.Y., Xue, C.L., 2009. Comparison of micron- and nanoparticle deposition patterns in a realistic human nasal cavity. Respir. Physiol. Neurobiol. 166, 142e151. Wierzbicka, A., 2008. What are the Characteristics of Airborne Particles that We are Exposed to? Focus on Indoor Environments and Emissions from Biomass Fired District Heating (Doctoral thesis). Lund University, Sweden. Wang, S.M., Inthavog, K., Wen, J., Tu, J.Y., Xue, C.L., 2009. Comparison of micron- and nanoparticle deposition patterns in a realistic human nasal cavity. Respir. Physiol. Neurobiol. 166, 142e151. Wierzbicka, A., 2008. What are the Characteristics of Airborne Particles that We are Exposed to? Focus on Indoor Environments and Emissions from Biomass Fired District Heating (Doctoral thesis). Lund University, Sweden. References Pagels, J., Wierzbicka, A., Nilsson, E., Isaxon, C., Dahl, A., Gudmundsson, A., Bohgard, M., 2009. Chemical composition and mass emission factors of candle smoke particles. J. Aerosol Sci. 40, 193e208. Afshari, A., Matson, U., Ekberg, L.E., 2005. Characterization of indoor sources of ﬁne and ultraﬁne particles: a study conducted in a full-scale chamber. Indoor Air 15, 141e150. Thatcher, T., Lunden, M., Revzan, K., Sextro, R., Brown, N., 2003. A concentration rebound method for measuring particle penetration and deposition in the in- door environment. Aerosol Sci. Technol. 37, 847e864. Araujo, J.A., Baraja, B., Kleinman, M., Wang, X.P., Bennett, B.J., Gong, K.W., Navab, M., Harkema, J., Sioutas, C., Lusis, A.J., Nel, A., 2008. Small ambient particulate pollutants in the ultraﬁne range promote atherosclerosis and systemic oxida- tive stress. Circulation 102, 589e596. Turpin, B.J., Weisel, C.P., Morandi, M., Colome, S., Stock, T., 2007. Relationships of Indoor, Outdoor, and Personal Air (RIOPA): Part II. Analyses of Concentrations of Particulate Matter Species. Report 130, Part II. Health Effects Institute. Available at: www.healtheffects.org. Asbach, C., Kaminski, H., Von Barany, D., Kuhlbusch, T.A.J., Monz, C., Dziurowitz, N., Pelzer, J., Vossen, K., Berlin, K., Dietrich, S., Gotz, U., Kiesling, H.J., Schierl, R., Dahmann, D., 2012. Comparability of portable nanoparticle exposure monitors. Ann. Occup. Hyg. 56, 606e621. C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 Wallace, L., Ott, W., 2011. Personal exposure to ultraﬁne particles. J. Expo. Sci. En- viron. Epidemiol. 21, 20e30. h h l ﬁ l C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 C. Isaxon et al. / Atmospheric Environment 106 (2015) 458e466 466 Wallace, L., 2006. Indoor sources of ultraﬁne and accumulation mode particles: size distributions, size-resolved concentrations, and source strengths. Aerosol Sci. Technol. 40, 348e360. Wallace, L., Ott, W., 2011. Personal exposure to ultraﬁne particles. J. Expo. Sci. En- viron. Epidemiol. 21, 20e30. Wan, M.-P., Wu, C.-L., Sze To, G.-N., Chan, T.-C., Chao, C.Y.H., 2011. Ultraﬁne particles and PM2.5 generated from cooking in homes. Atmos. Environ. 45 (34), 6141e6148.
https://openalex.org/W4250015228	https://journal.binus.ac.id/index.php/comtech/article/download/3884/3137	English	null	Editorial Page and Table of Content	ComTech/Comtech	2,017	cc-by-sa	708	Secretariat Haryo Sutanto Bina Nusantara University, Indonesia Research and Technology Transfer Office, Bina Nusantara University, Anggrek Campus, Jl.Kebon Jeruk Raya 27, Kebon Jeruk, Jakarta Barat 11530, Tel. +621-5350660 ext. 1705/1708, Fax.+621-5300244, E-mail: asundjaja@binus.edu, hsutanto@binus.edu, comtech@binus.edu, Url: http://journal.binus. ac.id/index.php/comtech, FB: https://www.facebook.com/ComTechBinus ComTech ComTech Computer, Mathematics and Engineering Applications Computer, Mathematics and Engineering Applications Editor in Chief Ngarap Imanuel Manik Managing Editors Arta Moro Sundjaja Noerlina Editor in Chief Ngarap Imanuel Manik Mathematics and Statistics Department, Bina Nusantara University, Indonesia Information System Department, Bina Nusantara University, Indonesia Information System Department, Bina Nusantara University, Indonesia International Editor Board Cynthia Hayat Eddy Wiyanto Tri Pujadi Gisela Nina Sevani Daniel M. Wonohadidjojo Trianggono Wiradinata Yudhi Windarto Kiyota Hashimoto Lukas S. Tanutama Rino Nugroho Fergyanto E. Gunawan Leon Andretti Abdillah Firza Utama Irpan Hidayat Agustinus Oey Tota Pirdo Kasih Hardi Humiras Purba Dase Hunaefi Ingrid S. Surono Tati Nurhayati Wikaria Gazali Information System Department, Krida Wacana Christian University, Indonesia Information System Department, Krida Wacana Christian University, Indonesia Information Systems Department, Bina Nusantara University, Indonesia Information Technology Department, Krida Wacana Christian University, Indonesia Information Technology Department, Ciputra University, Indonesia Information Technology Department, Ciputra University, Indonesia Information Technology Department, Krida Wacana Christian University, Indonesia Technology and Environment Faculty, Prince of Songkla University, Thailand Information Technology Department, Bina Nusantara University, Indonesia Research and Development in Computer Centre, Sebelas Maret University, Indonesia Master of Information System Management, Bina Nusantara University, Indonesia Monitoring Academic and Management Information Systems, Bina Darma University, Indonesia Architecture Department, Bina Nusantara University, Indonesia Civil Engineering Department, Bina Nusantara University, Indonesia Mechanical Engineering Department, Korea Advanced Institute of Science and Technology (KAIST), South Korea Industrial Engineering Department, Bina Nusantara University, Indonesia Industial Engineering Department, Mercu Buana University, Indonesia Research Center for Agriculture and Forestry, Free University of Bozen-Bolzano, Italy and Laimburg Food Technology Department, Bina Nusantara University, Indonesia Aquatic Product Technology Department, Bogor Agricultural Institute, Indonesia Mathematics and Statistics Department, Bina Nusantara University, Indonesia Language and Layout Editor Eka Yanti Pangputri Dina Nurfitria Holil Atmawati Bina Nusantara University, Indonesia Bina Nusantara University, Indonesia Bina Nusantara University, Indonesia Bina Nusantara University, Indonesia Bina Nusantara University, Indonesia Bina Nusantara University, Indonesia Bina Nusantara University, Indonesia Bina Nusantara University, Indonesia Vol. 8 No. 2 June 2017 Vol. 8 No. 2 June 2017 ISSN 2087-1244 (Printed) ISSN 2476-907X (Online) ComTech ComTech Computer, Mathematics and Engineering Applications Description COMTECH is a quadannual journal, published in March, June, September, and December. COMTECH is a forum for lecturers, academics, researchers, practitioners, and students to convey and share knowledge in the form of empirical and theoretical research articles, case studies, and review of the literature. Editors invite professional education, research, entrepreneurs and students to participate in spreading the development of science in the fields of Information Systems, Architecture, Civil Engineering, Computer Engineering, Industrial Engineering, Food Technology, Computer Science, Mathematics, and Statistics through this scientific journal. Currently, ComTech has been indexed by Directory of Open Access Journal (DOAJ), Bielefeld Academic Search Engine (BASE), World Catalogue (WorldCat), Google Scholar and Academic Resource Index (ResearchBib). The journal can be accessed online on http://journal. binus.ac.id/index.php/comtech. Authors should refer to the guidelines when preparing their articles. Research and Technology Transfer Office, Bina Nusantara University, Anggrek Campus, Jl.Kebon Jeruk Raya 27, Kebon Jeruk, Jakarta Barat 11530, Tel. +621-5350660 ext. 1705/1708, Fax.+621-5300244, E-mail: asundjaja@binus.edu, hsutanto@binus.edu, comtech@binus.edu, Url: http://journal.binus. ac.id/index.php/comtech, FB: https://www.facebook.com/ComTechBinus ISSN 2087-1244 (Printed) ISSN 2476-907X (Online) Vol. 8 No. 2 June 2017 TABLE OF CONTENTS Jakfat Haekal; Herawan Setio Selection of Raw Material Suppliers Using Analytical Hierarchy Process in Food and Beverage Company, South Jakarta................................................................................. 63-68 Budi Yulianto; Rita Layona An Implementation of Location Based Service (LBS) for Community Tracking............................................. 69-75 Rhio Sutoyo; Andry Chowanda The Development of Indoor Object Recognition Tool for People with Low Vision and Blindness........................ 77-82 Dennise Adrianto; Marlene Martani; Danella Indriani; Risan Susanti Development of Online Learning System for Software Laboratory Center in Bina Nusantara University.............. 83-94 Hana Silvana; Nadia Hanoum The Implementation of Massive Open Online Courses (MOOCs)-Based E-Learning System for College Level Learners 95-100 Febriyanti Darnis Mobile Application for Inventory Control in a Minimart........................................................................ 101-106 Teny Handhayania; Lely Hiryanto Predicting and Analyzing The Students’ Length of Study-Time Using Support Vector Machine ......................... 107-114 Shanti Pujilestari; Diny Agustini Sandrasari; Rimmaria Marida Chemical Characteristics of Pumpkin Seed Tempeh from Soybean and Pumpkin Seeds.................................. 115-119
https://openalex.org/W3196748320	https://zenodo.org/records/5463851/files/8.%20Bector%20Food%20Specialties_Fullpaper.pdf	English	null	A Case Study of Mrs. Bector Food Specialties Ltd	International journal of case studies in business, IT, and education	2,021	cc-by	8,564	Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION A Case Study of Mrs. Bector Food Specialties Ltd Reema Jenifer D’Silva1, & Ganesh Bhat S.2 1Research Scholar, College in Management & Commerce, Srinivas University, Mangalore- 575001 OrcidID: 0000-0002-9374-7028; E-mail: reemajenifer@gmail.com 2Research Professor, College in Management & Commerce, Srinivas University, Mangalore OrcidID: 0000-0003-1950-8536; E-mail: ganbhatbvr@rediffmail.com Area of the Paper: Business Management. Type of the Paper: Research Case Study. Type of Review: Peer Reviewed as per \|C\|O\|P\|E\| guidance. Indexed In: OpenAIRE. DOI: Google Scholar Citation: IJCSBE International Journal of Case Studies in Business, IT and Education (IJCSBE) A Refereed International Journal of Srinivas University, India. Crossref DOI : https://doi.org/10.47992/IJCSBE.2581.6942.0123 © With Authors. This work is licensed under a Creative Commons Attribution Non-Commercial 4.0 International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. How to Cite this Paper: D’Silva, Reema Jenifer, & Ganesh Bhat, S., (2021). A Case Study of Mrs. Bector Food Specialties Ltd. International Journal of Case Studies in Business, IT, and Education (IJCSBE), 5(2), 100-110. DOI: International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION A Case Study of Mrs. Bector Food Specialties Ltd Reema Jenifer D’Silva1, & Ganesh Bhat S.2 1Research Scholar, College in Management & Commerce, Srinivas University, Mangalore- 575001 OrcidID: 0000-0002-9374-7028; E-mail: reemajenifer@gmail.com 2Research Professor, College in Management & Commerce, Srinivas University, Mangalore OrcidID: 0000-0003-1950-8536; E-mail: ganbhatbvr@rediffmail.com Area of the Paper: Business Management. Type of the Paper: Research Case Study. Type of Review: Peer Reviewed as per \|C\|O\|P\|E\| guidance. Indexed In: OpenAIRE. DOI: Google Scholar Citation: IJCSBE International Journal of Case Studies in Business, IT and Education (IJCSBE) A Refereed International Journal of Srinivas University, India. Crossref DOI : https://doi.org/10.47992/IJCSBE.2581.6942.0123 © With Authors. This work is licensed under a Creative Commons Attribution Non-Commercial 4.0 International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. How to Cite this Paper: D’Silva, Reema Jenifer, & Ganesh Bhat, S., (2021). A Case Study of Mrs. Bector Food Specialties Ltd. International Journal of Case Studies in Business, IT, and Education (IJCSBE), 5(2), 100-110. DOI: ABSTRACT Purpose: In these days, women are exhibiting their entrepreneurial spirit and competencies. They have come to the forefront of development process and have proved themselves successful in their multitasking roles at home and office. Entrepreneurship among women improves the wealth of their families and the nation as well. Some women have managed to break the proverbial glass ceiling against the odd and have established their businesses successfully in food processing sector. Women are more inclined for food processing enterprises since they spend most of their time in the kitchen normally. Many women have the expertise in preparing new cuisines, so they start small and grow further to become a much- acclaimed food processing entrepreneurs. The food processing industry in India has great potential and it brings about the synergy between the consumer, industry and agriculture. Therefore, there is a need to inspire, encourage, motivate and co-operate with women entrepreneurs for developing the spirit of enterprise among every segment of the society. The purpose of the paper is to highlight the success story of Mrs. Bector in the food processing sector and the challenges faced by her. This study proved that Mrs. Bectors Food Specialties Ltd. owned by Mrs. Bector, based at Punjab has enough growth opportunities but to sustain itself in the market, the company has to pursue more competitive strategies to widen the operations and customer base. Design: For the purpose of analysis, this study used secondary data sources - open access journals, Google, Google scholar and Mrs. Bectors Food Specialties Ltd. websites. Furthermore, the literature is used to analyze the position of this company within SWOC framework and Michael Porter’s Five Forces analysis. Findings: Based on the analysis, it is suggested that Mrs. Bectors Food Specialties Ltd. has to expand its business beyond northern India and initiate campaign for its brand awareness. The study concludes that Mrs. Bectors Food Specialties Ltd.’s competitive pricing strategy is clearly defined to capture the market, but more proper execution of strategies is required to thrive in a competitive climate. Value: This paper focuses on the growth of Mrs. Bector Food Specialties Ltd. in terms of its current status and future opportunities. Based on findings and their interpretation, new knowledge in the form of suggestions is presented. Paper Type: Case study-based Research Analysis Paper Type: Case study based Research Analysis Keywords: Cremica, Mrs. ABSTRACT Bectors Food Specialties Ltd, women entrepreneurship, food processing industry, marketing aspects, SWOC framework and Michael Porter’s five forces analysis. International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION A Case Study of Mrs. Bector Food Specialties Ltd Reema Jenifer D’Silva1, & Ganesh Bhat S.2 1Research Scholar, College in Management & Commerce, Srinivas University, Mangalore- 575001 OrcidID: 0000-0002-9374-7028; E-mail: reemajenifer@gmail.com 2Research Professor, College in Management & Commerce, Srinivas University, Mangalore OrcidID: 0000-0003-1950-8536; E-mail: ganbhatbvr@rediffmail.com Area of the Paper: Business Management. Type of the Paper: Research Case Study. Type of Review: Peer Reviewed as per \|C\|O\|P\|E\| guidance. Indexed In: OpenAIRE. DOI: Google Scholar Citation: IJCSBE International Journal of Case Studies in Business, IT and Education (IJCSBE) A Refereed International Journal of Srinivas University, India. How to Cite this Paper: D’Silva, Reema Jenifer, & Ganesh Bhat, S., (2021). A Case Study of Mrs. Bector Food Specialties Ltd. International Journal of Case Studies in Business, IT, and Education (IJCSBE), 5(2), 100-110. DOI: Google Scholar Citation: IJCSBE © With Authors. This work is licensed under a Creative Commons Attribution Non-Commercial 4.0 International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. International License subject to proper citation to the publication source of the work. Disclaimer: The scholarly papers as reviewed and published by the Srinivas Publications (S.P.), India are the views and opinions of their respective authors and are not the views or opinions of the S.P. The S.P. disclaims of any harm or loss caused due to the published content to any party. Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com PAGE 100 International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION A Case Study of Mrs. Bector Food Specialties Ltd Reema Jenifer D’Silva1, & Ganesh Bhat S.2 1Research Scholar, College in Management & Commerce, Srinivas University, Mangalore- 575001 OrcidID: 0000-0002-9374-7028; E-mail: reemajenifer@gmail.com 2Research Professor, College in Management & Commerce, Srinivas University, Mangalore OrcidID: 0000-0003-1950-8536; E-mail: ganbhatbvr@rediffmail.com International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION A Case Study of Mrs. Bector Food Specialties Ltd Reema Jenifer D’Silva1, & Ganesh Bhat S.2 1Research Scholar, College in Management & Commerce, Srinivas University, Mangalore- 575001 OrcidID: 0000-0002-9374-7028; E-mail: reemajenifer@gmail.com 2Research Professor, College in Management & Commerce, Srinivas University, Mangalore OrcidID: 0000-0003-1950-8536; E-mail: ganbhatbvr@rediffmail.com SRINIVAS PUBLICATION The major sectors of food processing are food, vegetables, meat, poultry, fisheries, milk, dairy, food grains, cereals, plantation crops and confectionary items. Significance of food processing in India helps in improving food security in India which includes removal of food toxins, preservation of food and the ease of transportation and distribution. It also eliminates the food wastages and shortages as processed food can be preserved for a longer period time. The biggest challenge for the food processing sector is the absence of proper infrastructure and supply chain and logistics facilities in India. This will cause a big problem in the future because of uncertainty to meet the growing population demand. To encourage and regulate food processing activity in India, the Food Safety and Security Act of India (FSSAI) act has been laid down with scientific standards for food articles that regulates the manufacturing, processing, distribution, sale, storage and import. It also ensures consumer’s safety and quality of food [6]. This study shows that Mrs. Bector, a homemaker initially transformed herself as a successful woman entrepreneur in the food processing sector. Her enterprise was started in her garage for making ice creams turned out to be a first choice within Punjab. She was the first entrepreneur without having any projected studies or family in this field of business. Surmounting all the hurdles, her company has carved a niche for itself as Mrs. Bectors Food Specialties Ltd., a company is one of the biggest companies having a turnover of Rs. 2 billion [7]. SRINIVAS PUBLICATION ( ), , , , p crossed that hurdle, they have to strike a balance between their social and professional roles. During the 1980’s, a decent number of women, were educated and technologically sound, and equally contributed and worked towards family businesses. Compared with earlier two decades, the women in the 1990’s were dynamic and confident towards achieving their accomplishments and preferred to live alone even if the failure of marriage or loss of life partner, they could fearlessly raise their children on their own as they were clear of their goals. The 21st century women have tasted of spirit of enterprise and hence, ventured into business to build the enterprises and became pioneers in their field of business. These days, women are stepping out of their comfort zones and emerging as leaders in retail, technology, education and various sectors of the industries [3]. They are more inclined for food processing sector since they spend most of the time in kitchen and they are experts in trying out new cuisines. Indian food processing industry accounts for roughly 14% of the country’s Gross Domestic Product (GDP) and it ranks fifth in terms of production, consumption and exports [4]. Food processing flourished due to Urbanization, Working Women, Rising Incomes and Popular Indian Ingredients. The potentials for growth in food processing are packaged food, aerated food drinks, packaged drinking water, and alcoholic beverages. Indian food processing market is estimated to grow with a compound yearly growth rate of 11.5% during the projected period 2018-2023 [5]. The major sectors of food processing are food, vegetables, meat, poultry, fisheries, milk, dairy, food grains, cereals, plantation crops and confectionary items. Significance of food processing in India helps in improving food security in India which includes removal of food toxins, preservation of food and the ease of transportation and distribution. It also eliminates the food wastages and shortages as processed food can be preserved for a longer period time. The biggest challenge for the food processing sector is the absence of proper infrastructure and supply chain and logistics facilities in India. This will cause a big problem in the future because of uncertainty to meet the growing population demand. 1. INTRODUCTION : Women’s entrepreneurship is becoming more common, with women accounting for more than one- third of all entrepreneurs [1]. Many women have become successful entrepreneurs in the world of business [2]. If one traces the evolution of women entrepreneurship in India, it gives some interesting readings. In the 1970’s, women were held back to their share of work and responsibilities at home though they had aspirations and ambitions for self-employment and employment generation. If a few Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com PAGE 101 PAGE 101 International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION To encourage and regulate food processing activity in India, the Food Safety and Security Act of India (FSSAI) act has been laid down with scientific standards for food articles that regulates the manufacturing, processing, distribution, sale, storage and import. It also ensures consumer’s safety and quality of food [6]. This study shows that Mrs. Bector, a homemaker initially transformed herself as a successful woman entrepreneur in the food processing sector. Her enterprise was started in her garage for making ice creams turned out to be a first choice within Punjab. She was the first entrepreneur without having any projected studies or family in this field of business. Surmounting all the hurdles, her company has carved a niche for itself as Mrs. Bectors Food Specialties Ltd., a company is one of the biggest companies having a turnover of Rs. 2 billion [7]. crossed that hurdle, they have to strike a balance between their social and professional roles. During the 1980’s, a decent number of women, were educated and technologically sound, and equally contributed and worked towards family businesses. Compared with earlier two decades, the women in the 1990’s were dynamic and confident towards achieving their accomplishments and preferred to live alone even if the failure of marriage or loss of life partner, they could fearlessly raise their children on their own as they were clear of their goals. The 21st century women have tasted of spirit of enterprise and hence, ventured into business to build the enterprises and became pioneers in their field of business. These days, women are stepping out of their comfort zones and emerging as leaders in retail, technology, education and various sectors of the industries [3]. They are more inclined for food processing sector since they spend most of the time in kitchen and they are experts in trying out new cuisines. Indian food processing industry accounts for roughly 14% of the country’s Gross Domestic Product (GDP) and it ranks fifth in terms of production, consumption and exports [4]. Food processing flourished due to Urbanization, Working Women, Rising Incomes and Popular Indian Ingredients. The potentials for growth in food processing are packaged food, aerated food drinks, packaged drinking water, and alcoholic beverages. Indian food processing market is estimated to grow with a compound yearly growth rate of 11.5% during the projected period 2018-2023 [5]. 2. MRS. BECTORS FOOD SPECIALTIES LTD : Mrs. Bectors Food Specialties Ltd. is a company founded by Mrs. Rajni Bector in 1978 in Ludhiana, Punjab out of her passion for cooking, baking and making ice creams. Cremica brand was established by Mrs. Bector and has become one of the India’s fastest-growing brands over a span of more than three decades. It offers a wide array of products of incomparable quality, innovative recipes and health- oriented ingredients targeting all ages and state-of-the-art standards. As a result of the split in 2013, Mrs. Bectors Food Specialties Ltd. is managed by the eldest and youngest of Mrs. Bectors three sons, Ajay and Anup Bector respectively and Cremica Foods is managed by Akshay Bector. Mrs. Bectors Food Specialties Ltd. comprises the biscuits and bakery segment and Cremica Foods comprises the condiments segment. The company markets its biscuits such as crackers, creams, cookies, glucose and digestives under the brand name ‘Mrs. Bectors Cremica’ and bakery products such as buns, breads, pizza bases and cakes under the brand name ‘English Oven’. Presently, the Cremica Group is Rs. 650 crore business empire and it includes group companies like Cremica Frozen Food, Mrs. Bector’s Desserts, Cremica Agro and EBI foods in UK. Moreover, it has strategic tie-ups with McDonald’s, Hindustan Unilever, Big Bazaar, Spencer’s, Taj Group, ITC, Jet Airways, Air India, Barista, Café Coffee Day, Pizza Hut, Domino’s and Papa John’s. Mrs. Bectors Food Specialties Ltd. collaborated with one of McDonald’s suppliers of Europe and expanded its businesses beyond baking to include providing bread and batters to McDonald’s India and other companies [8]. Presently, this company is listed in Bombay Stock Exchange and National Stock Exchange. The company had made its Initial Public Offering (IPO) in December 2020 to raise funding for the issue of equity shares, raising Rs. 540.54 crores [10]. Recent days, its market price is quoted at Rs. 401.25 in BSE and Rs. 401.60 in NSE [11]. The proceeds of the fresh issue is utilized to finance the project cost Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com PAGE 102 PAGE 102 International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION towards the expansion of the Rajpura Manufacturing facility in Punjab and to establish a production line for biscuits. Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com 2. MRS. BECTORS FOOD SPECIALTIES LTD : Table 2: Related publications on the food industry by different researchers. Sl. No. Area of Study Focus of the Research References 1 Food Industry Women Entrepreneurial opportunities in the food industry and the societal changes that create new business opportunities for women. Vanika, C., & Manish, J. (2013). [10] 2 Food Industry Identifying and assessing critical aspects for ICT (Information Communication Technology) applications in order to help SME’s (Small and Medium enterprises) expand more effectively and sustainably in their food supply chains. Singh, S. P., et al. (2012). [11] 3 Women Entrepreneurship Problems and prospects of Women Entrepreneurship, reasons women become entrepreneurs, reasons for growth, schemes for promotion and development of women entrepreneurship. Goyal, M., & Parkash, J. (2011). [12] 4 Marketing and Logistics The Supply Chain process increases the sourcing and manufacturing activities of companies making companies focus more on the supply chain aspect. Companies must gain control over logistics to be able to face a competitive advantage in the industry. Companies in the food industry use effective supply chain activities thereby capturing a good market share in the industry. Behera, B. (2009). [13] 5 Women development Understanding Indian women, their identity, their role-taking, status and problems faced by them. The paper discusses the most successful women entrepreneurs and the trends of development in their field. Arakeri Shanta, V. (2013). [14] 6 Marketing and Promotion Analysis of Food Industry where 360-degree promotion strategy along with equally effective distribution strategy ensures deeper market penetration and coverage for the most successful food industries. Narayan Sarkar, D. (2013). [15] Sl. No. QSR Chain Relation with Mrs. Bector’s Food 1 Connaught Plaza Restaurants Pvt. Ltd. Sole supplier of burger buns and pan muffins since 1995 2 Hardcastle Restaurants Private Limited (McDonald’s India) Preferred supplier of burger buns and pan muffins, for nearly 13 years 3 Burger King India Limited The main supplier of burger buns since 2014 4 Yum! Restaurants (India) Private Limited The main supplier of burger buns since 2013 5 PVR Limited The preferred and main supplier of frozen burger buns, garlic bread and panini International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION Table 1: Exhibits the names of reputed Quick Service Restaurant chains. Source: https://blog finology in/investing/mrs bectors food ipo Sl. No. QSR Chain Relation with Mrs. Bector’s Food 1 Connaught Plaza Restaurants Pvt. Ltd. 2. MRS. BECTORS FOOD SPECIALTIES LTD : Sole supplier of burger buns and pan muffins since 1995 2 Hardcastle Restaurants Private Limited (McDonald’s India) Preferred supplier of burger buns and pan muffins, for nearly 13 years 3 Burger King India Limited The main supplier of burger buns since 2014 4 Yum! Restaurants (India) Private Limited The main supplier of burger buns since 2013 5 PVR Limited The preferred and main supplier of frozen burger buns, garlic bread and panini Source: https://blog.finology.in/investing/mrs-bectors-food-ipo 2. MRS. BECTORS FOOD SPECIALTIES LTD : 2.1 Product Range: The company offers products such as tomato ketchup, food curry/gravies, a range of mayonnaise and spreads, mocktail syrups and toppings, range of biscuits and bread. Due to fast- changing customer needs and preferences, Mrs. Bectors Food Specialties Ltd. through research and development and positioning technology secured the prominence by providing a wide array of products which include Opera Chips, Mayonnaise (Tandoori, Mint and Desi Express flavors), spreads, sauces, jams, salad dressings, syrups, food crushes, French fries and ketchup. The detailed product range of Mrs. Bectors Food Specialties Ltd. is exhibited in Figure 1. Fig 1. Exhibits the products of Mrs. Bectors Food Specialties Ltd. Fig 1. Exhibits the products of Mrs. Bectors Food Specialties Ltd. Mrs. Bectors Food Specialties Ltd. is well established itself in the retail sector and its brand focus is on ‘Customer Centricity’, relying on customer feedback and innovative products customized for local taste buds. Earlier, people prefer to cultivate a substantial portion of their food, however, in today’s developed economies, even the sustenance food items are secured from retail outlets [9]. The company has expanded in the Business to Business (B2B) segment and Business to Consumer (B2C) segment to generate revenue through volume orders by targeting Food Service Segment and HORECA (Hotel/Restaurant/Café) Segment through product innovation and expansion. 2.2 Quick Service Restaurant Chains: Quick Service Restaurant or QSR is a restaurant that serves food that requires little preparation time and is served quickly. They have implemented a bundle pricing model that combines their menu items into a bundle of complementary meals. For example, McDonald’s combo meal of fries, a soft drink and a burger wherein the burger buns are prepared by Mrs. Bectors Food Specialties Ltd. The company is a supplier for many reputed Quick Service Restaurant (QSR) chains which have been listed below in Table 1. Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION Table 1: Exhibits the names of reputed Quick Service Restaurant chains. Source: https://blog.finology.in/investing/mrs-bectors-food-ipo 3. REVIEW OF LITERATURE ON FOOD PROCESSING SECTOR : Several researchers have studied the food processing industry and its different facets. Some of the scholarly papers published relating to women entrepreneurship and the food industry are listed in Table 2 with the area of study, the focus of the research along references. 5. RESEARCH METHODOLOGY : This study is based on secondary data sources that highlight Mrs. Bectors Food Specialties Ltd. as a case of women entrepreneurship in food processing sector. Apart from it, this study uses the SWOC framework and Michael Porter’s five forces analysis to determine the growth opportunity and competitive strategy to be followed by the company for sustainable growth. Detailed analysis has been presented with the help of information collected from journal articles, business websites and company websites. 4. OBJECTIVES : This study is focused on the comprehensive analysis of one of the top Indian food processing company: This study is focused on the comprehensive analysis of one of the top Indian food processing company: Mrs. Bectors Food Specialties Ltd. The main objectives of the study are: This study is focused on the comprehensive analysis of one of the top Indian food processing company: This study is focused on the comprehensive analysis of one of the top Indian food processing co Mrs. Bectors Food Specialties Ltd. The main objectives of the study are: 1. To assess the company’s position through Strengths, Weakness, Opportunities and Challenges (SWOC) framework. 1. To assess the company’s position through Strengths, Weakness, Opportunities and Challenges (SWOC) framework. 2. To analyze the position of Mrs. Bectors Food Specialties Ltd. through Michael Porter’s Five Forces Analysis. 2. To analyze the position of Mrs. Bectors Food Specialties Ltd. through Michael Porter’s Five Forces Analysis. 2. To analyze the position of Mrs. Bectors Food Specialties Ltd. through Michael Porter’s Five Analysis. y 3. To suggest new strategies to be formulated to retain the market position of the company 6. SWOC ANALYSIS OF MRS. BECTORS FOOD SPECIALTIES LTD : The SWOC framework is the most extensively utilized method for tracking and evaluating a company’s competitive role. Its main goal is to assess corporate strategies in order to produce a business strategy that aligns the organization’s resources and skill sets with the market’s requirements [18]. Normally, organizations use this framework to assess their firms’ strengths, weaknesses, opportunities and challenges, as well as those of their competitors and goods. SWOC is a comprehensive examination of the industry’s operating environment that aids in the projection of many aspects of the environment as well as their incorporation into the organizations decision-making [19]. After an in-depth analysis of extensive literature on this company, following Strengths, Weaknesses, Opportunities and Challenges were arrived at: 6.1 Strengths: Mrs. Bectors Food Specialties Ltd has the following strengths: 6.1 Strengths: Mrs. Bectors Food Specialties Ltd has the following strengths: (1) The company offers a diverse range of well-known medium and premium brands in India, particularly in biscuits and bakery products. (2) Being a renowned biscuit exporter with numerous quality certificates, the company’s products are export ready. Its biscuits export has a 12% market share and has a market share of 4.5% of the premium and mid-premium biscuits market in North India as of FY 2020 [20]. (3) A leading provider of buns to the fast-growing QSR industry, Mrs. Bector’s has enhanced brand visibility. (4) The company has an extensive and reputable sales and distribution network that spans India and 64 countries across six continents [21]. (5) The company has innovative offerings, packaging and commitment to quality keeping in mind the changing consumer preferences. (6) Being the largest supplier of biscuits to Canteen Stores Department of Government of India, this company is also an approved supplier for Indian Railways in North India. (7) Mrs. Bector’s is one of the top two Indian biscuit manufacturers in the premium and mid-premium segment in the states - Himachal Pradesh, Punjab, Ladakh, Jammu and Kashmir. g j 8) The company is listed in National Stock Exchange and Bombay Stock Exchange [22]. 6.2 Weaknesses: Mrs. Bectors Food Specialties Ltd has the following weaknesses: 6.2 Weaknesses: Mrs. Bectors Food Specialties Ltd has the following weaknesses: (1) Lack of brand awareness and availability of the company’s products among the general public outside Northern India. The company is much dependent on northern India especially Delhi, Punjab and Uttar Pradesh for its revenue growth. 3. REVIEW OF LITERATURE ON FOOD PROCESSING SECTOR : Several researchers have studied the food processing industry and its different facets. Some of the scholarly papers published relating to women entrepreneurship and the food industry are listed in Table 2 with the area of study, the focus of the research along references. Table 2: Related publications on the food industry by different researchers. Sl. No. Area of Study Focus of the Research References 1 Food Industry Women Entrepreneurial opportunities in the food industry and the societal changes that create new business opportunities for women. Vanika, C., & Manish, J. (2013). [10] 2 Food Industry Identifying and assessing critical aspects for ICT (Information Communication Technology) applications in order to help SME’s (Small and Medium enterprises) expand more effectively and sustainably in their food supply chains. Singh, S. P., et al. (2012). [11] 3 Women Entrepreneurship Problems and prospects of Women Entrepreneurship, reasons women become entrepreneurs, reasons for growth, schemes for promotion and development of women entrepreneurship. Goyal, M., & Parkash, J. (2011). [12] 4 Marketing and Logistics The Supply Chain process increases the sourcing and manufacturing activities of companies making companies focus more on the supply chain aspect. Companies must gain control over logistics to be able to face a competitive advantage in the industry. Companies in the food industry use effective supply chain activities thereby capturing a good market share in the industry. Behera, B. (2009). [13] 5 Women development Understanding Indian women, their identity, their role-taking, status and problems faced by them. The paper discusses the most successful women entrepreneurs and the trends of development in their field. Arakeri Shanta, V. (2013). [14] 6 Marketing and Promotion Analysis of Food Industry where 360-degree promotion strategy along with equally effective distribution strategy ensures deeper market penetration and coverage for the most successful food industries. Narayan Sarkar, D. (2013). [15] Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION , , (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 PUBLICATION umbrella. The company is subject to a Brand Separation MoU among its promoters because it shares the name ‘Mrs. Bectors Cremica’ with other family members of promoter Mrs. Bector. y p (4) Mrs. Bector’s premium category products may not be of interest to the rural and semi-urban market due to its pricing. ( ) h d h l i h i i k i li f (5) The company does not have long-term contracts with its Quick Service Restaurant clients for sustenance. (5) The company does not have long-term contracts with its Quick Service Restaurant clients for sustenance. 6.3 Opportunities: The opportunities of Mrs. Bectors Food Specialties Ltd are as follows: (1) The company already has an established pan-India distribution channel hence it has opportunities to make its diverse products available to a bigger population across the country. (2) The company has a significant export potential to reach out to clients throughout the world with its diverse line of products. (4) They have recently launched ‘BONHEUR’ Syrup in India which has been designed using the French aroma and is tailor-made for the Indian market. (5) Due to urbanization, a large number of working-class, men and women, and students living alone; and greater exposure to fast food patterns make Indian consumers prefer fried, baked or toasted food straight from the freezer; and are willing to spend on bakery products and new launches resulting in increased demand and growth in the bakery sector [23]. So, Mrs. Bectors Food Specialties Ltd. can target such consumers to increase their sales. 4 Challenges: Mrs. Bectors Food Specialties Ltd has the following challenges to face: (1) The company is up against stiff competition from both local and national brands, making it to maintain market share at reasonable pricing. (2) Due to a decrease in demand for the company’s products in rural areas, it will be difficult to restore sales volume. (3) The demand for local bakery products over the company’s products poses a huge challenge to its profits. (3) The demand for local bakery products over the company’s products poses a huge challenge to its profits. p (4) Increase in the cost of the supply chain and transportation for delivery of products out of northern India will be a challenge for the company, if it is interested to look beyond Northern India. 6. SWOC ANALYSIS OF MRS. BECTORS FOOD SPECIALTIES LTD : (1) Lack of brand awareness and availability of the company’s products among the general public outside Northern India. The company is much dependent on northern India especially Delhi, Punjab and Uttar Pradesh for its revenue growth. (2) To stimulate point of purchase (POP) promotion, the company relies on retail outlets and small supermarkets to display products on their shelves. (3) Due to a prohibition on using the brand name Mrs. Bectors Cremica for any food business other than biscuits, the company is unable to introduce a new category under the name ‘Cremica’ Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com P PAGE 105 International Journal of Case Studies in Business, IT, and Education International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION umbrella. The company is subject to a Brand Separation MoU among its promoters because it shares the name ‘Mrs. Bectors Cremica’ with other family members of promoter Mrs. Bector. SRINIVAS PUBLICATION (4) Increase in the cost of the supply chain and transportation for delivery of products out of northern India will be a challenge for the company, if it is interested to look beyond Northern India. 7. MICHAEL PORTER’S FIVE FORCES ANALYSIS OF MRS. BECTORS FOOD SPECIALTIES LTD : SRINIVAS PUBLICATION SRINIVAS PUBLICATION consumer tastes and behaviours [27]. Buyers influence has the capacity to cut prices and improve product quality so it accounts for a significant amount of organization’s overall sales. Mrs. Bectors Food Specialties Ltd offers products at a low price to customers as they design and produce their products in their own factories. Since consumers’ tastes and preferences change as a result of their increased purchasing power, brand loyalty for these products is low. So, the company has to increase customer loyalty by providing a positive shopping experience through good service and customer feedbacks. 7.3 Bargaining power of suppliers: Suppliers have the power of raising the price or reducing the quality of raw materials. In comparison with buyers, there are many suppliers in the food processing industry; as a result, supplier power is limited and low supplier power increases profit for buyers. Mrs. Bectors Food Specialties Ltd. adopted a strategy of building greater loyalty and long term-relationships with suppliers. Secondly, the bargaining power of suppliers of unorganized sector such as milk and vegetables is weak. g 7.4 Threat of substitute products or services: Economic liberalization, globalization and multi- national companies’ entry into the agro processing industry have increased market and competition largely giving customers a benefit [28]. If some products or services meet similar needs, customers are more likely to switch to substitutes for the increase of price of a product or service. Likewise, low switching cost makes it easy for consumers to switch from one supplier to another in the food industry. There are many substitutes for bread, jams, mayonnaise, sauces and biscuits such as Parle, Nestle, MTR and Britannia. However, Mrs. Bectors Food Specialties Ltd employs a competitive pricing strategy and sells on the promise of quality. This expertise made the company remain profitable and keep their market share. 7.5 Internal rivalry: When there is internal rivalry between food processing sectors, it will lead to a reduction of the industry’s profits mainly because of competitive pricing. Moreover, the presence unorganized sector within the industry also increases competition. In the biscuits market, Mrs. Bectors Food Specialties Ltd competes with Parle Biscuits, Britannia Industries, ITC, Anmol Industries and Surya Agro Food. While in the bakery market, they compete with Modern Food Industries (India), Harvest Gold Foods India and Bonn Nutrients. The intensity of rivalry among these firms in food industry is very high. 7. MICHAEL PORTER’S FIVE FORCES ANALYSIS OF MRS. BECTORS SPECIALTIES LTD : Michael Porters Five Forces Analysis is used to critically analyze the business model of Mrs. Bectors Food Specialties Ltd as per company analysis-based research case study analysis framework [24-25]. Normally, a business model is a set of ideas that makes a value proposition by achieving a long-term goal. It describes how it makes profit by defining an organization’s position in the value chain. And hence, the twin challenges before an organization are: one, choosing a suitable business model that increases customer value and corporate income; and two, updating such business model, whenever, required [26]. Keeping in mind these two issues, Michael Porter’s Five Forces model is used to analyze the competitive position of the company. This conceptual model was developed by Harvard University’s Professor Michael Porter, who used industrial economics and principles of strategic management, to analyze five interacting factors that are important for a company to become competitive. The five interacting factors are competitive rivalry, threat of new entrants, threat of substitutes, buyer bargaining power and supplier bargaining power. The analysis of Mrs. Bectors Food Specialties Ltd. is here as follows: p 7.1 Threat of new entrants: The threat of new entrants into the food sector is significant when new competitors decide to enter an industry, reducing revenues produced by existing businesses and decreasing client loyalty. Mrs. Bector faces few risks such as stiff competition, higher dependency on the North Indian market for growth and a premium category target market that might not appeal to the rural and semi-urban areas. However, with Mrs. Bectors Food Specialties Ltd’s product differentiation, high brand name recognition and customer loyalty has reduced the threat of new entrants into the market. Bargaining power of buyers: Consumer’s attitude toward food and beverages alter on a daily bas result of broader socio-economic and cultural changes which largely influence the changes i Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 8. FINDINGS : Bectors Food Specialties Ltd. provides low-cost products to clients since they design and manufacture their own products. As the buyer is able to get similar products from other suppliers, the company increases customer loyalty by attracting customers and offering a positive shopping experience through excellent customer service and feedback. (7) As there are many substitutes available for buyers, Mrs. Bectors Food Specialties Ltd. is using brand loyalty and long-term connections with its customers and retailers, as the channel partner. (7) As there are many substitutes available for buyers, Mrs. Bectors Food Specialties Ltd. is using brand loyalty and long-term connections with its customers and retailers, as the channel partner. (8) In the food market, consumers switch from one supplier to another due to low switching costs, therefore, Mrs. Bectors Food Specialties Ltd. employed a competitive pricing strategy and focus on quality allowed the company to remain profitable among its many competitors. (9) Mrs. Bectors Food Specialties Ltd. is facing high intensity rivalry among its competitors Parle Biscuits, Britannia Industries, ITC India, Anmol, etc. in food industry. Due to the existence of brand loyalty in certain products and continuous product differentiation helped the company to maintain its commanding position against the competitive rivalry. (9) Mrs. Bectors Food Specialties Ltd. is facing high intensity rivalry among its competitors Parle Biscuits, Britannia Industries, ITC India, Anmol, etc. in food industry. Due to the existence of brand loyalty in certain products and continuous product differentiation helped the company to maintain its commanding position against the competitive rivalry. 8. FINDINGS : The following are the findings based on extensive literature analysis: The following are the findings based on extensive literature analysis: (1) Mrs. Bectors Food Specialties’ Ltd. has substantial turnover, possess a distribution network, and hence it can offer innovative products, packaging and quality assurance while keeping in mind the changing consumer tastes and preferences. Brand popularity, packaging, promotional strategy, complementary products and affordability are the factors influencing the consumption of jams, ketchup, bread, sauces, etc. resulting in an increase in demand for the company products. (1) Mrs. Bectors Food Specialties’ Ltd. has substantial turnover, possess a distribution network, and hence it can offer innovative products, packaging and quality assurance while keeping in mind the changing consumer tastes and preferences. Brand popularity, packaging, promotional strategy, complementary products and affordability are the factors influencing the consumption of jams, ketchup, bread, sauces, etc. resulting in an increase in demand for the company products. (2) Though it is one of the leading food processing companies in India, it is not one among the top 20 food processing companies, mainly because it focuses on north India alone. In the case of bread and biscuits segment, the profit margin is low, but given the company’s vast output and consistent distribution, the company’s long-term viability is secured. (3) Through the innovation initiatives, the company has secured a significant export potential to reach out to clients throughout the world with its diverse line of products. (3) Through the innovation initiatives, the company has secured a significant export potential to reach out to clients throughout the world with its diverse line of products. (4) The company’s profitability is affected by - the consumer preference for local bakery products over the company’s products and increasing competition from both local and national brands. (4) The company’s profitability is affected by - the consumer preference for local bakery products over the company’s products and increasing competition from both local and national brands. 5) The threat of new entrants is less likely to affect Mrs. Bectors Food Specialties Ltd.’s pro differentiation, high brand name recognition and customer loyalty. (6) Mrs. Bectors Food Specialties Ltd. provides low-cost products to clients since they design and manufacture their own products. As the buyer is able to get similar products from other suppliers, the company increases customer loyalty by attracting customers and offering a positive shopping experience through excellent customer service and feedback. (6) Mrs. SRINIVAS PUBLICATION In Figure 2, shown below, it can be understood that most of the Indian biscuit space is dominated by Britannia Industries Ltd, Parle Products Ltd and ITC India which collectively own 66% of the market. Parle derives a large portion of its revenue from Parle-G while Britannia’s top line is led by its mid-premium and premium products. However, ‘Mrs. Bector’s Cremica’ owns a market share of 1% of the biscuit segment and is a strong competitor as the company concentrates only in Northern India. If it increases its foothold beyond northern India, its market share and profitability will increase in the future. Fig 2: Exhibits the Market share of brands in the biscuit market. Source: Mrs. Bectors’ Red Herring Prospectus (Bloomberg/ Quint) www.bloombergquint.com. Anmol 4% ITC 11% Parle 27% Britannia 28% Cremica 1% Others 29% Market Share Of Brands In Biscuit Market Anmol ITC Parle Britannia Cremica Others Anmol ITC Market Share Of Brands In Biscuit Market Anmol 4% Fig 2: Exhibits the Market share of brands in the biscuit market. g Source: Mrs. Bectors’ Red Herring Prospectus (Bloomberg/ Quint) www.bloombergquint.com g tors’ Red Herring Prospectus (Bloomberg/ Quint) www.bloombergquint.com. Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRI PUBLIC 8 FINDINGS : International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 9. SUGGESTIONS : As it can be seen how Mrs. Bectors Food Specialties Ltd. has expanded over the years and have proven to be a successful business model, the company is planning to expand its operations in all the metropolitan cities to seize its investment opportunities. Below are a few suggestions which have been listed that can be of help for further improvement: (1) The company must expand their business out of northern India, and they have to initiate the steps to grow internationally. ( ) i h di ib d il h h ff k d (1) The company must expand their business out of northern India, and they have to initiate the steps to grow internationally. (2) To motivate the distributors and retailers, the company has to offer some tour packages and extra benefits based on the targets achieved. This will help to increase their sales volume and profits. (2) To motivate the distributors and retailers, the company has to offer some tour packages and extra benefits based on the targets achieved. This will help to increase their sales volume and profits. (3) The company has to organize events and exhibitions in some corporate places which would help to increase their brand awareness. (4) In addition to increasing their brand awareness, distributing samples of their products in colleges and school campuses would help to penetrate their brand to new generations. (5) The company can have tie-ups with supermarkets and promote their products in small sachets to increase their availability across the markets especially out of Northern India. (6) Teaming up with a few health clubs to build goodwill as a supplier of health-conscious products would help to increase their customer base. These days, neo rich customers are very health conscious irrespective of age or gender while purchasing the product especially biscuits. (6) Teaming up with a few health clubs to build goodwill as a supplier of health-conscious products would help to increase their customer base. These days, neo rich customers are very health conscious irrespective of age or gender while purchasing the product especially biscuits. (7) While expanding their markets outside India, the company may have to face high transportation costs, increased cycle time, higher inventory investment and consumers’ demand for low price. Therefore, they must concentrate more on the supply chain management specifically on logistics to secure a competitive advantage in the industry. 10. CONCLUSIONS : The success of Mrs. Bectors Food Specialties Ltd. is inspirational especially to women entrepreneurs as it can be seen how Mrs. Bector, from being a homemaker rose to the level of being a successful woman entrepreneur in food processing industry. She had overcome many difficulties and established herself as the leading woman entrepreneur in Punjab. Presently, this company is one of the biggest food processing companies having a substantial turnover, financially sound and has been progressing since 1980’s. Apart from it, ‘Mrs. Bectors Cremica’ is the most visible brand in the condiments business. The company is following market penetration strategy to increase the retail sales and thereby accelerating the growth process. Accelerated investment in storage and transport capabilities has lowered wastage levels, improved nutrient retention during storage and transportation and improved the shelf life of products resulting in the growth of food processing sector. Increasing number of working women are coming to the forefront and the food processing sector is the one which many can bank on. A few of them who capitalized this opportunity have achieved success and recognition in the society. The progress in the food processing industry will add to the Indian GDP and if similar development space is given for other women entrepreneurs in India, it will give more chance for inclusive growth of Indian economy in the coming decades. 9. SUGGESTIONS : Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com PAGE 108 PAGE 108 International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 PU International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION (8) Brand positioning helps in increasing sales volume, the company can improve their promotional activities by increasing sponsorships, television advertisements and celebrity endorsement so the consumers will be aware and keen on buying the new brands. (8) Brand positioning helps in increasing sales volume, the company can improve their promotional activities by increasing sponsorships, television advertisements and celebrity endorsement so the consumers will be aware and keen on buying the new brands. y g (9) The company should provide gifts with all types of flavor of biscuits like stickers, stationery, tattoos which are useful for children [29]. (9) The company should provide gifts with all types of flavor of biscuits like stickers, stationery, tattoos which are useful for children [29]. (10) Appropriate policy assistance to develop women entrepreneurs in the processing sector by competing with successful models such as Samridhi Mahila Cooperative Society (Palampur, Kangra), M/s Mushran’s Bhuira Jams (Rajgarh, Sirmaur) and M/s Bector’s Cremica (Taliwal, Una) which are effectively operating in the State [30]. REFERENCES : [1] Mukherjee, S. (2010). Profiling the Urban Women Microentrepreneurs in India. IUP Journal of Entrepreneurship Development, 24(1), 23-37. [2] Kumbhar, V. M. (2013). Some critical issues of women entrepreneurship in rural India. European Academic Research, 1(2), 192-200. [3] Chimthanawala, S. M., Naidu, K., & Shah, N. V. (2015). Development and growth of women entrepreneurship of India. In International Conference on Technology and Business Management, 6(1), 109-118. [4] Ajeet Sarathe, Rajesh Gupta, A.L. Basediya, Venkata Satish Kuchi, (2018). Recent Trends and SWOT Analysis of Food-Processing Industry Infrastructure in India: A Review. Bulletin of Environment, Pharmacology and Life Sciences, 7(6), 107-116. [5] India Food Processing Market Expected to Grow with a CAGR of 11.5% During the Forecast Period, 2018-2023 – www. Research and Markets.com \| Business Wire retrieved on 06.01.2021 [6] Shukla, S., Shankar, R., & Singh, S. P. (2014). Food safety regulatory model in India. Food Control, 37(6), 401-413. [7] Pathak, S., & Munjal, M. (2017). Societal Perspective towards Women Entrepreneurs: An Eagle Eye’s view. International Journal of Research in IT and Management, 7(10), 14-22. ttps://apps.aima.in/ejournal_new/articlesPDF/379-TheCremicaStory.pdf retrieved on 07.06.2021 [9] Rowley, B., & McMurtrey, M. E. (2016). McDonald’s and the Triple Bottom Line: A Case Study of Corporate Sustainability. Journal of Strategic Innovation and Sustainability, 11(1), 33-37. [10] Mrs. Bectors Food Specialties Ltd. Daily Technical Analysis \| Stochastic RSI indicators (moneycontrol.com) Retrieved on 24 June 2021. Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com PAGE 109 PAGE 109 International Journal of Case Studies in Business, IT, and Education (IJCSBE), ISSN: 2581-6942, Vol. 5, No. 2, September 2021 SRINIVAS PUBLICATION SRINIVAS PUBLICATION [11] Mrs. Bectors Food IPO - Issue Date, Price, Review and Details (groww.in) Retrieved on 24 June 2021. [12] Vanika, C., & Manish, J. (2013). Women entrepreneurial opportunities in food industry: a case of cremica products. International Journal of Research in Commerce, Economics and Management, 3(12), 60-64. [13] Singh, S. P., Tegegne, F., & Ekanem, E. P. (2012). The food processing industry in India: challenges and opportunities. Journal of Food Distribution Research, 43(1), 81-89. [14] Goyal, M., & Parkash, J. (2011). Women entrepreneurship in India-problems and prospects. International journal of multidisciplinary research, 1(5), 195-207. [15] Behera, B. (2009). Supply chain best practices in India. ASBM Journal of Management, 2(1), 134- 142. [16] Arakeri Shanta, V. (2013). Women entrepreneurship in India. National Monthly Referred Journal of Research in Arts & Education, 1(3), 1-7. [17] Narayan Sarkar, D. (2013). REFERENCES : Storm in a Milk-Cup: Oreo in India. IUP Journal of Business Strategy, 10(1), 31-45. [18] Aithal, P. S., & Kumar, P. M. (2015). Applying SWOC Analysis to an Institution of Higher Education. International Journal of Management, IT and Engineering, 5(7), 231–247. [19] Aithal, P. S. (2017). Industry Analysis – The First Step in Business Management Scholarly Research. International Journal of Case Studies in Business, IT and Education (IJCSBE), 2(1), 1– 13. [20] blog.tickertape.in/talking-points-of-mrs-bectors-food-specialities-ipo Retrieved on 19 June 2021 [21] https://blog.finology.in/investing/mrs-bectors-food-ipo Retrieved on 23 May 2021 [22] https://www.financialexpress.com/market/mrs-bectors-ipo-listing-price Retrieved on 19 Ju [23] NPCS Team. (2014). Bakery Industry in India (Bread, Biscuits and Other Products) Present & Future Prospects, Market Size, Statistics, Trends, SWOT Analysis and Forecasts (Up to 2017). NIIR Project Consultancy Services, 6(1), 7-29. [24] Aithal, P. S. (2017). An effective method of developing business case studies based on company analysis. International Journal of Engineering Research and Modern Education (IJERME), 2(1), 16-27. [25] Aithal, P. S. (2017). Company Analysis–The Beginning Step for Scholarly Research. International Journal of Case Studies in Business, IT and Education (IJCSBE), 1(1), 1-18. [26] Aithal, P. S. (2016). Study on ABCD Analysis Technique for Business Models, business strategies, Operating Concepts & Business Systems. International Journal in Management and Social Science (IJMSS), 04(1), 98–115. [27] Sarin, S., & Barrows, C. (2005). An examination of current food and beverage trends in India and an assessment of potential demand for luxury food and beverage products: implications for managers. Journal of services research, 24(6), 217-237. [28] Kaur, V. (2021). Analysis of Agro Processing Industry in Punjab, India. Turkish Journal of Computer and Mathematics Education (TURCOMAT), 12(10), 525-543. [29] Kanimozhi, N., & Karthik, S. A study on the Brand awareness and consumer satisfaction towards sunfeast biscuits in Erode District, Suraj Punj Journal for Multidisciplinary Research, 9(5), 231- 240. [30] Sharma, K. D., Pathania, M. S., & Lal, H. (2010). Value chain analysis and financial viability of agro-processing industries in Himachal Pradesh. Agricultural Economics Research Review, 23(1), 515-522. Reema Jenifer D’Silva, et al, (2021); www.srinivaspublication.com PAGE 110 PAGE 110
https://openalex.org/W3099203669	https://www.researchsquare.com/article/rs-107448/v2.pdf?c=1631886354000	English	null	Finite element analysis of different locking plate fixation methods for the treatment of ulnar head fracture	Journal of orthopaedic surgery and research	2,021	cc-by	6,973	Finite element analysis of different locking-plate fixation methods for the treatment of ulnar head fracture Yue Zhang Department of Traumatic Surgery, Shan Qin Shao Shanghai East Hospital Chensong Yang Shanghai East Hospital Changqing Ai Shanghai East Hospital Di Zhou Shanghai East Hospital Yang Yu Walkman biomaterial CO.,LTD Guixin Sun (  sunguixin@sina.com ) Shanghai East Hospital Yue Zhang Department of Traumatic Surgery, Shan Qin Shao Shanghai East Hospital Chensong Yang Shanghai East Hospital Changqing Ai Shanghai East Hospital Di Zhou Shanghai East Hospital Yang Yu Walkman biomaterial CO.,LTD Guixin Sun (  sunguixin@sina.com ) Shanghai East Hospital Research article Keywords: Finite element method, ulnar head fracture, locking plate fixation, distal radius and ulnar joint, internal fixation Posted Date: January 26th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-107448/v2 DOI: https://doi.org/10.21203/rs.3.rs-107448/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. d ll License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on March 15th, 2021. See the published version at https://doi.org/10.1186/s13018-021-02334-4. Page 1/24 Page 1/24 Abstract Background: Ulnar head fractures are increasingly higher with the growing proportion of the elderly people. Failure to achieve a stable anatomic reduction of ulna head fracture may lead to a distal radioulnar joint (DRUJ) dysfunction and nonunion of the distal radius. Due to the lack of the postoperative reporting outcomes and the biomechanical studies, it has not been well established about the optimal management of the comminuted distal ulna head fracture. Hence, the purpose of this study is to use finite element analysis to explain the advantages and disadvantages of ulnar-side locking plate fixation compared with dorsal-side locking plate fixation and its screw arrangement in the treatment of ulnar head fractures. Methods: FE models of the ulnar head fracture and the models of ulnar-side locking plate and dorsal-side plate with two or three distal screws was constructed. In order to simulate forces acting on the ulnar and the osteosynthesis material during daily-life activity in subjects who underwent reconstructive surgery, we applied three loading conditions to each model, viz. 20 N axial compression, 50 N axial compression, 1 N∙m torsion moment, 1 N∙m lateral bending moments, and 1 N∙m extension bending moments. Under these conditions, values of the von Mises stress (VMS) distribution of the implant, peak VMS, the relative displacement of the head and shaft fragments between the fracture ends and the displacement and its direction of the models were investigated. Results: The stress values of ulnar-side plates were lower than those of dorsal-side plates. And the ulnar- plate fixation system also has smaller maximum displacement and relative displacement. When adding a screw in the middle hole of the ulnar head, the values of model displacement and the peak stress in fixation system are lower, but it may evidently concentrate the stress on the middle screw. Conclusions: In conclusion, our study indicated that ulnar-side locking plates resulted in a lower stress distribution in the plate and better stability than dorsal-side locking plates for ulnar head fracture fixation. Adding an additional screw to the ulnar head could increase the stability of the fixation system and provide an anti-torsion function. This study requires clinical confirmation of its practicality in the treatment of ulnar head fractures. This study requires clinical confirmation as to its practicality in the treatment of ulnar head fracture. Background The wrist joint is one of the main joints of the human body and has high activity frequency. Previous studies have suggested that the stability of the the distal radioulnar joint (DRUJ) greatly affects the function of the wrist joint, not only for forearm rotation, but also for load and force transmission[1, 2]. If a DRUJ fracture is not treated in time, it often leads to posttraumatic chronic pain and limited wrist joint activity, which greatly inconveniences the work and daily life of patients. Lack of understanding of the details of anatomy and biomechanics at the distal end of the ulna resulted in 75 years of simple resection of the distal ulna as treatment for most disabling pathologies in this part of the distal Page 2/24 forearm[3]. In the past 25 years, one of the most exciting areas in hand surgery has been the study of anatomy, biomechanics, and pathophysiology at the distal end of the ulna[4]. Ulnar head fracture may be seen in up to 6% of patients with unstable fractures of the distal radius[5], and this rate is ever increasing due to the growing proportion of elderly people[6]. Metaphyseal distal radial fractures associated with distal ulnar shaft fractures represent an unstable injury pattern, which may cause nonunion of the distal radius[7]. In addition, researchers suspected that a significant joint reaction force can develop between the sigmoid notch of the radius and the rotationally fixed ulnar seat[8]. Thus, ulnar head fracture may also decrease forearm rotation[9]. Failure to achieve stable anatomic reduction of ulnar head fractures leads to the loss of ulnar variance and the distal ulna nonunion. Thus, may cause DRUJ dysfunction, ulnar-side wrist pain, and posttraumatic arthrosis[7, 10-12]. forearm[3]. In the past 25 years, one of the most exciting areas in hand s anatomy, biomechanics, and pathophysiology at the distal end of the uln seen in up to 6% of patients with unstable fractures of the distal radius[5 due to the growing proportion of elderly people[6]. Metaphyseal distal rad distal ulnar shaft fractures represent an unstable injury pattern, which m radius[7]. In addition, researchers suspected that a significant joint react the sigmoid notch of the radius and the rotationally fixed ulnar seat[8]. T also decrease forearm rotation[9]. Failure to achieve stable anatomic red leads to the loss of ulnar variance and the distal ulna nonunion. Thus, m ulnar-side wrist pain, and posttraumatic arthrosis[7, 10-12]. Background Due to the lack of the postoperative outcome reports and the biomechanical studies[13], the optimal management of comminuted distal ulna articular head fractures has not been well established. Ring et al. [14] reported that condylar blade plate fixation could achieve healing with good alignment, satisfactory function, and an acceptable rate of secondary surgery. David et al.[15] revealed the benefits of the application of a locked plate, which included the locked or fixed angle support, the ability to insert variable lengths of locked pegs, and a low-profile design. But dorsal locking plates may cause soft tissue complications[16]. Recently, a distal ulna hook plate has been introduced for the treatment of distal ulna fractures; however, the limitation of the vertical arrangement of distal screws may result in instability of the construct [17, 18]. In the present study, we found that ulnar-side micro-locking plates could achieve good outcomes. However, the distal radius fracture combined with ulnar head fracture is not a common clinical case, and the application of ulnar-side locking plates has never been reported before. Since the number of cases is relatively small, postoperative research is hard to perform. Through technological advancements, computer modelling has become more accurate[19]. Finite element analysis provides a convenient and accurate research method for doctors. This approach can stimulate the actual force and stress of bone and joints[20]. The obtained results showed the potential application of finite element analysis in a wide range of numerical studies [21]. Therefore, such studies are not limited to the number of specific cases. Throughout the research history of distal ulna fractures, it has become indispensable to apply finite element analysis to evaluate the mechanical properties of implants. Hence, the purpose of this study is to use finite element analysis to explain the advantages and disadvantages of the ulnar-side locking plate fixation, compared with the dorsal-side locking plate fixation, and its screw arrangement in the treatment of the ulnar head fractures. Methods Establishment of the finite element models Establishment of the finite element models A 45-year-old healthy female volunteer without a history of wrist and systemic diseases was recruited for the study. A Canon Aquilion ONE ViSION Edition CT scanner was used to perform a high-resolution CT scan of her right forearm. The scanning layer thickness was 0.5 mm. The CT scan was stored as a Page 3/24 DICOM file in Mimics 19.0. The reconstruction slice thickness was 0.5 mm. A 3D model of the right forearm was obtained based on the grey value of the tissue and segmentation of the region, and was exported as an IGES file and then incorporated into Geomagic 12 for smoothing, meshing and fitting surface processing (Fig. 1). DICOM file in Mimics 19.0. The reconstruction slice thickness was 0.5 mm. A 3D model of the right forearm was obtained based on the grey value of the tissue and segmentation of the region, and was exported as an IGES file and then incorporated into Geomagic 12 for smoothing, meshing and fitting surface processing (Fig. 1). Then, the model was incorporated into the Creo Parametric 2.0. In this study, we used an OsteoMED 2.0 HPS Y-plate system. Thus, cannulated screws with a diameter of 2.0 mm and Y-steel plates were fabricated using Creo Parametric 2.0. A model of the ulnar head was established and stabilized with an ulnar-side plate and a dorsal-side plate respectively according to a practical surgical method with no interfragmentary gap (Fig. 2). The implant material was modelled as titanium alloy Ti6Al4V with the following material constants: elastic modulus E= 110 Gpa and Poisson’s ratio μ = 0.33. Subsequently, the models were incorporated into ANSYS Workbench 15.0 for meshing. and the fracture line of the ulnar head fracture was cut as described by Paksima[13]. When there are more than two geometric models, the relative relationship between the models should be set according to the actual situation, so we set the contact setting to a bonded relation in this report. (Fig. 3) The bone was defined with linear elastic material properties using a Young’s modulus of 17 GPa for cortical bone and 1.5 GPa for cancellous bone. The Poisson’s ratio for both cortical and cancellous bones was 0.3 [22]. The elastic modulus and Poisson's ratio of various structural materials are shown in Table 1. A three-dimensional model of cortical bone and cancellous bone was developed by Boolean operations, and the proximal femoral bone model was built for reassembly. The von Mises stress (VMS) Distribution The VMS patterns of the five loading settings—20 N axial compression, 50 N axial compression, 1 N∙m torsion moment, 1 N∙m lateral bending moment, and 1 N∙m extension bending moments—in the four fixation systems are shown in Fig. 5, Fig. 6, Fig. 7, Fig. 8, and Fig. 9, respectively. The stress values of the ulnar-side plate were lower than those of the dorsal-side plate. In 5 loading settings, obvious stress concentrations were found near the fracture line in the 4 models. The maximum von Mises stress on the fixation plate are recorded in Table 1 and Fig 10. Thus, the maximum von Mises peak stress of the ulnar- side fixation plate were lower, which indicated that the ulnar-side fixation plate could smoothly transfer the load to the proximal cortical bone. The peak stress in the fixation system under 3 rotating moments were apparently reduced by adding a screw in the middle hole of the ulnar head. Although the peak stress decreased only in ulnar-side plate fixation under axial compression, it may evidently concentrate the stress on the middle screw in four fixation systems. Establishment of the finite element models Table 1. The maximum Von Mises peak stresses on fixation plate dorsal-side plate (2 screws) dorsal-side plate (3 screws) ulnar-side plate (2 screws) ulnar-side plate (3 screws) 20N axial compression 392.53MPa 397.17Mpa 383.90Mpa 313.25Mpa 50N axial compression 981.31Mpa 992.93Mpa 958.80Mpa 636.35Mpa 1 N∙m torsion moments 201.05Mpa 158.10Mpa 192.08Mpa 151.94Mpa 1 N∙m lateral bending moments 851.93 MPa 643.20MPa 719.91 MPa 535.43 MPa 1 N∙m extension bending moments 774.30 MPa 632.65 MPa 468.12 MPa 311.61 MPa Loading force settings . The maximum Von Mises peak stresses on fixation plate Table 1. The maximum Von Mises peak stresses on fixation plate In vivo loading conditions in the human DRUJ have not been completely determined. Bernal et al. [23] found that the mean grip force was 18.6 N when performing a daily life activity by measuring different subjects through wearable capacitive pressure sensors in the fingers. Putnam et al.[24] reported that each 10 N of grip force would transmit 26 N of force through the distal ulna metaphysis in the wrist neutral position. If the wrist was no longer in extension and ulnar deviation owing to the variation in hand position during a power grip, the amount of force through the distal ulna metaphysis would decrease. Shaaban et al. [25] reported that the loading of the hand could create an anterior bending force in the distal ulna in half of the forearm and a posterior bending force in the remaining half. Gordon et al. found that a positive bending moment about the medial-lateral axis results from a posteriorly would decrease. Shaaban et al. [25] reported that the loading of the hand could create an anterior bending force in the distal ulna in half of the forearm and a posterior bending force in the remaining half. Gordon et al. found that a positive bending moment about the medial-lateral axis results from a posteriorly Page 4/24 Page 4/24 directed joint reaction force, whereas a positive bending moment about the anterior-posterior axis results from a medially directed joint reaction force[3]. Thus, to simulate forces acting on the ulnar and the osteosynthesis material during daily life activity in subjects who underwent reconstructive surgery, we applied the following loading conditions to each model: 20 N axial compression, 50 N axial compression, 1 N∙m torsion moment, 1 N∙m lateral bending moment, and 1 N∙m extension bending moment. (Fig. 4) [26-28]. Evaluation criteria of the system First, the maximum displacement and its direction of the model were measured. Second, the von Mises stress (VMS) distribution and peak VMS of both the fixation plates and the internal fixation system were observed for four models [29]. Then, the relative displacement of the head and shaft fragments between the fracture ends, which was used to evaluate the support effects, was calculated by measuring the displacement in each direction of the XYZ axis. Finally, the von Mises stress (VMS) distribution and the displacement of the four different models were plotted as a nephogram[30-32].These parameters were used to capture the mechanical factors involved in the fixation stability and fracture healing[33]. Fracture displacement changes The model displacement patterns in the four fixation systems under the two axial compression and three rotating loading settings are shown in Fig. 11 and Fig. 12, respectively. The maximum displacement and the relative displacement of the 4 models are shown in Table 2, Table 3, and Fig. 13. It is clear that the ulnar plate fixation system has smaller maximum displacement and relative displacement, which reflects Page 5/24 Page 5/24 not only better system stability but also less friction and movement between the head and shaft fragments. Table 2. The maximum displacement of the 4 models Table 2. The maximum displacement of the 4 models dorsal-side plate (2 screws) dorsal-side plate (3 screws) ulnar-side plate (2 screws) ulnar-side plate (3 screws) N axial compression 0.403mm 0.386mm 0.301mm 0.248mm N axial compression 1.007mm 0.966mm 0.837mm 0.431mm ∙m torsion moments 0.176mm 0.137mm 0.156mm 0.043mm N∙m lateral bending moments 1.081mm 0.947mm 0.480mm 0.412mm m extension bending moments 0.799mm 0.676mm 0.391mm 0.248mm Table 3. The relative displacement of the head and shaft fragments in 4 models dorsal-side plate (2 screws) dorsal-side plate (3 screws) ulnar-side plate (2 screws) ulnar-side plate (3 screws) xial compression 0.317mm 0.248mm 0.163mm 0.123mm xial compression 0.675mm 0.562mm 0.388mm 0.263mm torsion moments 0.126mm 0.081mm 0.108mm 0.033mm lateral bending moments 0.598mm 0.467mm 0.275mm 0.184mm extension bending moments 0.475mm 0.346mm 0.135mm 0.065mm Table 3. The relative displacement of the head and shaft fragm d l id d l id l id Discussion It is challenging to perform an internal fixation of distal ulnar metaphyseal fractures because the distal fracture fragment is small, comminuted, osteoporotic, and covered with an articular surface over a 270° arc, and surgical exposure of the distal ulna for hardware placement introduces the possibility of damaging the dorsal sensory branch of the ulnar nerve[45]. The most widely used fixation methods are dorsal micro-locking plates and anatomical hook plates, but their merits, drawbacks and mechanical properties remain unclear. Although the hook plate conforms to the ulnar anatomical structure of the distal ulna, there are few screw holes in the head that are arranged vertically, and the screw placement is limited during the operation. On the other hand, the locking plate has more screw holes and the characteristics of pre- bending. Considering that the horizontal arrangement of screws has a higher anti-rotation ability, we propose a method of placing the micro-locking plate on the ulnar side. Nevertheless, limited by the number of clinical cases, retrospective studies are difficult to carry out. Therefore, a new method of analysis is urgently needed. Currently, thanks to the latest development of finite element model generation, such as improved quality of CT imaging, segmentation algorithms and computing power, the accuracy of finite element modelling has been greatly increased[46]. With the maturity of technology, 3D finite element analysis (FEA) can simulate the biomechanical analysis of complex orthopaedic diseases and eliminate the limitation of the lack of cases. In this study, we chose to use FEA to determine whether placing an ulnar-side locking plate has better biomechanical properties than the current choice of a dorsal-side locking plate. We hope the mechanical results of this study provide experimental guidance for its application in clinical surgeries. As shown in Table 2, Table 3 and Fig. 13, the ulnar-side locking plate models provided more stable fixation than the dorsal-side models, and the stability increased from the increased number of head screws. According to the direction of the displacement shown in Figs. 11 and 12, axial compression is more likely to produce palmar, proximal and lateral movement. Under a torsion moment, the radial side of the ulnar head produces more radial-palmar displacement. When the lateral bending force is applied on the ulnar side, the ulnar head fragments move diagonally towards the distal side to the ulnar and dorsal sides. Discussion The static stability of the DRUJ is achieved by the bony congruity between the sigmoid notch of the radius and the ulnar head and by the ligaments that hold the joint together[34]. Part of the ligaments constitute the main stabilizer of the DRUJ, which runs from the fovea of the ulnar head to the dorsal and palmar edges of the sigmoid notch on the distal radius. [35-37]. The distal interosseous membrane (DIOM) of the forearm acts as a secondary soft tissue stabilizer of the DRUJ. The DIOM originates from the distal ulna 54 mm (on average) proximal to the ulnar head and runs distally to insert on the dorsal inferior rim of the sigmoid notch of the radius, which is at the end of the central band of the interosseous membrane [38-40]. Therefore, when the ulnar head breaks, the ligament will lose its stable attachment point, resulting in the instability of the DRUJ. Distal ulnar metaphyseal fracture can be deemed as a fracture ranging from the ulnar neck to within 5 cm of the distal dome of the ulnar head and its high incidence of it is related to osteoporosis[41]. Since 2000, with the development of internal fixation technology and the increasing population of elderly people, the requirements for the recovery of wrist joint function have gradually increased. An increasing number of surgeons choose open reduction and internal fixation to treat unstable distal ulnar fractures[42, 43]. Palmer and Werner[44] showed that up to 42% of force passes through the ulna, in which the axial force passing down the ulnar head fracture end was closer to 20%[44]. The above studies indicated that the loss of the ulna head would disrupt the Page 6/24 Page 6/24 biomechanics and load-bearing capacity of the DRUJ. Therefore, the demand for internal fixation treatment has increased owing to the biomechanical characteristics of ulnar head fractures. However, the number of reported cases and literature studies is rather sparse, which is mostly limited by the low incidence, merely 5-6%, of distal radius fractures accompanied by a distal ulnar metaphyseal fracture.[5]. At present, the treatment of distal ulnar head fractures remains controversial. Conclusions In conclusion, our study indicated that ulnar-side locking plates resulted in a lower stress distribution in the plate and better stability than dorsal-side locking plates for ulnar head fracture fixation. Adding an additional screw to the ulnar head could increase the stability of the fixation system and provide an anti- torsion function. This study requires clinical confirmation of its practicality in the treatment of ulnar head fractures. Discussion Proportionately, when the extension bending force is applied, the ulnar head fragments move to the ulnar and dorsal sides. Fig. 5 through 9 illustrate that the stress of the four fixation systems was concentrated at the fracture line. Both the stress concentration zone and the maximum displacement were decreased in ulnar-side locking plate fixation. As shown in Table 1 and Fig. 10, under torsion, extension and lateral bending moments, the peak VMS in the four fixation models decreased with the addition of the ulnar head screw, which evidently indicated the anti-torsion function of the ulnar head screw. Under axial loading, the peak VMS increased in the dorsal-side fixation models and concentrated at the additional middle screw, whereas it decreased in the ulnar-side fixation models. The Page 7/24 Page 7/24 abovementioned results indicate that ulnar-side locking plate fixation provided better stability, resulting in a lower stress distribution in the plate and greater security of the fixation system. Ulnar-side plate fixation could generate a rigid, stable mechanism and provide a strong resistance to counter compression and torsion effects. Adding the additional screw enabled the fixation models to generate better stability but concentrated the stress on the middle screw, which will guide the design of subsequent plate improvement. This study is the first FEA comparing the mechanical efficiency of dorsal-side locking plates and ulnar-side locking plates in the fixation of ulnar head fractures. However, with the limitation that no experimental validation was conducted and no soft tissue structure was included in the models, the application of these fixation plates still requires more research. This finite element simulation may facilitate further mechanical researches and provide guidance for the clinical treatment of the ulnar head fractures. Consent for publication Not applicable Ethics approval and consent to participate This study was approved by the ethics committee of Shanghai East Hospital affiliated to Tongji University School of Medicine. Informed consent was obtained from all the participants. Abbreviations DRUJ, Distal radius and ulna joint; CT, Computed tomography; VMS, Von Mises Stress; DIOM, Distal interosseous membrane; FEA, Finite element analysis; Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests Competing interests Page 8/24 Funding This study was supported by the Natural Science Foundation of China (81704101), the Emergency and Critical Care Medicine project of Pudong (PWYgf2018-05) and the Project of Shanghai Science and Technology Commission (17411968400). Authors’ contributions Guixin Sun and Yue Zhang designed the study. Yue Zhang and Qin Shao performed the data analysis, and review and submission and was a major contributor in writing the manuscript. Chensong Yang and Yang Yu performed finite element analysis. Changqin Ai performed the manuscript redaction and review. Di Zhou performed the collection of radiography data. Guixin Sun approved the final version of the manuscript. All authors have read and approved the final manuscript. The authors would like to acknowledge Haojun Liu for his help in the picture production. The authors would like to acknowledge Haojun Liu for his help in the picture production. The authors would like to acknowledge Haojun Liu for his help in the picture production. References 1. Shaaban, H., et al., the distal radioulnar joint as a load-bearing mechanism—a biomechanical study1 1No benefits in any form have been received or will be received by a commercial party related directly or indirectly to the subject of this article. The Journal of Hand Surgery, 2004. 29(1): p. 85-95. 2. Naito, K., et al., Assessment of dorsal instability of the ulnar head in the distal radioulnar joint: comparison between normal wrist joints and cases of ruptured extensor tendons. European Journal of Orthopaedic Surgery & Traumatology, 2016. 26(2): p. 161-166. 3. Gordon, K.D., et al., Design and implementation of an instrumented ulnar head prosthesis to measure loads in vitro. J Biomech, 2006. 39(7): p. 1335-41. 4. Kleinman, W.B., Stability of the distal radioulna joint: biomechanics, pathophysiology, physical diagnosis, and restoration of function what we have learned in 25 years. J Hand Surg Am, 2007. 32(7): p. 1086-106. 5. Biyani, A., A.J. Simison, and L. Klenerman, Fractures of the distal radius and ulna. J Hand Surg Br, 1995. 20(3): p. 357-64. 6. Boretto, J.G., et al., Comparative Study of Internal Fixation of the Ulna and Distal Ulna Resection in Patients Older Than 70 Years With Distal Radius and Distal Metaphyseal Ulna Fractures. Hand (N Y), 2019. 14(4): p. 540-546. 7. McKee, M.D., et al., Nonunion of distal radial fractures associated with distal ulnar shaft fractures: a report of four cases. J Orthop Trauma, 1997. 11(1): p. 49-53. Page 9/24 Page 9/24 8. Schuind, F., et al., The distal radioulnar ligaments: a biomechanical study. J Hand Surg Am, 1991. 16(6): p. 1106-14. 9. Nemeth, N. and R.R. Bindra, Fixation of distal ulna fractures associated with distal radius fractures using intrafocal pin plate. J Wrist Surg, 2014. 3(1): p. 55-9. 10. Fernandez, D.L., D. Ring, and J.B. Jupiter, Surgical management of delayed union and nonunion of distal radius fractures. J Hand Surg Am, 2001. 26(2): p. 201-9. 11. Ring, D., Nonunion of the distal radius. Hand Clin, 2005. 21(3): p. 443-7. 11. Ring, D., Nonunion of the distal radius. Hand Clin, 2005. 21(3): p. 443-7. 12. Ruchelsman, D.E., K.B. Raskin, and M.E. Rettig, Outcome following acute primary distal ulna resection for comminuted distal ulna fractures at the time of operative fixation of unstable fractures of the distal radius. Hand (New York, N.Y.), 2009. 4(4): p. 391-396. 12. Ruchelsman, D.E., K.B. Raskin, and M.E. References Rettig, Outcome following acute primary distal ulna resection for comminuted distal ulna fractures at the time of operative fixation of unstable fractures of the distal radius. Hand (New York, N.Y.), 2009. 4(4): p. 391-396. 13. Paksima, N., et al., Fracture of the Distal Ulna Metaphysis in the Setting of Distal Radius Fractures. Bull Hosp Jt Dis (2013), 2017. 75(2): p. 104-108. 13. Paksima, N., et al., Fracture of the Distal Ulna Metaphysis in the Setting of Distal Radius Fractures. Bull Hosp Jt Dis (2013), 2017. 75(2): p. 104-108. 14. Ring, D., et al., Condylar blade plate fixation of unstable fractures of the distal ulna associated with fracture of the distal radius 1 1No benefits in any form have been received or will be received by a commercial party related directly or indirectly to the subject of this article. The Journal of Hand Surgery, 2004. 29(1): p. 103-109. 14. Ring, D., et al., Condylar blade plate fixation of unstable fractures of the distal ulna associated with fracture of the distal radius 1 1No benefits in any form have been received or will be received by a commercial party related directly or indirectly to the subject of this article. The Journal of Hand Surgery, 2004. 29(1): p. 103-109. 15. Dennison, D.G., Open Reduction and Internal Locked Fixation of Unstable Distal Ulna Fractures With Concomitant Distal Radius Fracture. The Journal of Hand Surgery, 2007. 32(6): p. 801-805. 16. Hazel, A., N. Nemeth, and R. Bindra, Anatomic Considerations for Plating of the Distal Ulna. J Wrist Surg, 2015. 4(3): p. 188-93. 17. Lee, S.K., et al., Distal ulna hook plate fixation for unstable distal ulna fracture associated with distal radius fracture. Orthopedics, 2012. 35(9): p. e1358-64. 18. Nunez, F.A., Jr., et al., Distal ulna hook plate: angular stable implant for fixation of distal ulna. Journal of wrist surgery, 2013. 2(1): p. 87-92. 19. Esmaeili, S., et al., A porous polymeric-hydroxyapatite scaffold used for femur fractures treatment: fabrication, analysis, and simulation. Eur J Orthop Surg Traumatol, 2020. 30(1): p. 123-131. 20. Yekta, H.J., et al., Mathematically and experimentally defined porous bone scaffold produced for bone substitute application. Nanomedicine Journal, 2018. 5(4): p. 227-234. 21. Montazeran, A.H., S. Saber-Samandari, and A. Khandan, Artificial intelligence investigation of three silicates bioceramics-magnetite bio-nanocomposite: Hyperthermia and biomedical applications. Nanomedicine Journal, 2018. 5(3): p. 163-171. 22. 8. Schuind, F., et al., The distal radioulnar ligaments: a biomechanical study. J Hand Surg Am, 1991. 16(6): p. 1106-14. References Cun, Y., et al., Traditional and bionic dynamic hip screw fixation for the treatment of intertrochanteric fracture: a finite element analysis. Int Orthop, 2020. 22. Cun, Y., et al., Traditional and bionic dynamic hip screw fixation for the treatment of intertrochanteric fracture: a finite element analysis. Int Orthop, 2020. 23. Cepria-Bernal, J., et al., Grip force and force sharing in two different manipulation tasks with bottles. Ergonomics, 2017. 60(7): p. 957-966. 23. Cepria-Bernal, J., et al., Grip force and force sharing in two different manipulation tasks with bottles. Ergonomics, 2017. 60(7): p. 957-966. 24. Putnam, M.D., et al., Distal radial metaphyseal forces in an extrinsic grip model: Implications for postfracture rehabilitation. The Journal of Hand Surgery, 2000. 25(3): p. 469-475. Page 10/24 Page 10/24 25. Shaaban, H., et al., The load-bearing characteristics of the forearm: pattern of axial and bending force transmitted through ulna and radius. J Hand Surg Br, 2006. 31(3): p. 274-9. 26. Cheng, H.Y., et al., Biomechanical evaluation of the modified double-plating fixation for the distal radius fracture. Clin Biomech (Bristol, Avon), 2007. 22(5): p. 510-7. 27. Nagata, H., S. Hosny, and G.E. Giddins, In-vivo measurement of distal radio-ulnar joint translation. Hand Surg, 2013. 18(1): p. 15-20. 28. Collins, M., et al., Distal Ulna Fractures: A Biomechanical Comparison of Locking Versus Nonlocking Plating Constructs. J Orthop Trauma, 2014. 28(8): p. 470-5. 29. Tianye, L., et al., Finite element analysis of different internal fixation methods for the treatment of Pauwels type III femoral neck fracture. Biomed Pharmacother, 2019. 112: p. 108658. 30. Salmani, M.M., M. Hashemian, and A. Khandan, Therapeutic effect of magnetic nanoparticles on calcium silicate bioceramic in alternating field for biomedical application. Ceramics International, 2020. 46(17): p. 27299-27307. 31. Khandan, A., et al., Fabrication and Characterization of Porous Bioceramic-Magnetite Biocomposite for Maxillofacial Fractures Application. Dental Hypotheses, 2020. 11(3): p. 74-85. 32. Saber-Samandari, S., et al., Micro- Finite Element Model to Investigate the Mechanical Stimuli in Scaffolds Fabricated via Space Holder Technique for Cancellous. International Journal for Simulation and Multidisciplinary Design Optimization, 2020: p. 47-54. 33. Zhang, H., et al., Finite Element Analysis of Different Double-Plate Angles in the Treatment of the Femoral Shaft Nonunion with No Cortical Support opposite the Primary Lateral Plate. Biomed Res Int, 2018. 2018: p. 3267107. 34. Garcia-Elias, M., Soft-tissue anatomy and relationships about the distal ulna. Hand Clin, 1998. 14(2): p. 165-76. 35. af Ekenstam, F. and C.G. References Hagert, The distal radio ulnar joint. The influence of geometry and ligament on simulated Colles' fracture. An experimental study. Scand J Plast Reconstr Surg, 1985. 19(1): p. 27- 31. 36. Palmer, A.K., Triangular fibrocartilage complex lesions: a classification. J Hand Surg Am, 1989. 14(4): p. 594-606. 37. Shaw, J.A., A. Bruno, and E.M. Paul, Ulnar styloid fixation in the treatment of posttraumatic instability of the radioulnar joint: a biomechanical study with clinical correlation. J Hand Surg Am, 1990. 15(5): p. 712-20. 38. Moritomo, H., The distal interosseous membrane: current concepts in wrist anatomy and biomechanics. J Hand Surg Am, 2012. 37(7): p. 1501-7. 39. Moritomo, H., The distal oblique bundle of the distal interosseous membrane of the forearm. J Wrist Surg, 2013. 2(1): p. 93-4. 40. Moritomo, H. and S. Omori, Influence of ulnar translation of the radial shaft in distal radius fracture on distal radioulnar joint instability. J Wrist Surg, 2014. 3(1): p. 18-21. Page 11/24 Page 11/24 41. Logan, A.J. and T.R. Lindau, The management of distal ulnar fractures in adults: a review of the literature and recommendations for treatment. Strategies Trauma Limb Reconstr, 2008. 3(2): p. 49- 56. 42. Chen, W.P., et al., Selection of fixation devices in proximal femur rotational osteotomy: clinical complications and finite element analysis. Clin Biomech (Bristol, Avon), 2004. 19(3): p. 255-62. 43. Azad, A., et al., Epidemiological and Treatment Trends of Distal Radius Fractures across Multiple Age Groups. J Wrist Surg, 2019. 8(4): p. 305-311. 44. Palmer, A.K. and F.W. Werner, Biomechanics of the distal radioulnar joint. Clin Orthop Relat Res, 1984(187): p. 26-35. 44. Palmer, A.K. and F.W. Werner, Biomechanics of the distal radioulnar joint. Clin Orthop Relat Res, 1984(187): p. 26-35. 45. Richards, T.A. and D.N. Deal, Distal ulna fractures. J Hand Surg Am, 2014. 39(2): p. 385-91. 45. Richards, T.A. and D.N. Deal, Distal ulna fractures. J Hand Surg Am, 2014. 39(2): p. 385-91 46. Poelert, S., et al., Patient-specific finite element modeling of bones. Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine, 2012. 227(4): p. 464-478. 46. Poelert, S., et al., Patient-specific finite element modeling of bones. Proceedings of the Institution Mechanical Engineers, Part H: Journal of Engineering in Medicine, 2012. 227(4): p. 464-478. Figures 41. Logan, A.J. and T.R. Lindau, The management of distal ulnar fractures in adults: a review of the literature and recommendations for treatment. Strategies Trauma Limb Reconstr, 2008. 3(2): p. 49- 56. Figures Page 12/24 Page 13/24 Figure 1 3D model of the right ulna built by Geomagic 12. Figure 1 3D model of the right ulna built by Geomagic 12. Page 13/24 Page 13/24 Figure 2 A) Model of dorsal side locking plate with two distal screws. (B) M three distal screws. (C) Model of ulnar side locking plate with two Figure 2 A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 2 Figure 2 A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Page 14/24 Page 14/24 Page 14/24 Figure 3 Stablishing the internal fixation models of ulnar head fracture and importing the models into Abaqus software for meshing Figure 3 Stablishing the internal fixation models of ulnar head fracture and importing the models into Abaqus ft f hi software for meshing. Figure 4 Loading force settings:(A) 20N Axial compression. (B) 50N Axial compression. (C) 1 N∙m Torsion moments . (D) 1 N∙m lateral bending moments. (E) 1 N∙m extension bending moments. Figure 4 Loading force settings:(A) 20N Axial compression. (B) 50N Axial compression. (C) 1 N∙m Torsion moments . (D) 1 N∙m lateral bending moments. (E) 1 N∙m extension bending moments. Page 15/24 Figure 5 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 20N axial compression. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 5 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 20N axial compression. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Page 16/24 Figure 6 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 50N axial compression. Figure 1 (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 6 Figure 6 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 50N axial compression. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Model of ulnar side locking plate with three distal screws. Page 17/24 Page 17/24 Figure 7 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 1 N∙m Torsion moments. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 7 Von Mises Stress distribution of models and fixation plates in four different fixation systems u N∙m Torsion moments. (A) Model of dorsal side locking plate with two distal screws. (B) Mod Figure 7 Figure 7 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 1 N∙m Torsion moments. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. (D) Model of ulnar side locking plate with three distal screws. Page 18/24 Page 18/24 Figure 8 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 1 N∙m lateral bending moments. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 8 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 1 N∙m lateral bending moments. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 6 Page 19/24 Figure 9 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 1 N∙m extension bending moments. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 9 Von Mises Stress distribution of models and fixation plates in four different fixation systems under 1 N∙m extension bending moments. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Page 20/24 Figure 10 The peak Von Mises Stress distribution of four different fixation systems under 5 loading settings. Figure 10 The peak Von Mises Stress distribution of four different fixation systems under 5 loading settings. The peak Von Mises Stress distribution of four different fixation systems under 5 loading settings. The peak Von Mises Stress distribution of four different fixation systems under 5 loading settings. Page 21/24 Figure 11 The model displacement patterns with direction of four different fixation plates under 20N and 50N axia compression loading settings. (A) Model of dorsal side locking plate with two distal screws. (B) Model o dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Figure 11 The model displacement patterns with direction of four different fixation plates under 20N and 50N axial compression loading settings. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Page 22/24 Figure 12 gure 12 e model displacement patterns with direction of four different fixation plates under 1 N∙ oments, 1 N∙m lateral bending moments and 1 N∙m extension bending moments loadin odel of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking stal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ul ate with three distal screws Figure 12 Figure 12 The model displacement patterns with direction of four different fixation plates under 1 N∙m torsion moments, 1 N∙m lateral bending moments and 1 N∙m extension bending moments loading settings. (A) Model of dorsal side locking plate with two distal screws. (B) Model of dorsal side locking plate with three distal screws. (C) Model of ulnar side locking plate with two distal screws. (D) Model of ulnar side locking plate with three distal screws. Page 23/24 Figure 13 (A) The maximum displacement of 4 models under 5 loading settings. (B) The relative displacement of the head and shaft fragments in 4 models under 5 loading settings. Figure 13 Figure 13 (A) The maximum displacement of 4 models under 5 loading settings. (B) The relative displacement of the head and shaft fragments in 4 models under 5 loading settings. Page 24/24
https://openalex.org/W2123561782	https://uu.diva-portal.org/smash/get/diva2:690635/FULLTEXT01	English	null	Suboptimal care and maternal mortality among foreign-born women in Sweden: maternal death audit with application of the ‘migration three delays’ model	BMC pregnancy and childbirth	2,014	cc-by	9,598	© 2014 Esscher et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE Open Access Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Suboptimal care and maternal mortality among foreign-born women in Sweden: maternal death audit with application of the ‘migration three delays’ model Annika Esscher1, Pauline Binder-Finnema1, Birgit Bødker2, Ulf Högberg1, Ajlana Mulic-Lutvica1 and Birgitta Essén1 Abstract Background: Several European countries report differences in risk of maternal mortality between immigrants from low- and middle-income countries and host country women. The present study identified suboptimal factors related to care-seeking, accessibility, and quality of care for maternal deaths that occurred in Sweden from 1988–2010. Methods: A subset of maternal death records (n = 75) among foreign-born women from low- and middle-income countries and Swedish-born women were audited using structured implicit review. One case of foreign-born maternal death was matched with two native born Swedish cases of maternal death. An assessment protocol was developed that applied both the ‘migration three delays’ framework and a modified version of the Confidential Enquiry from the United Kingdom. The main outcomes were major and minor suboptimal factors associated with maternal death in this high-income, low-maternal mortality context. Results: Major and minor suboptimal factors were associated with a majority of maternal deaths and significantly more often to foreign-born women (p = 0.01). The main delays to care-seeking were non-compliance among foreign-born women and communication barriers, such as incongruent language and suboptimal interpreter system or usage. Inadequate care occurred more often among the foreign-born (p = 0.04), whereas delays in consultation/referral and miscommunication between health care providers where equally common between the two groups. Conclusions: Suboptimal care factors, major and minor, were present in more than 2/3 of maternal deaths in this high-income setting. Those related to migration were associated to miscommunication, lack of professional interpreters, and limited knowledge about rare diseases and pregnancy complications. Increased insight into a migration perspective is advocated for maternity clinicians who provide care to foreign-born women. Keywords: Maternal migration effect, Foreign-born, Structured implicit review, Maternal death audit, Low-income country Correspondence: annika.esscher@kbh.uu.se 1Department of Women’s and Children’s Health, International Maternal and Child Health (IMCH), Uppsala University, SE-751 85 Uppsala, Sweden Full list of author information is available at the end of the article Background women cannot be easily explained by well-known ob- stetric and socioeconomic risk factors [1,8,9]. However, maternity care provided to foreign-born women is fre- quently reported as substandard, especially when it comes to availability of medical translation services and culturally competent care providers, and for underestima- tion of an existing condition due to communication barriers [1,2,8,10,11]. Averting maternal mortality may thus be dependent upon provision of optimal emergency care [12] that is provided in a timely way [13]. The necessity of recording the number and causes of death is not in question [14]. Accurate maternal mortality surveillance is considered an important tool for providing appropriate and effective patient care [8]. However, in HIC maternal mortality is rare, and it can take years to ap- proach a clear understanding about risks and how to im- prove health care conditions in a single, particular setting [7,15]. Scrutinising the road to death may therefore clarify why the incidence is higher for different groups of women [4,16,17] and why women representing high risk groups face barriers to care-seeking or regular utilisation of avail- able maternity care services [8,18,19]. Assessing both the quality of maternity care that a woman received and her own pregnancy care strategies may be essential [20]. Two obstetricians independently reviewed each case file using a version of the “Maternal Deaths Enquiry” de- veloped by CMACE, which was modified for this study to reflect the Swedish context. Maternal death in Sweden has not been systematically reviewed by the Swedish Maternal Mortality Group prior to 2007, when it was formed. The present dataset therefore reflect only those data based on medical records and not, for example, on interviews conducted with hospital staff or those medical professionals associated with a particular case. Following the initial review, the additional members of the audit group were provided a copy of the case assess- ments and a narrative summary of each case, which was anonymised by the first author. Each narrative summary contained a description of time-related events relevant to pregnancy, delivery and the postpartum period, and included a woman’s medical history, the available informa- tion about her social conditions and cultural background, her language skills, as well as the surveillance and inter- ventions she received, and the death outcome. These nar- ratives were derived precisely from the records and any missing pertinent information was duly noted. Background for Swedish-born women 5.9, for women born in LIC 21.1, MIC 4.7, and other high-income countries (HIC) 5.5 [5,6]. Over the past decade, Confidential Enquiries into maternal deaths in the United Kingdom (UK) have consistently shown that maternal deaths among ‘Black’ African mothers, including women from LIC settings in sub-Saharan Africa, are significantly more prevalent and have more frequently resulted from direct causes relative to ‘White’ British-born women [7,8]. Differences in maternal mortality between immigrants and host country women are observed in several European countries, often with elevated risk for women coming from low-income countries (LIC) or middle-income countries (MIC) [1-4]. In Sweden during 1988–2007, the maternal mortality ratio for all women was 6 per 100 000 live births, * Correspondence: annika.esscher@kbh.uu.se 1Department of Women’s and Children’s Health, International Maternal and Child Health (IMCH), Uppsala University, SE-751 85 Uppsala, Sweden Full list of author information is available at the end of the article Why women from lower income and low-resource settings are at higher risk of dying than host European Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 2 of 11 were excluded because they either came from other Nordic countries, which have similar languages and health care sys- tems to the Swedish, or from Anglophone countries, which implied that language barriers would be difficult to detect because most Swedish care providers are proficient in English. For each of the remaining 25 foreign-born mater- nal deaths, two Swedish-born cases were matched by ges- tational age (with a cut-off point at 24 weeks) and day of death. That is, if a foreign-born woman died before reach- ing gestational age 24 weeks, she was matched to two Swedish-born cases having died before week 24, as in the case closest before and after the death of the index person. Since there was a cluster of foreign-born women who died towards the end of the study period, we could not always find a Swedish-born woman dying after the index case. To compensate, we chose two cases that died earlier. The medical records comprised all available information about a deceased woman’s treatment and came from the hospi- tals where she either died or received treatment before her death. The latter included information from the antenatal and delivery records from the index pregnancy. Data used for analysis comprised 75 maternal death records. Background In this study, we aimed to identify suboptimal factors of maternity care related to maternal death as it occurred in Sweden over a period of increased migration of childbearing women from LIC and MIC [21]. Our specific objectives were to quantify the medical causes of maternal death, and to explore these in relation to clinical care and sociocultural influences. This approach attempts to unravel the paradox of maternal death in a high-income, social medical system that offers open access to all pregnant women who live in the country. Data collection Maternal death data were collected from Swedish official and national registries (n = 123) for 1988 to 2007 [5], and cases from the Swedish Society of Obstetrics and Gynaecology (SFOG) Maternal Mortality Group for 2008 to 2010 (n = 17). We have defined maternal mortality according to the ICD-9 and ICD-10 definition as “the death of a woman while pregnant or within 42 days (that is, between Day 0 and 41) of termination of preg- nancy – irrespective of the duration and site of the pregnancy – from any cause related to or aggravated by the pregnancy or its management, but not from accidental or incidental causes” [22]. Women’s country of birth was classified according to the World Bank [23]. We then selected all maternal deaths from LIC and MIC during 1988–2010 (n = 26). One of these cases was ex- cluded because the record was not retrievable from the hospital archive. Immigrants from high-income countries Audit protocol: medical and sociocultural factors The structured implicit review [24], which identified major and minor factors of suboptimal care, began with the development of a coded audit protocol. This protocol applied a modified version of the ‘three delays’ model [25], referred to as the ‘migration three delays’ model of Binder et al. [26]. The model assumes a ‘maternal migration effect’ where a woman’s pre-migration experiences in LIC may negatively influence her care-seeking and utilisation Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 3 of 11 following migration to a HIC host country [27]. Phase 1 involves all the social factors underlying a woman’s decision to seek emergency or non-emergency care. Phase 2 facilitates a woman’s ability to identify and reach a medical facility. Phase 3 comprises all the factors that allow a woman to receive adequate and appropriate treat- ment for a suspected obstetric complication. The context in Sweden is that facility-based care and childbirth were the norms of childbearing during the years represented by the dataset. The model thus provides a theoretical concep- tual framework to understand how immigrant women’s sociocultural aspects might have created barriers to their receipt of optimal care in this setting. Moreover, the model can also help to facilitate insight into how HIC maternity care professionals provided care to immigrant women dur- ing these years. The flexibility of the model is that factors identified by it can be made context-specific to any high- income setting. Figure 1 illustrates the sociocultural factors that can influence maternity care-seeking and utilisation in European high-income settings [27]. most likely been avoided by different management of the case [8]. According to the migration ‘three delays’ model, one woman could have experienced several sub- optimal events within the same level of delay. For a case to be assessed as major suboptimal care, at least one of these had to be a major contributing factor. Likewise, suboptimal care would be assessed as minor if there were no major but at least one minor suboptimal factor. If the summary transcripts failed to allow unanimous consensus of suboptimal factors, then the original medical record was read through by the entire audit group. The audit group comprised senior obstetricians. However, when a more complete picture of normative care in other specialties was needed, the audit group consulted relevant specialists in cardiology, neurology, infectious diseases, and pathology. The procedure Th i The severity of suboptimal factors was assessed as fol- lows. A factor was labelled as minor if it was a relevant contributory factor and an alternative management strategy might have made a difference to the outcome, but the mother’s survival was unlikely in any case. A fac- tor was labelled as major if it contributed significantly to the death of the mother, and if the death could have Audit protocol: medical and sociocultural factors Special regard was taken to the year of the death since knowledge, prophy- laxis, and standard treatment for some conditions, such as optimal blood pressure levels, thrombosis prophylaxis, eclampsia prophylaxis, and perimortem caesarean section have been modified over the course of the study period. The analysis and interpretation of sociocultural factors involved a medical anthropologist. Statistical analyses Fisher’s exact test was used to compare suboptimal factors (major + minor) between Swedish-born and foreign- born women considering p-value <0.05 as statistically significant. Figure 1 Factors influencing care-seeking and utilisation of facility-based care and obstetric outcome in high-income western settings [26]. Figure 1 Factors influencing care-seeking and utilisation of facility-based care and obstetric outcome in high-income western settings [26]. Figure 1 Factors influencing care-seeking and utilisation of facility-based care and obstetric outcome in high-incom Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 4 of 11 Details of ethics approval and clinicians involved in the case did not agree on the cause of death. The most common direct cause of death overall was amniotic fluid embolism (AFE); however no instances of major suboptimal care were evident in these cases. All remaining cases resulting from direct death had major contributing suboptimal care factors, includ- ing genital sepsis (3/5), venous thromboembolism (3/5), pre-eclampsia (3/4), and complications resulting from surgery (4/4). Both direct deaths resulting from haemor- rhage were associated with major suboptimal factors of care. Among the overall indirect deaths, the majority re- sulted from cardiovascular conditions, which represent a heterogeneous group of diseases with a fairly even dis- tribution of both major and minor suboptimal care fac- tors. In all cases of non-genital sepsis, avoidable major suboptimal factors were identified, whereas courses of death from conditions of the CNS showed no associations to suboptimal care. Ethics approval for this study was not needed according to Swedish laws on ethical review, since all women were deceased. The regional ethics committee in Uppsala, Sweden, confirmed that the study did not fall into the category of research requiring ethical clearance [2008/381, 2009-01-14]. All heads of clinical departments where a woman had been cared for were asked for consent to share a copy of the medical records. Factors of suboptimal care h f l Overall, 52/73 cases were delivered by caesarean sec- tion, and 12 of those were performed during on-going resuscitation. In two attempted resuscitations the oppor- tunity to perform perimortem caesarean was missed be- cause it was not recommended at the time in Sweden (in 1996 and 2005) as an important part of resuscitation in pregnant women. Only 4 women died outside hospital whereas 14 had failed resuscitation for circulatory arrest during transportation and were dead upon arrival at the hospital. Information on BMI was missing in 20 cases. Overall, five women had BMI ≥35. If a death was sudden or could not be explained by the preceding clinical course of disease, or if the death could be suspected as associated with omissions or incorrect care treatment, a forensic autopsy should be performed. Among the 38 deaths that underwent a clinical autopsy, we identified conditions for which a forensic autopsy could have been performed, but was not. Autopsy was not performed at all in 11 cases. One case was missing information about autopsy. The majority of cases where relatives of the deceased woman opposed autopsy were foreign-born. In the majority of maternal death cases (51/73), suboptimal maternity care was associated with the mother’s death. In 36 of these, at least one major suboptimal factor was identified whereas in the remaining 15 cases, there were no major but at least one minor relevant factor was identified. Table 3 shows that there was a statistically significant difference in numbers of sub- optimal factors, major and minor, between foreign- born and Swedish-born mothers. When calculating LIC and MIC separately, this statistically significant difference remained for women born in LIC, but not for the group of women born in MIC. Delay-causing factors were significantly different between the groups for care-seeking and receipt of quality medical care. However, there were no factors related to care accessibility among the Swedish-born deaths. All of the accessibility- related, delay-causing barriers were identified in the foreign- born group. Cases of maternal death No cases were excluded due to lack of consensus among the audit group. However, of the total maternal death cases, two Swedish-born cases were excluded as coin- cidental deaths. Of the remaining 73 cases, 13 women were foreign-born in LIC (Ethiopia, Eritrea, Somalia, Democratic Republic of Congo, Zimbabwe, Gambia, and Pakistan) and 12 in MIC (Poland, Former Yugoslavia, Turkey, Iran, Iraq, Morocco, Philippines, and Thailand). One foreign-born woman was an adoptee who had originated in a MIC but was brought up in Sweden from early childhood. The me- dian age of all women was 32 years (range 21–45). For the foreign-born, the median age was 29 years (range 21–42), and for the Swedish-born 32.5 years (21–45). The case histories, including mode of pregnancy termination, are summarised in Table 1. Four of the deaths were related to diseases originating in low-income settings. Two of these cases resulted from tuberculosis (TB), one case presented with severe anaemia, and one woman who had HIV died from rheumatic heart disease (discovered at autopsy). In both of the TB cases and the anaemia case, the women received major sub- optimal care. The woman with rheumatic heart disease received minor suboptimal care. Phase 1: Care-seeking No Phase 1 delay alone was assessed as a major or minor contributor to suboptimal care. However, among the foreign-born women, non-compliance was the most common Phase 1 delay to care-seeking. In the example below, Phase 1 factors were assessed as major for non- compliance (woman refused hospital care), Phase 2 factors Table 2 shows causes of death by classification into direct (n = 37) and indirect (n = 35), and includes the number of cases having major and minor factors asso- ciated with suboptimal care. One Swedish case was assessed as a maternal death but it was not possible to classify direct or indirect status because the pathologist Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 5 of 11 Table 1 Case histories, including mode of pregnancy termination, of maternal deaths in Sweden 1988–2010 Total (n = 73) Foreign-born (n = 25) Swedish-born (n = 48) Median age at death in years (range) 32.0 (21–45) 29.0 (21–42) 32.5 (21–45) Died during first pregnancy or after first delivery 32 11 21 Early pregnancy death Miscarriage 8 4 4 Ectopic pregnancy 4 1 3 Mode of delivery Unassisted vaginal delivery 13 7 6 Vacuum extraction 6 2 4 Elective caesarean 2 1 1 Urgent/emergency caesarean 20 4 16 Peri-mortem caesarean1 12 4 8 Death during pregnancy <24 weeks 5 1 4 ≥24 weeks 3 1 2 Place of death Outside hospital 4 1 3 Declared dead at hospital2 14 4 10 District hospital 6 2 4 County hospital 16 6 10 University hospital 33 12 21 BMI (kg/m2) <18.5 4 1 3 18.5–34.9 44 16 28 ≥35 5 1 4 Information missing 20 7 13 Autopsy Clinical 38 8 30 Forensic 23 10 13 Relatives opposed 7 6 1 Not performed other reasons 4 0 4 Information missing 1 1 0 1During on-going resuscitation. 2F il d i i d i Table 1 Case histories, including mode of pregnancy termination, of maternal death Total (n 73) Foreign born including mode of pregnancy termination, of maternal deaths in Sweden 1988–2010 were deemed minor, and the major contributing factor for Phase 3 was due to inadequate care (insufficient surveillance and delayed treatment). stayed with her baby at the neonatology ward. She was advised to come to the obstetric ward to record blood pressure, but no such check-ups were recorded. Phase 1: Care-seeking However, on Day 13, she presented at the obstetric ward as an emergency patient with heavy vaginal bleeding and had an immediate surgical evacuation of the uterus. The lowest Hb recorded, before transfusion, was 3.6 g/dL. One day later she had an irreversible cardiac arrest. Clinical autopsy showed heart infarction. Case A: A 21-year-old primigravida from the Middle East developed pre-eclampsia in gestational week 35 + 2. The patient declined hospital admittance because of communi- cation problems, and was managed instead as an outpatient until the labour was induced. She delivered at hospital at week 36 + 2 with complications of atonic bleeding (2 L). Three days postpartum the woman requested discharge against the recommendations of the doctor. She was still hypertensive (140/100 mmHg), but was discharged and Most examples of Phase 1 delays leading up to the mo- ment of death were minor contributing factors, such as failure to return for follow-up appointments. Other minor contributing factors included late-booking (after 20 weeks) Esscher et al. Phase 1: Care-seeking BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 6 of 11 Table 2 Suboptimal factors associated with maternal death by cause of death in Sweden 1988–2010 Causes of death Total Foreign-born Swedish-born n = 73 Cases with suboptimal factors (major + minor) n = 25 Cases with suboptimal factors (major + minor) n = 48 Cases with suboptimal factors (major + minor) Direct 37 28 (20 + 8) 16 14 (11 + 3) 21 14 (9 + 5) AFE1 7 5 (0 + 5) 2 2 (0 + 2) 5 3 (0 + 3) Genital sepsis 5 4 (3 + 1) 2 1 (1 + 0) 3 3 (2 + 1) VTE2 5 3 (3 + 0) 2 1 (1 + 0) 3 2 (2 + 0) Pre-eclampsia3 4 4 (3 + 1) 2 2 (1 + 1) 2 2 (2 + 0) Surgery4 4 4 (4 + 0) 3 3 (3 + 0) 1 1 (1 + 0) Ectopic pregnancy 3 2 (1 + 1) 0 0 3 2 (1 + 1) Haemorrhage5 2 2 (2 + 0) 2 2 (2 + 0) 0 0 Other direct6 7 4 (4 + 0) 3 3 (3 + 0) 4 1 (1 + 0) Indirect 35 23 (16 + 7) 9 8 (4 + 4) 26 15 (12 + 3) Cardiovascular7 21 15 (8 + 7) 5 5 (1 + 4) 16 10 (7 + 3) Non-genital sepsis 6 6 (6 + 0) 3 3 (3 + 0) 3 3 (3 + 0) CNS8 6 0 1 0 5 0 Other indirect9 2 2 (2 + 0) 0 0 2 2 (2 + 0) Unclear10 1 0 0 0 1 0 1Amniotic fluid embolism. 2Venous thromboembolism. 3Pre-eclampsia, eclampsia, HELLP syndrome. 4Complication of caesarean section (n = 2), complication of anaesthesia (n = 2). 5Cervix rupture (n = 1), postpartum haemorrhage (n = 1). 6Acute fatty liver of pregnancy (n = 1), peripartum cardiomyopathy (n = 2), anaemia (n = 1), unclear cause of death assessed as direct by the audit group (n = 3). 7Myocarditis (n = 3), myocardial infarction (n = 4), arrthymia/SADS (sudden arrhythmic death syndrome) (n = 3), aortic aneurysm (n = 8), rupture of splenic artery (n = 1), rheumatic valve disease (n = 1), congenital heart disease (n = 1). 8Central nervous system; intracranial haemorrhage (n = 5), epilepsy (n = 1). Phase 1: Care-seeking 9Pulmonary oedema (n = 1), ileus (n = 1). 10Assessed as a maternal death, however it is unclear if it was direct or indirect. for antenatal care (n = 3) and delayed care-seeking when an early pregnancy complication occurred (n = 2). diagnosed her signs and symptoms as anxiety. During the morning of the surgical evacuation procedure at Hospital 1 she was assessed by an anaesthesiologist with help of a professional interpreter. As stated in the record, the patient said she was feeling well. However, there is no indication she mentioned about the emergency visit at Hospital 2 the night before. Immediately following the evacuation procedure, she had a cardiac arrest. Autopsy showed pulmonary embolism. Phase 2: Accessibility of services Limited language congruence created one or more Phase 2 barriers to accessing health care services among foreign- born women and their care providers. Where language incongruence was identified as the major barrier to acces- sibility (n = 3), the failure to communicate limited the woman’s ability to explain her health concerns to the care provider or from being forthcoming about additional care she had received elsewhere. The following example repre- sents a death that was assessed as minor Phase 1 delay for non-compliance (withholding information), but major Phase 2 for limited language congruence and major Phase 3 for inadequate care (wrong diagnosis). Phase 3: Quality of medical care I d h Phase 3: Quality of medical care Inadequate care was the most common contributing factor to maternal death in both groups. However, among the foreign-born women, the influence of delay-causing barriers from Phases 1 and 2 could not be ignored. For example, out of 14 cases of major inadequate care, 10 cases also had Phase 2 barriers from, e.g. limited language congruence, and 8 of these also had Phase 1 barriers, including non-compliance or late-booking. The following example describes contributing factors as minor Phase 1 for late-booking, minor Phase 2 for limited language congruence, and major Phase 3 for inadequate care (delayed treatment) and delayed referral. Case B: A 42-year old sub-Saharan African multipara, with two childbirths in Sweden but whose Swedish was recorded as poor, had an surgical evacuation of the uterus procedure planned at Hospital 1 for a miscarriage. The night before surgery she sought care at Hospital 2 for chest pains. No interpreter use was indicated. The doctor Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 7 of 11 Table 3 Suboptimal factors associated with maternal death by phase of delay in Sweden 1988–2010. Phase 3: Quality of medical care I d h Table 3 Suboptimal factors associated with maternal death by phase of delay in Sweden 1988–2010. Fisher’s exact test (p < 0.05) 3 Suboptimal factors associated with maternal death by phase of delay in Sweden 1988–2010. 0 05) actors associated with maternal death by phase of delay in Sweden 1988–2010. Fisher’s exact se of delay in Sweden 1988–2010. Fisher’s exact 1The care on each level was assessed as major suboptimal if there was at least one major suboptimal factor, and minor if there were no major but at least one minor suboptimal factor. Note that the specified suboptimal factors within one level could add up to more than the total, because one woman could have several suboptimal factors within the same level, i.e. the numbers within each level could be more than by level. 2Recently arrived refugee (n = 1); lived in Sweden instead of Denmark due to Danish immigration law (n = 1). 3Referral from primary care to hospital, or between departments within a hospital. 4Lack of beds in intensive care unit (n = 4); delayed surgical procedure due to prioritisation of another patient (n = 1). resulted from inadequate care included misdiagnosis, lack of reacting to typical symptoms, or insufficient surveillance. The influence of Phase 1 delays among the Swedish- born women included non-compliance and influence of unhealthy lifestyle. Case C: A sub-Saharan African woman who had given birth to one child in Sweden had lived in the country for more than four years, but was recorded as speaking poor Swedish. No interpreter use was documented. Antenatal care was booked late at gestation week 25. At that time the blood pressure was 110/60 mmHg. One week later, she presented at the same local clinic with nausea, abdominal and back pain, and a blood pressure of 155/85 mmHg. The next day she returned and tested positive for white and red blood cells, nitrite, and proteinuria (3+). Blood pres- sure was 130/70. She was prescribed treatment for a urinary tract infection and sent home. According to the investigation after her death, the woman had sought her general practitioner the day before her death, and again earlier on the same day, for headache. Sometime after her visit to the general practitioner but on the same day, she was admitted to the hospital ICU, had an eclamptic fit, and developed intracerebral haemorrhage. The patient was moved to the Neuro-ICU. Phase 3: Quality of medical care I d h Fisher’s exact test (p < 0.05) Suboptimal factor Total (N = 73) Foreign-born (N = 25) Swedish-born (N = 48) Cases with suboptimal factors (major + minor)1 Cases with suboptimal factors (major + minor)1 Cases with suboptimal factors (major + minor)1 p value (Fisher’s exact test) Total 51 (36 + 15) 22 (15 + 7) 29 (21 + 8) 0.01 Phase 1: Care-seeking 18 (6 + 12) 11 (1 + 10) 7 (5 + 2) 0.01 Non-compliance 10 (3 + 7) 6 (1 + 5) 4 (2 + 2) 0.08 ns Late-/non-booking 5 (0 + 5) 5 (0 + 5) 0 Unhealthy lifestyle (substance abuse) 3 (3 + 0) 0 3 (3 + 0) Phase 2: Accessibility of services 14 (3 + 11) 14 (3 + 11) 0 Limited language congruence 13 (3 + 10) 13 (3 + 10) 0 Incomplete legal status2 2 (0 + 2) 2 (0 + 2) 0 Delayed transport 1 (0 + 1) 1 (0 + 1) 0 Phase 3: Quality of medical care 50 (34 + 16) 21 (15 + 6) 29 (19 + 10) 0.03 Inadequate care 49 (31 + 18) 21 (14 + 7) 28 (17 + 11) 0.02 Delay in consultation or referral3 24 (16 + 8) 10 (8 + 2) 14 (8 + 6) 0.2 ns Appropriate care, but too late 16 (11 + 5) 5 (4 + 1) 11 (7 + 4) 0.5 ns Miscommunication between providers 9 (5 + 4) 5 (3 + 2) 4 (2 + 2) 0.1 ns Limited use/priority of resources4 5 (1 + 4) 2 (1 + 1) 3 (0 + 3) 0.6 ns 1The care on each level was assessed as major suboptimal if there was at least one major suboptimal factor, and minor if there were no major but at least one minor suboptimal factor. Note that the specified suboptimal factors within one level could add up to more than the total, because one woman could have several suboptimal factors within the same level, i.e. the numbers within each level could be more than by level. 2Recently arrived refugee (n = 1); lived in Sweden instead of Denmark due to Danish immigration law (n = 1). 3Referral from primary care to hospital, or between departments within a hospital. 4Lack of beds in intensive care unit (n = 4); delayed surgical procedure due to prioritisation of another patient (n = 1). Strength and limitations A major strength of the study design is that the majority of cases were linked to three sources of register data: the Swedish cause of death register, the medical birth register, and the national patient register [5]. We further managed to obtain complete medical records for all but one foreign-born woman, who was excluded from the data- set. During the performance of the structured implicit review, our panel of clinical experts reached unanimous consensus per case while assessing the maternal death files, which strengthens validity [24]. We created a platform of normative knowledge that approximated the clinical con- text for when the maternal deaths occurred by conducting the audit with clinical experts who were employed as obste- tricians during the time period of the dataset. The group of expert auditors might have included a wider base of obstet- ric specialists, e.g., midwives and obstetric anaesthesiolo- gists. However, since the number of maternal death cases was small, it was preferred to consult with these specialists as the need arose. Similarly, experts from complementary specialities, i.e. cardiology and neurology, were consulted to fill gaps in knowledge about particular clinical events that occurred in the records. The introduction of a medical an- thropologist was further necessary to broaden the holistic interpretation of the sociocultural aspects, and for expand- ing a theoretical base in maternal mortality research. These steps were necessary for applying the ‘migration three de- lays’ model, and for explaining how and why medical and social factors might relate to immigrant women’s experi- ences of maternity care in this HIC setting. Priority of adequate consultation and referral is cited among the top-10 recommendations of the UK Confiden- tial Enquiries into maternal deaths [8]. Enquiry into adverse maternal outcomes and patient safety has also shown that failures in teamwork can contribute to maternal death [7,8,31]. In our study, some level of provider-provider miscommunication contributed as a failure to optimal care. This includes information transfer between specialists but also between clinical departments. Furthermore, much focus has been on developing teamwork skills within a specific emergency situation, as in the delivery room [32]. Our results support that teamwork, both when taking care of a severely ill pregnant woman and when emergency situations arise in the labour room, should be multidisciplinary and include the timely en- trance of complementary specialties, such as anaesthe- sia, cardiology, and surgical ICU [33]. Suboptimal quality of maternal care d l d d d l d y Inadequate care included delayed or misdiagnosis. Several of these cases strongly suggest errors from premature satisfaction of the search, which is when the care pro- vider becomes satisfied with an investigation once a sin- gle diagnosis is identified, even if it is not the root cause of the problem [28]. Other examples include inadequate and delayed treatment for hypertensive disorders and sepsis. This finding is consistent with substandard care reported from other European countries [8,29]. However, in the two cases that missed performing perimortem caesarean section, the audit group was unable to clas- sify the providers’ limited knowledge about standards for resuscitation of pregnant women as suboptimal be- cause the European guidelines were not yet published at the time the deaths occurred [30]. Major findings Almost all (69/73; 95%) women died at or on their way to a hospital. In two-thirds of cases suboptimal factors were identified. In half of these cases at least one major subopti- mal factor contributed to the woman’s death irrespective of country of birth. Nevertheless, suboptimal care was a significantly more frequent contributing factor of maternal death for the foreign-born women than for the Swedish women. Among the foreign-born cases, many of these deaths were associated with communication-related bar- riers and delays to care-seeking. For example, failed ability to access clinical services was mainly due to language barriers and substandard interpretation services. The main delay to care-seeking was women’s non-compliance of care provider advice or treatment recommendations. At the medical facility, inadequate care occurred more often among the foreign-born, whereas delays in consultation/ referral and miscommunication between providers showed no significant differences. Phase 3: Quality of medical care I d h Both treatment with magnesium and caesarean section were delayed for more than 12 hours because the obstetrician in charge did not attend the patient at the Neuro-ICU unit. Two weeks later the woman died. Case D: A 23 year old Swedish woman relocated during an unplanned pregnancy from a rural Swedish community to live with her boyfriend in one of the major urban centres. The boyfriend had been imprisoned due to drug criminality and continued being criminal during the course of the pregnancy. No suspicion was ever raised that the woman was also using drugs. She had a cardiac arrest at 36 weeks, and the autopsy showed sepsis with staphylococcus aureus. Amphetamine was found in both hair and blood samples. The Swedish women encountered no Phase 2 delays, including those that might occur in relation to receipt of optimal care in Phase 3. The remaining Phase 3 barriers to optimal care were not significantly different between the two groups. These included delayed consultation or referral, delayed receipt of appropriate care, and lack or prioritization of available resources. Examples of bar- riers between providers included transfer of information between specialists, for example between an obstetrician and anaesthesiologist, as well as failure to recognise needing assistance. Although inadequate care was more frequent among the foreign-born women, this was also the most commonly occurring barrier for the Swedish-born. The deaths that Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 8 of 11 Discussion Major findings deaths nationally and by representing nearly completed record materials. The recruitment of cases represents an extended calendar period, which can be regarded as a limitation because both recording processes and clinical routines evolved over the study period. However, the long period of time was necessary in order to gain a util- isable sample size in this small population of Sweden. Additionally, the entrance of the foreign-born sample increased greatly during the later years of the study period, and constituted nearly half of the cases during the years 2006–2010. This potential limitation complicated the original matching procedure, but was overcome by matching two Swedish-born women who died before the index case. A weakness of the analysis and interpretation of sociocultural factors was the lack of interviews with professionals, as performed by the CMACE procedures or with relatives as performed during verbal autopsy, which could have minimised the risk for representation errors. Strength and limitations Lack of intensive care beds was assessed as a factor contributing to four maternal deaths in this study, although we cannot say The main limitation is that our outcome interpreta- tions are dependent upon a small sample size. However, these unique data are validated by having tracked the Page 9 of 11 Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 9 of 11 if it was a matter of de facto insufficient resources or insufficient communication between specialists, overview and triage of critically ill patients at a hospital level [34]. Reliance on interpreter service Interpreter use or the recording of interpreter usage was poor across the dataset despite that, over this time period, Swedish Law established the right to equitable health care for all inhabitants and that a patient has the right to a professional interpreter [35,36]. However, underuse of interpretation services was implied when the case file stated that the husband or other relative acted as trans- lator during some component of the care leading up to the maternal death event. Standard guidelines for opti- mal medical interpretation services in a maternity care setting are limited [11]. Since the time period repre- sented in this dataset, some European countries have implemented guidelines discouraging the use of per- sonally known interpreters [8]. The implicit problem with lack of optimal interpretation services is that the language barriers between a woman and her care provider limit her ability to access and understand biomedical knowledge and similarly for the care provider to impart it [26]. Our findings are consistent with communication re- lated problems for pregnant immigrant women in other western settings who experienced adverse birth outcomes [7,8,37-39]. In Sweden, one study also showed that lack of interpreter use contributed to perinatal deaths occurring between 1990 and 1996 [10]. The importance of high-standard maternal autopsies to assure correct causes of death was emphasized in the latest UK maternal mortality report, and the recommen- dation is that maternal autopsies should be centralised [8]. We identified cases for which the quality of autopsy could be questioned. Some cases of sudden, unexpected death also fulfilled the criteria for forensic autopsy according to Swedish law, but then were not performed [46]. This sug- gests some clinicians have limited knowledge about laws regulating post-mortem examinations. Some of the rela- tives of the deceased women opposed autopsy. However, if the cause of death is unknown then the legal right to per- form it remains intact even if the relatives oppose [47]. Clinical implications l h It is essential that maternal care providers are prepared to identify and meet the needs of all women, especially immigrant women, who may be at increased risk for ma- ternal death or for experiencing other adverse pregnancy outcomes. This study emphasizes the obvious importance of using professional interpreters to avert adverse obstetric outcomes [8,10,11]. Regulations stating the right to pro- fessional interpretation for non-native speaking persons, such as those found in Sweden [35,36,47], should be incorporated into care management strategies across immigrant-receiving countries. Education of health care professionals should also not stereotype women’s care needs based upon presumed sociocultural barriers. Im- migrant women want competent maternity care, just as all women, and the ‘culture-sensitive model’ needs to be founded on insight gleaned from research [11]. Gaining insight into the modified migration model used here could be useful to care professionals in other HIC settings for identifying context-dependent knowledge about any sociocultural factors relevant for immigrant women in their communities, and also for expanding sociocultural variables into maternal death reporting. Additionally, all patients receiving care in Sweden have the legal right to decline care [35]. However, limited guidelines exist for training professionals when it occurs [48]. Similarly, it is Causes of death The most frequent causes of direct maternal death among the foreign-born women are comparable to those reported for maternal deaths across Europe [1,3,7,8,33,42]. Four women who died from diseases not usually seen in Sweden or Europe were from LIC, primarily sub-Saharan Africa. The association of limited professional insight into to those diseases illustrates the importance of improving knowledge about rare diseases complicating pregnancies, such as tuberculosis, rheumatic heart disease and severe anaemia, which have lately returned to European obstetrics [43-45]. Limited care provider knowledge about such pregnancy complications may also conflict with women’s limited insight into identifying risks to her pregnancy [27]. Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 10 of 11 The Netherlands 1983–1992. Eur J Obstet Gynecol Reprod Biol 1998, 79(1):57–62. The Netherlands 1983–1992. Eur J Obstet Gynecol Reprod Biol 1998, 79(1):57–62. The Netherlands 1983–1992. Eur J Obstet Gynecol Reprod Biol 1998, 79(1):57–62. The Netherlands 1983–1992. Eur J Obstet Gynecol Reprod Biol 1998, 79(1):57–62. also important to identify management strategies for non- booking and non-compliance, and to appreciate that failure to comply is the problem of both women and care pro- fessionals. Western maternal care providers also need to increase their skills in recognition and interpretation of symptoms and appreciate that immigrant women may have other medical needs than those expected for host country women. Since suboptimal medical care contrib- utes to many deaths that are simultaneously considered rare, it is important not to overlook ways to learn from adverse events and improve clinical management. Philibert M, Deneux-Tharaux C, Bouvier-Colle MH: Can excess maternal mortality among women of foreign nationality be explained by suboptimal obstetric care? BJOG 2008, 115(11):1411–1418. Philibert M, Deneux-Tharaux C, Bouvier-Colle MH: Can excess maternal mortality among women of foreign nationality be explained by suboptimal obstetric care? BJOG 2008, 115(11):1411–1418. Schutte JM, Steegers EA, Schuitemaker NW, Santema JG, de Boer K, Pel M, Vermeulen G, Visser W, van Roosmalen J: Rise in maternal mortality in The Netherlands. BJOG 2010, 117(4):399–406. 4. Esscher A, Haglund B, Högberg U, Essén B: Excess mortality in women of reproductive age from low-income countries: a Swedish national register study. Eur J Public Health 2013, 23(2):274–279. y 5. Esscher A, Högberg U, Haglund B, Essén B: Maternal mortality in Sweden 1988–2007: more deaths than officially reported. Acta Obstet Gynecol Scand 2013, 92(1):40–46. 6. Esscher A: Maternal Mortality in Sweden. Classification, Country of Birth, and Quality of Care. Uppsala: Uppsala University; 2014. Author details 1 1Department of Women’s and Children’s Health, International Maternal and Child Health (IMCH), Uppsala University, SE-751 85 Uppsala, Sweden. 2Department of Obstetrics and Gynaecology, Hillerød Hospital, Hillerød, Denmark. 21. Högberg U: An “American dilemma” in Scandinavian childbirth: unmet needs in healthcare? Scand J Public Health 2004, 32(1):75–77. 22. World Health Organization: ICD-10: International statistical classification of diseases and related health problems: tenth revision. Volume 2. Instruction manual, Volume 2. 2nd edition. Geneva: World Health Organization; 2004. Received: 1 November 2013 Accepted: 9 April 2014 Published: 12 April 2014 Received: 1 November 2013 Accepted: 9 April 2014 Published: 12 April 2014 23. World Bank Country Classification. http://data.worldbank.org/. 24. Ashton CM, Kuykendall DH, Johnson ML, Wray NP: An empirical assessment of the validity of explicit and implicit process-of-care criter for quality assessment. Med Care 1999, 37(8):798–808. Competing interests The authors declare that they have no competing interests. 15. Liljestrand J: Strategies to reduce maternal mortality worldwide. Curr Opin Obstet Gynecol 2000, 12(6):513–517. Authors’ contribution BE had the original idea. AE, PBF and BE prepared the protocol. AE and AML scrutinised the medical records. AE, BB, AML, UH and BE constituted the audit group. All co-authors participated in data analysis, and AE and PBF interpreted the data and wrote the manuscript in collaboration with the other co-authors. All authors have approved the final manuscript. 16. Elebro K, Rööst M, Moussa K, Johnsdotter S, Essén B: Misclassified maternal deaths among East African immigrants in Sweden. Reprod Health Matters 2007, 15(30):153–162. 17. Saucedo M, Deneux-Tharaux C, Bouvier-Colle MH: Understanding regional differences in maternal mortality: a national case–control study in France. BJOG 2012, 119(5):573–581. Acknowledgements 18. Maine D, Rosenfield A: The AMDD program: history, focus and structure. Int J Gynaecol Obstet 2001, 74(2):99–103. discussion 104. The authors express appreciation to Christina Christersson MD PhD, Torsten Danfors MD PhD, Katharina Ericson MD, Maria Lundberg MD, Uppsala University for consulting about their individual medical specialities. This work was supported by a grant from the Swedish Council for Working Life and Social Research [FAS 2007–2026] and by the Medical Faculty, Uppsala University. The authors express appreciation to Christina Christersson MD PhD, Torsten Danfors MD PhD, Katharina Ericson MD, Maria Lundberg MD, Uppsala University for consulting about their individual medical specialities. 19. National Collaborating Centre for Women’s and Children’s Health: Antenatal care. Routine care for the healthy pregnant woman. In NICE Clinical Guideline 62. London: National Intitute for Health and Clinical Excellence; 2008. This work was supported by a grant from the Swedish Council for Working Life and Social Research [FAS 2007–2026] and by the Medical Faculty, Uppsala University. 20. Essén B: Perinatal mortality among immigrants from Africa’s Horn. The importance of experience, rationality, and tradition for risk assessment in pregnancy and childbirth. Malmö: Lund University; 2001. Conclusions 7. Confidential Enquiry into Maternal and Child Health (CEMACH): In Saving mothers’ lives: Reviewing maternal deaths to make motherhood safer, 2003–2005. The Seventh Report on Confidential Enquiries into Maternal Deaths in the United Kingdom. Edited by Lewis G. London: CEMACH; 2007. Combining a maternal death audit procedure with a theoretical conceptual framework has helped to iden- tify migration-related barriers to optimal care that are associated with maternal death in this high-income set- ting. The majority of maternal deaths were related to sub- optimal care factors, but also to women’s care-seeking strategies and their limited ability to access quality mater- nity services. Immigrants from LIC and MIC are at in- creased risk of maternal death and this study advocates better insight into a migration perspective for clinicians who provide care to foreign-born women. 8. Centre for Maternal and Child Enquiries (CMACE): Saving mothers’ lives: Reviewing maternal deaths to make motherhood safer, 2006–2008. The Eighth Report of the Confidential Enquiries into Maternal Deaths in the United Kingdom. BJOG 2011, 118(Suppl 1):1–203. 8. Centre for Maternal and Child Enquiries (CMACE): Saving mothers’ lives: Reviewing maternal deaths to make motherhood safer, 2006–2008. The Eighth Report of the Confidential Enquiries into Maternal Deaths in the United Kingdom. BJOG 2011, 118(Suppl 1):1–203. 9. Ibison JM, Swerdlow AJ, Head JA, Marmot M: Maternal mortality in England and Wales 1970–1985: an analysis by country of birth. Br J Obstet Gynaecol 1996, 103(10):973–980. 9. Ibison JM, Swerdlow AJ, Head JA, Marmot M: Maternal mortality in England and Wales 1970–1985: an analysis by country of birth. Br J Obstet Gynaecol 1996, 103(10):973–980. 10. Essén B, Bødker B, Sjöberg NO, Langhoff-Roos J, Greisen G, Gudmundsson S, Östergren PO: Are some perinatal deaths in immigrant groups linked to suboptimal perinatal care services? BJOG 2002, 109(6):677–682. 10. Essén B, Bødker B, Sjöberg NO, Langhoff-Roos J, Greisen G, Gudmundsson S, Östergren PO: Are some perinatal deaths in immigrant groups linked to suboptimal perinatal care services? BJOG 2002, 109(6):677–682. 11. Binder P, Borne Y, Johnsdotter S, Essén B: Shared language is essential: communication in a multiethnic obstetric care setting. J Health Commun 2012, 17(10):1171–1186. 11. Binder P, Borne Y, Johnsdotter S, Essén B: Shared language is essential: communication in a multiethnic obstetric care setting. J Health Commun 2012, 17(10):1171–1186. Abbreviations BMI B d BMI: Body mass index; CMACE: Centre for Maternal and Child Enquiries; Hb: Haemoglobin; HIC: High-income country; HIV: Human immunodeficiency virus; ICD: International statistical classification of diseases and related health problems; ICU: Intensive care unit; LIC: Low-income country; MIC: Middle-income country; SFOG: Swedish Society of Obstetrics and Gynaecology; TB: Tuberculosis; UK: United Kingdom. 12. Freedman LP, Graham WJ, Brazier E, Smith JM, Ensor T, Fauveau V, Themmen E, Currie S, Agarwal K: Practical lessons from global safe motherhood initiatives: time for a new focus on implementation. Lancet 2007, 370(9595):1383–1391. 12. Freedman LP, Graham WJ, Brazier E, Smith JM, Ensor T, Fauveau V, Themmen E, Currie S, Agarwal K: Practical lessons from global safe motherhood initiatives: time for a new focus on implementation. Lancet 2007, 370(9595):1383–1391. 13. Campbell OM, Graham WJ: Strategies for reducing maternal mortality: getting on with what works. Lancet 2006, 368(9543):1284–1299. 13. Campbell OM, Graham WJ: Strategies for reducing maternal mortality: getting on with what works. Lancet 2006, 368(9543):1284–1299. 14. Pattinson RC, Say L, Makin JD, Bastos MH: Critical incident audit and feedback to improve perinatal and maternal mortality and morbidity. Cochrane Database Syst Rev 2005, 4, CD002961. 14. Pattinson RC, Say L, Makin JD, Bastos MH: Critical incident audit and feedback to improve perinatal and maternal mortality and morbidity. Cochrane Database Syst Rev 2005, 4, CD002961. Negative responses to care-seeking A number of cases related to care-seeking were influenced by such factors as late-booking and non-compliance. However, it is difficult to reconstruct a woman’s decision- making processes around her choices to seek care. The medical record can only document her registered contacts with a care centre, which become the perspective of the care provider, and from these we can presume whether or not a woman chose to comply. Sociocultural expla- nations for delays to care-seeking have been theorised as a ‘maternal migration effect’, where factors related to the pre-migration experience, have the potential to in- fluence women’s obstetric choices after migrating to a new setting [27]. Other probable factors include mutual broken trust during the care encounter, which is also ex- hibited as women’s non-compliance/limited adherence to treatment advice and refusal of care as well as care provider frustration at not being able to impart quality care as a matter of course [26,40]. These negative ap- proaches to care-seeking have the potential to delay the provision of optimal, preventive advice as well as timely referral. Mutual trust therefore requires that a woman’s care-seeking strategies are synonymous with the provision of trustworthy care [41]. Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Page 11 of 11 48. Essén B, Johnsdotter S, Binder P: Maternal mortality in Sweden–comprehending the incomprehensible refusal to accept acute caesarean section. In Women’s Health to Gender Medicine, Volume 75. Stockholm: The Swedish Council for Working Life and Social Research (FAS); 2011. doi:10.1186/1471-2393-14-141 Cite this article as: Esscher et al.: Suboptimal care and maternal mortality among foreign-born women in Sweden: maternal death audit with application of the ‘migration three delays’ model. BMC Pregnancy and Childbirth 2014 14:141. 48. Essén B, Johnsdotter S, Binder P: Maternal mortality in Sweden–comprehending the incomprehensible refusal to accept acute caesarean section. In Women’s Health to Gender Medicine, Volume 75. Stockholm: The Swedish Council for Working Life and Social Research (FAS); 2011. 48. Essén B, Johnsdotter S, Binder P: Maternal mortality in Sweden–comprehending the incomprehensible refusal to accept acute caesarean section. In Women’s Health to Gender Medicine, Volume 75. Stockholm: The Swedish Council for Working Life and Social Research (FAS); 2011. 26. Binder P, Johnsdotter S, Essén B: Conceptualising the prevention of adverse obstetric outcomes among immigrants using the ‘three delays’ framework in a high-income context. Soc Sci Med 2012, 75(11):2028–2036. 27. Binder P: The maternal migration effect. Exploring maternal healthcare in diaspora using qualitative proxies for medical anthropolgy. Uppsala: Uppsala University; 2012. doi:10.1186/1471-2393-14-141 Cite this article as: Esscher et al.: Suboptimal care and maternal mortality among foreign-born women in Sweden: maternal death audit with application of the ‘migration three delays’ model. BMC Pregnancy and Childbirth 2014 14:141. 28. Croskerry P: The importance of cognitive errors in diagnosis and strategies to minimize them. Acad Med 2003, 78(8):775–780. 29. Schutte JM, Schuitemaker NW, van Roosmalen J, Steegers EA: Substandard care in maternal mortality due to hypertensive disease in pregnancy in The Netherlands. BJOG 2008, 115(6):732–736. 30. Soar J, Deakin CD, Nolan JP, Abbas G, Alfonzo A, Handley AJ, Lockey D, Perkins GD, Thies K: European Resuscitation Council guidelines for resuscitation 2005. Section 7. Cardiac arrest in special circumstances. Resuscitation 2005, 67(Suppl 1):S135–S170. 31. Cornthwaite K, Edwards S, Siassakos D: Reducing risk in maternity by optimising teamwork and leadership: an evidence-based approach to save mothers and babies. Best Pract Res Clin Obstet Gynaecol 2013. 32. Siassakos D, Draycott TJ, Crofts JF, Hunt LP, Winter C, Fox R: More to teamwork than knowledge, skill and attitude. BJOG 2010, 117(10):1262–1269. 33. References 1. Schuitemaker N, van Roosmalen J, Dekker G, van Dongen P, van Geijn H, Bennebroek Gravenhorst J: Confidential enquiry into maternal deaths in 25. Thaddeus S, Maine D: Too far to walk: maternal mortality in context. Soc Sci Med 1994, 38(8):1091–1110. Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Bødker B, Hvidman L, Weber T, Møller M, Aarre A, Nielsen KM, Sørensen JL: Maternal deaths in Denmark 2002–2006. Acta Obstet Gynecol Scand 2009, 88(5):556–562. 34. Sinuff T, Kahnamoui K, Cook DJ, Luce JM, Levy MM: Rationing critical care beds: a systematic review. Crit Care Med 2004, 32(7):1588–1597. 35. Swedish Ministry of Social Welfare: Health Care Act 1982:763. Stockholm: Hälso- och sjukvårdslagen; 1982. j g Swedish Ministry of Justice: Administrative Act 1986:223, Volume vol. § 36. Swedish Ministry of Justice: Administrative Act 1986:223, Volume vol. § 8. Stockholm: [Förvaltningslagen]; 1986. 36. Swedish Ministry of Justice: Administrative Act Stockholm: [Förvaltningslagen]; 1986. Stockholm: [Förvaltningslagen]; 1986. 37. Essén B, Johnsdotter S, Hovelius B, Gudmundsson S, Sjöberg NO, Friedman J, Östergren PO: Qualitative study of pregnancy and childbirth experiences in Somalian women resident in Sweden. BJOG 2000, 107(12):1507–1512. 38. Small R, Gagnon A, Gissler M, Zeitlin J, Bennis M, Glazier R, Haelterman E, Martens G, McDermott S, Urquia M, Vangen S: Somali women and their pregnancy outcomes postmigration: data from six receiving countries. BJOG 2008, 115(13):1630–1640. 39. Bray JK, Gorman DR, Dundas K, Sim J: Obstetric care of new European migrants in Scotland: an audit of antenatal care, obstetric outcomes and communication. Scott Med J 2010, 55(3):26–31. 40. Essén B, Binder P, Johnsdotter S: An anthropological analysis of the perspectives of Somali women in the West and their obstetric care providers on caesarean birth. J Psychosom Obstet Gynaecol 2011, 32(1):10–18. 41. Tanassi LM: Compliance as strategy: the importance of personalised relations in obstetric practice. Soc Sci Med 2004, 59(10):2053–2069. 42. Högberg U, Innala E, Sandström A: Maternal mortality in Sweden, 1980–1988. Obstet Gynecol 1994, 84(2):240–244. 42. Högberg U, Innala E, Sandström A: Maternal mortality in Sweden, 1980–1988. Obstet Gynecol 1994, 84(2):240–244. 43. Knight M, Kurinczuk JJ, Nelson-Piercy C, Spark P, Brocklehurst P: Tuberculosis in pregnancy in the UK. BJOG 2009, 116(4):584–588. 43. Knight M, Kurinczuk JJ, Nelson-Piercy C, Spark P, Brocklehurst P: Tuberculosis in pregnancy in the UK. BJOG 2009, 116(4):584–588. 44. Farah MG, Tverdal A, Selmer R, Heldal E, Bjune G: Tuberculosis in Norway by country of birth, 1986–1999. Int J Tuberc Lung Dis 2003, 7(3):232–235. 44. Farah MG, Tverdal A, Selmer R, Heldal E, Bjune G: Tuberculosis in Norway by country of birth, 1986–1999. Int J Tuberc Lung Dis 2003, 7(3):232–235. 45. Esscher et al. BMC Pregnancy and Childbirth 2014, 14:141 http://www.biomedcentral.com/1471-2393/14/141 Huisman CM, Zwart JJ, Roos-Hesselink JW, Duvekot JJ, van Roosmalen J: Incidence and predictors of maternal cardiovascular mortality and severe morbidity in the Netherlands: a prospective cohort study. PLoS One 2013, 8(2):e56494. 45. Huisman CM, Zwart JJ, Roos-Hesselink JW, Duvekot JJ, van Roosmalen J: Incidence and predictors of maternal cardiovascular mortality and severe morbidity in the Netherlands: a prospective cohort study. PLoS One 2013, 8(2):e56494. 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: 46. Swedish National Board of Health and Welfare: National Board of Health and Welfare's regulations and general advice on clinical autopsies [Socialstyrelsens föreskrifter och allmänna råd om kliniska obduktioner m.m. SOSFS 1996:28. In SOSFS 1996:28. Edited by National Board of Health and Welfare. Stockholm: Swedish National Board of Health and Welfare; 1996. • Convenient online submission 47. Swedish National Board of Health and Welfare: In National Board of Health and Welfares regulations and general advice on death in health care settings [Socialstyrelsens föreskrifter och allmänna råd om vissa åtgärder inom hälso- och sjukvården vid dödsfall SOSFS 1996:29]. Edited by National Board of Health and Welfare. Stockholm: Swedish National Board of Health and Welfare; 1996.
https://openalex.org/W4377565567	https://www.frontiersin.org/articles/10.3389/fendo.2023.1175213/pdf	English	null	Editorial: Revisiting cellular metabolism and type 1 diabetes	Frontiers in endocrinology	2,023	cc-by	1,364	Editorial on the Research Topic Revisiting cellular metabolism and type 1 diabetes Editorial on the Research Topic Revisiting cellular metabolism and type 1 diabetes COPYRIGHT © 2023 Chandra, Rachdi and Gopalakrishnan. 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 1 diabetes (T1D) is a chronic autoimmune condition characterized by islet inﬂammation resulting from poorly deﬁned combinations of genetic, metabolic, immunologic, and environmental factors. The pathology elicits profound changes in energy metabolism and mitochondrial function in insulin-deprived conditions of the affected individuals. Several studies point to changes in systemic metabolites other than glucose levels in T1D patients. Reduced serum levels of succinic acid and phosphatidylcholine in neo-natal patients of T1D highlight the precedence of metabolic perturbation in the body before the full onset of b-cell autoimmunity. T1D patients are also reported to have differential fatty acid metabolism as compared to their healthy counterparts. Further, methionine deﬁciency in early childhood is proposed to be a possible risk factor to develop T1DM. Even though many studies highlight the changes in body metabolism in T1D conditions, we know very little about the cause and effect of changes in b-cell energy metabolism induced by risk factors a major one being the inﬂammatory milieu in TID. In the present Research Topic, we aimed to provide a collection of papers on the emerging concepts on modelling of T1D pathophysiology for example, the effect of pro-inﬂammatory cytokines on b-cells, how cellular communication with immune cells affect metabolic reprogramming in b-cells. In this topic call, a total ﬁve articles have been published of which two are original research articles, two reviews and one perspective. In the ﬁrst perspective article, Wong et al. compared 10 different machine learning algorithms which are currently used in big data analysis and identiﬁed set of genes associated with insulin transcription and validate it in the established T1D preclinical mouse model (Ire1ab-/-) and T2D individuals big data sets. Interestingly, they found candidate genes including metabolic genes APP, ADCYAP1, LDHA and SST that were down regulated in the T1D preclinical mouse model (Ire1ab-/-) suggesting a possible role of de-differentiation of b-cells in this model. TYPE Editorial PUBLISHED 04 May 2023 DOI 10.3389/fendo.2023.1175213 TYPE Editorial PUBLISHED 04 May 2023 DOI 10.3389/fendo.2023.1175213 OPEN ACCESS OPEN ACCESS EDITED AND REVIEWED BY Jared Rutter, The University of Utah, United States CORRESPONDENCE Vikash Chandra vikash.chandra@helsinki.ﬁ Swetha Gopalakrishnan swetha.gopalakrishnan@helsinki.ﬁ RECEIVED 27 February 2023 ACCEPTED 21 April 2023 PUBLISHED 04 May 2023 CITATION Chandra V, Rachdi L and Gopalakrishnan S (2023) Editorial: Revisiting cellular metabolism and type 1 diabetes. Front. Endocrinol. 14:1175213. doi: 10.3389/fendo.2023.1175213 EDITED AND REVIEWED BY Jared Rutter, The University of Utah, United States CORRESPONDENCE Vikash Chandra vikash.chandra@helsinki.ﬁ Swetha Gopalakrishnan swetha.gopalakrishnan@helsinki.ﬁ Vikash Chandra 1, Latif Rachdi 2 and Swetha Gopalakrishnan 3,4 1Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland, 2Institut Cochin, Universite´ Paris Cite´ , L’Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM) U1016, Centre National de la Recherche Scientiﬁque (CNRS) UMR 8104, Paris, France, 3Molecular and Integrative Biosciences Program (MIBS), Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland, 4Institute of Biotechnology, University of Helsinki, Helsinki, Finland COPYRIGHT © 2023 Chandra, Rachdi and Gopalakrishnan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS type 1 diabetes, T1D autoimmunity, T1D mouse model, stem cell modeling, pancreatic beta (b) cells Chandra V, Rachdi L and Gopalakrishnan S (2023) Editorial: Revisiting cellular metabolism and type 1 diabetes. Front. Endocrinol. 14:1175213. doi: 10.3389/fendo.2023.1175213 Funding VC and SG would like to acknowledge the personal grant support from Maud Kuistila Memorial Foundation 2022. The antigen-speciﬁc immunotherapy that can restore the immune tolerance to disease-relevant antigen has been long sought for the treatment of autoimmune diseases however there are several limitations to this therapy like timing, dose, route of administration etc. In the fourth original research article Martens et al. investigated the insulin peptide antigen-speciﬁc immunotherapy for T1D and shown how the addition of adjuvants like alum enhanced the efﬁcacy of insulin B:8-24 peptide treatment in NOD mice model. Furthermore, they showed that inclusion of alum as adjuvant enhanced the tolerogenic response to the antigen accompanied with increased frequencies of insulin-reactive FoxP3+ Tregs and reduced frequencies of activated pathogenic CD4+ and CD8+ T cells in the pancreas of treated mice. 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. Editorial on the Research Topic Revisiting cellular metabolism and type 1 diabetes It will be interesting to see if changes in metabolic genes is a cause or consequence to the proposed de-differentiation of b-cells. Additionally, this work also provides technical input to the b-cell community with publicly Frontiers in Endocrinology 01 frontiersin.org Chandra et al. 10.3389/fendo.2023.1175213 available codes/scripts for other researcher to use in existing as well as emerging datasets to identify genes or genetic pathways associated with diabetes. microenvironments to understand their crosstalk. Furthermore, the authors discuss the optimum readout parameters for both T-cells and pancreatic b-cells and the prospective of developing such in vitro human b-cell killing platforms which will not only help in identifying novel therapeutic targets to halt the T1D progression but also advantageous for the b-cell replacement therapy. The second article from Houeiss et al. is a comprehensive article that reviews our current understanding on different humanized HLA and autoantigen transgenic mice models and discussed how such models can be used to develop antigen speciﬁc immunotherapy for the restoration of immune tolerance in T1D patients. This strategy will aid in identifying new biomarkers and design new screening assays for early detection of the disease. Moreover, these personalized in vivo models will be an ideal tool to test the cross-talk between differentiated human stem cells derived islets (SC-islets) and hemopoietic stem progenitors of the same patient. In summary, this Research Topic presents ﬁve different papers on understanding and modelling of T1D pathophysiology. It includes two very timely review on the progress of humanized HLA and autoantigen transgenic mice and on human b-cell-T-cell in vitro crosstalk platforms for the modelling of human T1D pathogenesis. ER stress has long been considering as a major contributor for the b-cell failure in T1D and T2D. In the third original research article by Vig et al., highlights the previously unrecognized role of mitochondrial DNA (mtDNA) leakage in the cytosol of stressed b- cell which triggers IFN-I responses and consequently stimulates IL8 secretion resulting in neutrophil migration. They have employed a valid human b-cell line model EndoC-bh1 to demonstrate this phenomenon. This observation further strengths the involvement of b-cell ER stress in activating the innate immune responses which triggers immune-cells inﬁltration and sparked the autoimmunity. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The purpose of the 5th review paper was to summarize the progress and challenges in studying the in vitro b-cell killing models using immune cells and SC-islets (Halliez et al.). Here, authors elegantly compared all the available pancreatic islet/b-cell models and the possible islet-reactive cytotoxic CD8+ T-cell models and in vitro 02 Frontiers in Endocrinology frontiersin.org
https://openalex.org/W4385635575	https://bmcpediatr.biomedcentral.com/counter/pdf/10.1186/s12887-023-04219-3	English	null	Development of the national Dutch PEWS: the challenge against heterogeneity and implementation difficulties of PEWS in the Netherlands	BMC pediatrics	2,023	cc-by	6,829	RESEARCH Open Access Abstract Background For the early recognition of deteriorating patients several Pediatric Early Warning Score (PEWS) systems have been developed with the assumption that early detection can prevent further deterioration. Although PEWS are widely being used in hospitals in the Netherlands, there is no national consensus on which score to use and how to embed the score into a PEWS system. This resulted in a substantial heterogeneity of PEWS systems, of which many are unvalidated or self-designed. The primary objective of this study was to develop a pragmatic consensus-based PEWS system that can be utilized in all Dutch hospitals (University Medical Centers, teaching hospitals, and general hospitals). Methods This study is an iterative mixed-methods study. The methods from the Core Outcome Measures in Effectiveness Trials (COMET) initiative were used and consisted of two Delphi rounds, two inventories set out to all Dutch hospitals and a focus group session with parents. The study was guided by five expert meetings with different stakeholders and a final consensus meeting that resulted in a core PEWS set. Results The first Delphi round was completed by 292 healthcare professionals, consisting of pediatric nurses and physicians. In the second Delphi round 217 healthcare professionals participated. Eventually, the core PEWS set was been developed comprising of the parameters work of breathing, respiratory rate, oxygen therapy, heart rate and capillary refill time, and AVPU (Alert, Verbal, Pain, and Unresponsive). In addition, risk stratification was added to the core set with standardized risk factors consisting of [1] worried signs from healthcare professionals and parents and [2] high-risk treatment, with the option to add applicable local defined risk factors. Lastly, the three categories of risk stratification were defined (standard, medium, and high risk) in combination with standardized actions of the professionals for each category. Conclusion This study demonstrates a way to end a country’s struggle with PEWS heterogeneity by co-designing a national Dutch PEWS system. Currently, the power of the system is being investigated in a large multi-center study in the Netherlands. Correspondence: Joris Fuijkschot joris.fuijkschot@radboudumc.nl Full list of author information is available at the end of the article Full list of author information is available at the end of the article © The Author(s) 2023. BMC Pediatrics BMC Pediatrics Fuijkschot et al. BMC Pediatrics (2023) 23:387 https://doi.org/10.1186/s12887-023-04219-3 Development of the national Dutch PEWS: the challenge against heterogeneity and implementation difficulties of PEWS in the Netherlands Joris Fuijkschot1, Jikke Stevens1, Lara Teheux1, Erica de Loos2, Hester Rippen3, Maaike Meurs4 and Janke de Groot4,5 © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Abstract 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. Background While pediatric hospital mortality and cardiac arrests have a low incidence, outcomes are poor and have a high impact on family and caregivers [1]. At the same time, literature shows that adverse events in pediatric care seem to occur less frequently compared to adult care, 50–60% of these events are considered prevent able [2–4]. For the early recognition of deteriorating patients several Pediatric Early Warning Scores (PEWS) have been developed [5, 6]. PEWS generally consist of a predefined set of vital parameters, such as heart rate, respiratory rate, body temperature, blood pressure, and oxygen saturation, using age-specific cut-off points. Most PEWS also contain parameters based on behavioral items and concerns of healthcare professionals. Parameters are either added up to a numerical score that defines which response is appropriate or may directly trigger an alarm. Alarming PEWS should preclude an early intervention by healthcare professionals to prevent further deteriora tion. Therefore, it is essential that PEWS are part of an integrated PEWS track and trigger system in which, sub sequent to the monitoring of PEWS, appropriate rules of escalation and communication are in place [7, 8]. The challenge with PEWS is that clear evidence of effects upon quantitative outcome measures such as pediatric hospital mortality or unplanned admissions to Pediatric Intensive Care Units (PICU) is still lacking. Also, almost all validation studies were performed in Uni versity Medical Centers which are known for their com plex population of patients with high co-morbidity and sophisticated infrastructures for the escalation of care [15]. Besides, the addition of risk stratification to PEWS has already been proven to be a powerful instrument to improve PEWS sensitivity in a University Medical Center in the Netherlands, indicating that the performance of a PEWS is related to the way it is used in clinical practice [16]. Performance studies of PEWS systems in the setting of their largest user group, general (teaching) hospitals, are largely lacking and will be very difficult to perform due to the present vast heterogeneity of systems used in countries. This will remain so unless standardization on a national level ends this heterogeneity first. Keywords Dutch PEWS, Pediatric early warning score, Risk stratification, Watcher signs, Worried sign Keywords Dutch PEWS, Pediatric early warning score, Risk stratification, Watcher signs, Worried si Keywords Dutch PEWS, Pediatric early warning score, Risk stratification, Watcher signs, Worried sign hospitals would use a standardized PEWS system when available [12]. Methodsh The main objectives of the study were to: The main objectives of the study were to: 1. reach consensus of a Core Set-PEWS (CS-PEWS) to be used in all Dutch hospital settings, add relevant risk factors (so called watcher signs) and (possibly) risk stratification to develop a PEWS; i 2. determine a set of minimal standard operating procedures for early intervention; 3. determine and limit options for adaptation of the system to the local hospital setting. (2023) 23:387 Fuijkschot et al. BMC Pediatrics (2023) 23:387 Page 2 of 9 Fuijkschot et al. BMC Pediatrics Background i Supported by relevant Dutch scientific societies, nurs ing societies, and patient representatives, as well as the Dutch Healthcare Inspectorate and the Ministry of Health, we decided to start a national PEWS study.h Although PEWS are widely used in almost all Dutch hospitals there is no national consensus on which sys tem to use [9]. Also, none of the PEWS reported in the literature has proven to be superior in the recognition of clinical deterioration [10, 11]. This may relate difficul ties validating systems using quantitative endpoints such as the classically low pediatric hospital mortality This resulted in the use of a wide variety of PEWS in the Neth erlands, of which many are unvalidated or self-designed. We found that 45 different systems are being used in 68 hospitals including 20 different parameters of which none is being used in all systems [12]. This large hetero geneity and use of unvalidated systems within one coun try are also seen in other European countries such as the United Kingdom (UK) [7]. Furthermore, while healthcare professionals in the Netherlands intuitively believe PEWS may be helpful in early recognition, they have also raised doubt due to the lack of validation and evidence of the effectiveness of PEWS and this hampers the successful implementation of PEWS in clinical practice [9, 13, 14].h The aimed outcome of this study was a pragmatic con sensus-based system for daily practice of PEWS in Dutch hospitals. Design and context An iterative mixed methods study using the Core Out come Measures Trials (COMET) initiative methodology (chosen because of its structuralized method to develop a consensus based core set) [17–19]. The study was con ducted by the independent Netherlands Institute for Health Services Research in close cooperation with the Dutch Society for Pediatrics, the Dutch Society for Nurs ing, and Dutch Foundation Child & Hospital. The vast heterogeneity of PEWS in the Netherlands also results in a missed opportunity to validate the PEWS in different hospital settings. Due to this lack of valida tion, healthcare professionals in the Netherlands found it difficult to choose or develop a PEWS suitable for their local setting [9]. At the same time, a recent inventory in all hospitals in the Netherlands revealed that 98% of the Fuijkschot et al. BMC Pediatrics (2023) 23:387 Page 3 of 9 Page 3 of 9 Fuijkschot et al. BMC Pediatrics (2023) 23:387 Different methods and data sources were used and at times integrated. A summary of the used data sources for each objective is presented in Appendix 1. The context for this study was limited to develop a sys tem for all children (0–18 years of age) admitted to gen eral pediatric wards. The system is not designed for use in pediatric and neonatal intensive or high care depart ments and emergency departments. Ethics According to the Dutch Medical Research Involving Human Subject Act, this study does not require ethics approval by an independent Medical Ethics Commit tee. In accordance with the principles of the Declaration of Helsinki, set up by the World Medical Association (WMA), we did ask participants to give informed con sent to use the data obtained during the expert meetings in an anonymous way. Study protocol A summary of the iterative study protocol is summarized in Fig. 1. Appendix 2 provides a more detailed insight of the used methods. Fig. 1 Study protocol Relevant stakeholders of PEWS involve professionals in pediatric hospital care (e.g. pediatricians, pediatric nurses, surgeons treating children), parents of children who are admitted to the hospitals, and quality improve ment professionals. During the research period, an expert group of stake holders was formed with the responsibility to reach con sensus in discussion and support the development of the Delphi rounds. This group consisted of experienced healthcare professionals (three pediatricians representing all different hospital settings, one pediatric surgeon, two pediatric nurses), one patient organization representa tive, one implementation expert, and two researchers. All pediatricians, general surgeons treating chil dren, and pediatric nurses in the Netherlands as well as selected patient representatives were invited through a mass mailing by their professional organizations and social media channels to participate in two online Delphi rounds. Reminder emails were sent to those who failed to complete any round. Parents of children were selected and invited by the Dutch Foundation Child & Hospital to participate in a focus group session. The final phase of the Delphi study involved a national consensus meeting with representatives of all stakeholder groups including physicians, nurses, implementation professionals, parent representatives, and members of the expert group. Delphi round 1 and survey In total 212 pediatric nurses and 80 physicians completed the survey 1 (n = 292). 54 of the participants came from University Medical Centers, 85 came from teaching hos pitals, and 153 came from general hospitals. Appendix 4 describes the results per item and the potential param eters to be included in the core set. domain were considered to be part of the neurologi cal domain and subjective signs were already taken into account with the worried signs. Therefore, no comple mentary domains were added to the structure of the core set. Concerning the structure of the core set, it was found that all domains (respiratory, cardiovascular and neuro logical; ABCD) met the present criteria for consensus inclusion (Table 1). This indicates that a core set should address all these three domains of the ABCD approach. Additional domains that were suggested by participants were ‘clinical view’, ‘cognition’, and the ‘psychological domain’. Relating to worried signs, it was decided that the wor ries by physicians, nurses or parents should not be priori tized in the second Delphi but were considered eligible to act as a separate risk factor in the system. Its exact posi tion and role were determined later in the study. From the initial set, the following parameters met the criteria to be included in Delphi round 2: work of breath ing; respiratory rate; consciousness; heart rate; worries by physician, nurse and/or parents; pulse oxygen saturation; respiratory retractions; apnea; agitation; blood pressure; capillary refill time; skin color; body temperature, and supplemental oxygen therapy. Expert meeting 1: inventory currently used PEWS parametershi The literature search resulted in five published system atic reviews on PEWS [11, 20, 21]. An inventory of PEWS parameters used in Dutch hospitals was done and has been published elsewhere [12]. Discussing both the liter ature and the twenty parameters currently used in Dutch Fig. 1 Study protocol Fig. 1 Study protocol Fig. 1 Study protocol Fuijkschot et al. BMC Pediatrics (2023) 2 Page 4 of 9 Fuijkschot et al. BMC Pediatrics Page 4 of 9 Table 1 Potential ABCD domains to be included in CS-PEWS (% of answers) 1 2 3 1 + 2 + 3 4 5 6 7 8 9 7 + 8 + 9 Respiratory (AB) 0.4 0 0 0.4 0 0 0 2.8 8,5 88.4 99.7 Cardiovascular (C) 0.3 0.3 0.3 0.9 0.3 0.7 0.7 4.7 15.3 71.8 91.8 Neurological (D) 0.7 0.3 1.3 2.3 0.7 3.7 2.7 12.6 20.6 51.8 85 9-point Likert scale: 1 ‘not important’ to 9 ‘critically important’. Items in bold met criteria for inclusion Table 2 Ranking of parameters of respiratory domain (%) 1 2 3 4 5 Work of breathing 8.8 12.6 14.9 20.9 42.8 Respiratory rate 11.2 15.8 27.9 27.0 18.1 Pulse oxygen saturation 5.1 18.1 38.1 26.0 12.6 Apnea 35.5 22.9 8.9 13.6 19.2 02therapy 39.5 30.7 10.2 12.6 7.0 hospitals, the expert group made a long list of 33 items to be included in the Delphi Survey (see Appendix 3). Part 2: prioritizing within three domains Participants were also asked to rank the remaining parameters included in survey 1 within the respiratory, cardiovascular and neurological domains.i In the respiratory domain five parameters were being prioritized. Work of breathing was considered the most important parameter, followed by respiratory rate, apnea, oxygen saturation, and supplemental oxygen therapy (see Table 2). Delphi round 2 and survey Part 1: prioritizing shortlist h d In the second survey participants (n = 217 - a full sub set of survey 1) were asked to further prioritize param eters to be able to create a core set of eight items (see Appendix 5 for full results). This resulted in the following high to low order meeting inclusion criteria: heart rate (95.5%), work of breathing (92%), pulse oxygen satura tion (89.9%), respiratory rate (89%), consciousness (88%), supplemental oxygen therapy (78.3%), capillary refill time (74.6%), and agitation (71%). Participants were also asked to suggest new parameters if they felt some were missing. Additional parameters that were mentioned by participants included petechiae (mentioned twice), experienced shortness of breath (mentioned once), glucose level (mentioned once), and pupillary reflexes (mentioned once). l Furthermore, up to 79.1% of the participants indicated that “worried signs” should be considered as a separate acting alarm sign and by itself be able to directly trigger a response. In regards to the optimal number of items in a PEWS, the participants agreed that the final core set should consist of five to seven preferable non-invasive parameters. Expert meeting 2: prioritizing items for the next round p g p g Based on the survey with only limited suggestions for additional parameters, the expert group concluded that the long list was complete and results from Delphi round 1 were considered valid. All of the included parameters therefore were eligible for Delphi round 2. The impor tance to address all three of the ABCD domains in the core set was confirmed. Cognition and psychological Within the cardiovascular four parameters were being prioritized. Heart rate was considered most important, followed by capillary refill time, blood pressure, and skin color (see Table 3). Within the neurologic domain (consisting of agitation and consciousness), consciousness was considered the most important parameter. The temperature was not Fuijkschot et al. BMC Pediatrics (2023) 23:387 Fuijkschot et al. BMC Pediatrics Page 5 of 9 Fig. 2 First draft of the Dutch PEWS taken into account as this is not a parameter relating to the ABCD but to the E domain of the ABCDE approach. Table 3 Ranking of parameters of the cardiovascular domain (%) 1 2 3 4 Heart rate 14.4 10.7 14.0 60.9 Capillary refill time 10.2 33.0 40.5 16.3 Blood pressure 37.2 26.0 24.2 12.6 Color of skin 38.1 30.2 21.4 10.2 taken into account as this is not a parameter relating to the ABCD but to the E domain of the ABCDE approach. Expert meeting 3: first draft of Dutch PEWSh This meeting focused on constructing a first draft of PEWS. The primary goal was the concept of a lean, mini mal invasive, child-friendly and nurses workload reduc ing system. Firstly, based on the second Delphi round and the focus group session, the Dutch PEWS core set was formulated. It was decided to use eight included parame ters but also to differentiate between ‘early’ and ‘late’ pre dictors of clinical deterioration. The differentiation was based on physiological principles of bodily responses to clinical deterioration. For the respiratory domain, it was decided that the work of breathing and respiratory rate are the most important ‘early’ predictors. Pulse oxygen saturation usually only decreases after attempts to com pensate for respiratory failure have failed. In the absence of increased respiratory rate and/or work of breathing, it is unlikely that pulse oxygen saturation is decreased. Therefore, pulse oxygen saturation is considered a late predictor. For the cardiovascular domain, the early pre dictors were considered to be the heart rate and capillary refill time since these parameters are already deviant in early, compensated shock. In critically ill children, blood pressure often is compensated for a long time hence this was considered to be a late predictor of clinical deteriora tion [22]. For the neurological domain, a differentiation between early and late parameters could not be made so both parameters were included in this draft of the core set. This resulted in the following first draft of the PEWS core set, summarized in Fig. 2. Most respondents agreed PEWS should ideally be assessed three times per 24 h, at the beginning of each nursing shift, while again a large group (30.6%) answered this should depend on factors such as diagnosis, indica tion for admission, disease severity, time after admis sion, clinical point of view, previous PEWS scores and whether a child is sleeping. With regard to sleep, only 2.4% thought children should always be awakened for the assessment of PEWS, while over 80% thought this should depend on the circumstances. Survey on scoring, implementation and organization of working with a PEWS Next to prioritizing items, the survey also included ques tions on risk factors to be taken into account and how to use PEWS in different hospital settings. Respondents prioritized the following items as important risk fac tors: medical history of the patient, high-risk medica tion, abnormal airway, and high-risk interventions (see Appendix 6). Fig. 2 First draft of the Dutch PEWS a deviant clinical course during the admissions of their children. Their ‘gut-feeling’ was often felt as a strong pre dictor for clinical deterioration and therefore should play part in a PEWS. When asked about which patients, how often, and when to measure PEWS (Appendix 7), most respon dents answered PEWS should be applied to all hospital ized children (52.4%), with a large group (31.6%) thinking this should depend on factors such as diagnosis, disease severity, high-risk patient, threatened vital signs, gut feeling, clinical point of view, surgery, worried parents, admission in the maternity departments. More specifi cally there were questions about whether PEWS are rel evant for children admitted for psychosocial reasons or observations. Expert meeting 4: national consensus meetingi The final expert meeting focused on the last issues to complete the Dutch PEWS. For the core set part of the system, based upon input from the consensus meeting, it was decided to use the data from the Parshuram bedside PEWS (normal values for different age groups) as this was considered to be the most validated set available at that time. Numeric scoring thresholds for the sum of the respiratory and cardiovascular domains were defined and divided into three scoring groups (0–3/4–6/≥7) with an alarming score threshold at seven points. The threshold is based on previous experiences with other PEWS sys tems in the Netherlands that also used these parameters [16].h During this consensus meeting, the first draft of the Dutch PEWS and data from the survey was presented and discussed with a large multidisciplinary group of pro fessionals from twenty different hospitals (including four University Medical Centers, seven teaching hospitals, and nine general hospitals) and patient representatives. Using the methodology of ‘world café’ [23] the group was asked in several rounds to comment and (dis)agree upon the first draft, advice on risk factors and risk stratifica tion, formulate which children should be assessed, how often and when, and what actions should be part of the rapid response to an alarming PEWS score. Outcomes resulted in the following recommendations for the expert group:hi The use of the AVPU score for the neurological domain was approved. It was decided to position this score as a risk factor. This was decided because the sensitivity of PEWS to timely detect patients with abnormal con sciousness is notoriously low [16]. With the change to risk factor, abnormal consciousness will automatically move a patient from the standard to high-risk assessment independent from other parameters. – The first draft of the core set was fully supported by the participants. For the neurological domain, it was advised to use the AVPU (Alert, Verbal, Pain, and Unresponsive) score for consciousness. Agitation was not recognized as a crucial PEWS component since this usually coincides with alternations in consciousness. It was advised to use data from Parshuram’s Bedside PEWS to determine cut-off points for different PEWS parameters and age- groups since this is a rather sufficiently validated set. Further attention was paid to the role of risk factors and the application of risk stratification. Focus group sessioni A total of five parents (representing five children) par ticipated in the focus group session. They expressed the wish for a lean system using mostly observations while limiting the number of invasive measurements to lower the impact of the repeated PEWS measurements on their children. Wherever possible they would like to be part of the PEWS measurement to help their children to feel as comfortable as possible and maybe improve diagnos tic accuracy. The participants of the focus group session stated that the worried sign given by parents is of utmost importance to be taken into account when developing a new system. They shared their experiences in recognizing Secondly, the expert group discussed the position of risk factors (watcher signs) that may or may not directly trigger an alarm and the principle of risk stratification. Page 6 of 9 Fuijkschot et al. BMC Pediatrics (2023) 23:387 Fuijkschot et al. BMC Pediatrics (2023) 23:387 Fuijkschot et al. BMC Pediatrics (2023) 23:387 Fuijkschot et al. BMC Pediatrics Risk factors often are present early during admission and may improve PEWS’ sensitivity to detect clinical dete rioration if used to stratify patients into risk categories. Risk stratification enables professionals to follow up on watcher patients more closely with more checks and measurements whilst non-watcher patients receive stan dard care. This may lead up to a more refined system, refined effort of human resources, and patient friendli ness. Previous experience with risk factors and risk strati fication in the Netherlands found a significant increase in PEWS sensitivity as well as positive effects on com munication and situational awareness [13, 16]. Based upon Delphi rounds data, literature and national experi ences, it was decided that risk factors and risk stratifica tion are crucial components of the Dutch PEWS system. To determine its exact position and usage in the system the subsequent consensus meeting was needed to further receive input from the working field. recommendations for minimal actions of the professionals were defined for each risk category. recommendations for minimal actions of the professionals were defined for each risk category. i – PEWS measurements apply to every patient admitted to the general pediatric department (including the day care unit) as part of standard care. Exclusions can be made if deemed necessary by the healthcare professionals. – The standard care will consist of three PEWS measurements per 24 h, including the assessment of ‘watcher signs’ once per day. Focus group sessioni The timing of the PEWS measurements can be integrated with the nurses rounds and ideally patients are not wakened during the night. – Parents should be informed about PEWS and asked about possible worries they have. – Subsequent validation of Dutch PEWS was highly recommended. Expert meeting 4: national consensus meetingi In the final ver sion of the Dutch PEWS, the numeric PEWS score and the existence of watcher signs (including AVPU and wor ried signs) adds up to a risk score, ranging from standard, medium to high, with specific recommendations for action (see Fig. 3). fi – Risk factors (watcher signs) should consist of standardized factors (such as worried signs and high-risk treatment) and part of the core set but also supplemented by locally defined non-core set risk factors to improve the adaptability of the system to the local hospital. For further approval of this newly designed and unique system, the results were presented and discussed at the annual meeting of the Dutch Pediatric Society (2019) and the Dutch Society for Pediatric Nurses (2020) and presented to the Dutch Healthcare Inspectorate (2020). In all these meetings relevant stakeholders approved the – Risk stratification should be applied to identify watcher patients. Three risk categories (high, medium and standard) were defined and Page 7 of 9 Page 7 of 9 Fuijkschot et al. BMC Pediatrics (2023) 23:387 (2023) 23:387 Fuijkschot et al. BMC Pediatrics Fig. 3 Dutch PEWS and actions Fig. 3 Dutch PEWS and actions stable patients is eliminated. It is expected that this helps to improve protocol adherence, which is a notorious problem in PEWS system implementation [14]. system for usage with a strong recommendation for sub sequent validation of the system in Dutch hospitals. For implementation and promotion purposes an info graphic was made and a website with information about the Dutch PEWS (www.dutchpews.com) to enable the usage of the new system in the Netherlands. To facilitate implementation and protocol adher ence, preparations have been made by the study group to incorporate the Dutch PEWS system in the electronic health record systems of the two largest providing com panies in the Netherlands; Epic and ChipSoft. Working together with representatives of these providers and the national network of chief medical information officers in the Netherlands in 2020, led to the direct availability of this national system for approximately 85% of all hospi tals. In its design, much attention has been paid to reduc ing registration load and improving the usability of the data in a user-friendly interface. Discussion h In the context of the heterogeneity of currently used PEWS systems in different countries over the world, we demonstrate a way to end this struggle by co-designing a consensus-based PEWS suitable for all hospital settings. To the best of our knowledge, this study is unique using Delphi methodology with a large group of profession als and patients/parents to construct a national PEWS system and not reported by other countries. Unique in relation to other national systemsis the participation of a large number of pediatric healthcare profession als using Delphi methods and paying attention to local hospital context differences that may influence PEWS performance and implementation. Further, the consen sus-based PEWS is expected to fit well within the cur rent practice in Dutch pediatric care. The approach of this study can serve as an example to solve problems with heterogeneity in PEWS systems in other countries. To the best of our knowledge, the Dutch PEWS sys tem is the first nationwide PEWS that incorporates risk stratification and explicitly shifts focus to those patients of whom a deviant clinical course can be expected (so- called ‘watcher patients’). Risk stratification can help identify these watcher patients and enables profession als to pro-actively follow up on their clinical course more closely and respond to deterioration more quickly. Acknowledgements None. f Furthermore, there is a risk of selection bias amongst participants of the Delphi rounds by including mainly believers or non-believers of a PEWS system. However, due to the rather large amount of respondents, selection bias probably had limited influence on the results of the Delphi rounds. Limitationsh In the design of the system a lot of attention has been given to feasibility in daily practice (e.g. the time consum ing and child unfriendly parameters blood pressure and pulse oxygen saturation are only to be measured when other less challenging PEWS parameters are abnormal). This increases the expectations of the Dutch PEWS to meet its original objectives and at the same time be child friendly and reduce nurses’ workload. The time-con suming use of technical medical equipment in otherwise The largest limitation of this study is that by designing a new system it is unclear what the power of the system is in terms of sensitivity and diagnostic accuracy. Param eters from Delphi round 1 were scored upon clinical relevance in participants’ opinion/experience and this may not reflect importance in terms of validity in its assessment [24]. Although elements of the existing and validated Bedside PEWS were used and the basis of the system resembles a well-studied system that has been Fuijkschot et al. BMC Pediatrics (2023) 23:387 Fuijkschot et al. BMC Pediatrics Page 8 of 9 used for many years in a University Medical Center in the Netherlands (Radboud University Medical Center), it is of utmost importance that the system is validated in the different hospital settings for which it is designed. Author details 1 Author details 1Radboud University Medical Center, Radboudumc Amalia Childrens Hospital, Nijmegen, The Netherlands 2Dutch Hospital Association, Utrecht, The Netherlands 3Dutch Foundation Child & Hospital, Utrecht, The Netherlands 4Netherlands Institute for Health Services Research, Utrecht, The Netherlands 5Knowledge Institute for Medical Specialists Utrecht The Netherlands Author details 1Radboud University Medical Center, Radboudumc Amalia Childrens Hospital, Nijmegen, The Netherlands 2Dutch Hospital Association, Utrecht, The Netherlands 3Dutch Foundation Child & Hospital, Utrecht, The Netherlands 4Netherlands Institute for Health Services Research, Utrecht, The Netherlands 5Knowledge Institute for Medical Specialists Utrecht The Netherlands Funding Thi d his study was funded by the Dutch Institute for Medical Specialists. Data Availability Th d d Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Currently, a large multi-center study is being performed in the Netherlands testing the system in twelve hospi tals and one ambulance service. These twelve hospitals include three University Medical Centers (comprising one national pediatric oncology center), five teaching hospitals, and four general hospitals. The main objec tives are to validate the system using different end points customized for hospital setting and to determine its effectiveness in improving patient safety. The results and findings of this study are expected in 2024. Experi ences from hospitals participating in this study need to be actively shared with all hospitals in the Netherlands to provide the opportunity to learn and improve together. More information regarding this study is available at the website https://dutchpews.com. Authors’ contributions JF, JS, MM and JG wrote the main manuscript text. JF, LT, EL, HR, MM, JG participated in the original study group. JS extensively reviewed the main manuscript, edited main text and prepared all figures. All authors critically reviewed the mansucript. Competing interests The authors declare no competing interests. Conclusion By co-designing a nationwide and consensus-based PEWS suitable for all hospital settings, this study con tributes to the quality and patient safety of pediatric care. The study protocol can serve as a blueprint and support other countries to solve problems with heterogeneity in PEWS systems and enable subsequent studies focusing on the validity and effectiveness of the system nation wide. Currently, the power of the Dutch PEWS is being investigated by this research group in a large multi-center study in the Netherlands. nowledge Institute for Medical Specialists, Utrecht, The Netherlands Received: 1 July 2022 / Accepted: 28 July 2023 Received: 1 July 2022 / Accepted: 28 July 2023 Received: 1 July 2022 / Accepted: 28 July 2023 Ethics approval and consent to participate According to the Dutch Medical Research Involving Human Subject Act, this study does not require ethics approval by an independent Medical Ethics Committee. In accordance with the principles of the Declaration of Helsinki, set up by the World Medical Association (WMA), we did ask and obtain informed consent from all participants in this study to anonymously use the data obtained. References Paediatric early warning systems for detecting and responding to clinical deterioration in children: a systematic review. Vol. 7, BMJ Open. 2017. 21. Heward Y. Advanced practice in paediatric intensive care: a review. Paediatr Nurs. 2009;21(1):18–21. 21. Heward Y. Advanced practice in paediatric intensive care: a review. Paediatr Nurs. 2009;21(1):18–21. y 12. Van Sambeeck SJ, Vos G, Theeuwes B, Starre C, Fuijkschot J. De Pediatric Early Warning Score (PEWS) en veilige (re) zorg in Nederland. 2016;(1). 22. Advanced Life Support Group. Advanced paediatric life support: the practical approach to emergencies. Sixth edit. Wiley-Blackwell; 2016. p. 362. 22. Advanced Life Support Group. Advanced paediatric life support: the practical approach to emergencies. Sixth edit. Wiley-Blackwell; 2016. p. 362. 23. Brown J, Isaacs D. The World Cafe: shaping our futures through conversations that matter. 1st ed. San Francisco: Berrett-Koehler; 2005. pp. 1–264. 23. Brown J, Isaacs D. The World Cafe: shaping our futures through conversations that matter. 1st ed. San Francisco: Berrett-Koehler; 2005. pp. 1–264. 13. de Vries A, Draaisma JMT, Fuijkschot J. Clinician Perceptions of an Early Warning System on Patient Safety. Hosp Pediatr [Internet]. 2017 Oct 1 [cited 2022 Jun 12];7(10):579–86. Available from: https://pubmed.ncbi.nlm.nih. gov/28928156/ 24. Boulkedid R, Abdoul H, Loustau M, Sibony O, Alberti C. Using and reporting the Delphi method for selecting healthcare quality indicators: a systematic review. PLoS ONE. 2011;6(6). 24. Boulkedid R, Abdoul H, Loustau M, Sibony O, Alberti C. Using and reporting the Delphi method for selecting healthcare quality indicators: a systematic review. PLoS ONE. 2011;6(6). 14. van Sambeeck SJ, Fuijkschot J, Kramer BW, Vos GD. Pediatric Early Warning System Scores: Lessons to be Learned. J Pediatr Intensive Care [Inter net]. 2018 Mar [cited 2022 Jun 12];7(1):27. Available from: /pmc/articles/ PMC6260327/ References List of abbreviations ABCDE Airway, Breathing, Circulation, Disability, and Exposure AVPU Alert, Verbal, Pain, and Unresponsive COMET Core Outcome Measures in Effectiveness Trials Kind&Ziekenhuis Child and Hospital Foundation Nivel Netherlands Institute for Health Services Research NVK Dutch society for pediatrics PEWS Pediatric Early Warning System PICU Pediatric Intensive Care Unit PMET Pediatric Medical Emergency Team V&VN Dutch Society for Nursing VMS National safety program Supplementary Information 1. Girotra S, Spertus JA, Li Y, Berg RA, Nadkarni VM, Chan PS. Survival trends in pediatric in-hospital cardiac arrests an analysis from get with the guidelines- resuscitation. Circ Cardiovasc Qual Outcomes. 2013;6(1):42–9. 1. Girotra S, Spertus JA, Li Y, Berg RA, Nadkarni VM, Chan PS. Survival trends in pediatric in-hospital cardiac arrests an analysis from get with the guidelines- resuscitation. Circ Cardiovasc Qual Outcomes. 2013;6(1):42–9. 2. Fajreldines A, Schnitzler E, Torres S, Panattieri N, Pellizzari M. Measurement of the incidence of care-associated adverse events at the Department of Pediatrics of a teaching hospital. Arch Argent Pediatr. 2019;117(2):E106–9. g g 3. Starmer AJ, Sectish TC, Simon DW, Keohane C, McSweeney ME, Chung EY, et al. Rates of medical errors and preventable adverse events among hospital ized children following implementation of a resident handoff bundle. JAMA. 2013;310(21):2262–70. 4. Kahn S, Abramson EL. What is new in paediatric medication safety? Arch Dis Child. 2019;104(6):596–9. 5. Smits M, Langelaan M, De Groot J, Wagner C. Examining causes and prevention strategies of adverse events in deceased hospital patients: a retrospective patient record review study in the netherlands. J Patient Saf. 2021;17(4):282–9. Supplementary Information 6. Parshuram CS, Duncan HP, Joffe AR, Farrell CA, Lacroix JR, Middaugh KL, et al. Multicentre validation of the bedside paediatric early warning system score: Page 9 of 9 Page 9 of 9 Fuijkschot et al. BMC Pediatrics (2023) 23:387 Fuijkschot et al. BMC Pediatrics (2023) 23:387 Fuijkschot et al. BMC Pediatrics (2023) 23:387 16. Teheux L, Verlaat CW, Lemson J, Draaisma JMT, Fuijkschot J. Risk stratification to improve Pediatric early warning Systems: it is all about the context. Eur J Pediatr. 2019;178(10):1589–96. a severity of illness score to detect evolving critical illness in hospitalised children. Crit Care. 2011;15(4):R184. a severity of illness score to detect evolving critical illness in hospitalised children. Crit Care. 2011;15(4):R184. a severity of illness score to detect evolving critical illness in hospitalised children. Crit Care. 2011;15(4):R184. 7. Roland D, Oliver A, Edwards ED, Mason BW, Powell CVE. References Use of paediatric early warning systems in Great Britain: has there been a change of practice in the last 7 years? Arch Dis Child. 2014;99(1):26–9. 17. Prinsen CAC, Vohra S, Rose MR, Boers M, Tugwell P, Clarke M, et al. How to select outcome measurement instruments for outcomes included in a “Core Outcome Set” - a practical guideline. Trials. 2016;17(1):1–10. y 8. Fuijkschot J, Vernhout B, Lemson J, Draaisma JMT, Loeffen JLCM. Validation of a paediatric early warning score: first results and implications of usage. Eur J Pediatr. 2015;174(1):15–21. 18. Leotsakos A, Zheng H, Croteau R, Loeb JM, Sherman H, Hoffman C, et al. Standardization in patient safety: the WHO High 5s project. Int J Qual Heal Care. 2014;26(2):109–16. 9. de Groot JF, Damen N, de Loos E, van de Steeg L, Koopmans L, Rosias P et al. Implementing paediatric early warning scores systems in the Netherlands: future implications. BMC Pediatr. 2018;18(1). 9. de Groot JF, Damen N, de Loos E, van de Steeg L, Koopmans L, Rosias P et al. Implementing paediatric early warning scores systems in the Netherlands: future implications. BMC Pediatr. 2018;18(1). 19. Davies A, Teare L, Falder S, Coy K, Dumville JC, Collins D, et al. Protocol for the development of a core indicator set for reporting burn wound infection in trials: Icon-b study. BMJ Open. 2019;9(5):1–6. 19. Davies A, Teare L, Falder S, Coy K, Dumville JC, Collins D, et al. Protocol for the development of a core indicator set for reporting burn wound infection in trials: Icon-b study. BMJ Open. 2019;9(5):1–6. 10. Sinitsky L, Reece A. Question 2: can paediatric early warning systems predict serious clinical deterioration in paediatric inpatients? Arch Dis Child. 2016;101(1):109–13. 10. Sinitsky L, Reece A. Question 2: can paediatric early warning systems predict serious clinical deterioration in paediatric inpatients? Arch Dis Child. 2016;101(1):109–13. 20. Brown SR, Garcia DM, Agulnik A. Scoping review of pediatric early warning Systems (PEWS) in resource-limited and humanitarian settings. Front Pediatr. 2019;6(JAN):410. 20. Brown SR, Garcia DM, Agulnik A. Scoping review of pediatric early warning Systems (PEWS) in resource-limited and humanitarian settings. Front Pediatr. 2019;6(JAN):410. 11. Lambert V, Matthews A, MacDonell R, Fitzsimons J. Paediatric early warning systems for detecting and responding to clinical deterioration in children: a systematic review. Vol. 7, BMJ Open. 2017. 11. Lambert V, Matthews A, MacDonell R, Fitzsimons J. Publisher’s Note 15. Trubey R, Huang C, Lugg-Widger FV, Hood K, Allen D, Edwards D et al. Validity and effectiveness of paediatric early warning systems and track and trigger tools for identifying and reducing clinical deterioration in hospitalised chil dren: a systematic review. BMJ Open. 2019. 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. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2574986000	https://www.zora.uzh.ch/id/eprint/143104/1/PhysRevLett.118.111803.pdf	English	null	Observation of <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msubsup><mml:mi>B</mml:mi><mml:mi>c</mml:mi><mml:mo>+</mml:mo></mml:msubsup><mml:mo stretchy="false">→</mml:mo><mml:msup><mml:mi>D</mml:mi><mml:mn>0</mml:mn></mml:msup><mml:msup><mml:mi>K</mml:mi><mml:mo>+</mml:mo></mml:msup></mml:math> Decays	Physical review letters	2,017	cc-by	8,687	Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2017 DOI: 10.1103/PhysRevLett.118.111803 The decay Bþ →¯D0πþ is used for normalization. Since the ratio of production rates for Bþc and Bþ mesons within the LHCb acceptance, fc=fu, is unknown, the measured observables are The Bþc meson is the only ground-state meson consisting of two heavy quarks of different flavor, namely a ¯b and a c quark. As such, its formation in pp collisions is suppressed relative to the lighter B mesons. Unlike B0, Bþ and B0s mesons, the b-quark decay accounts for only ∼20% of the Bþc width [1]. Around 70% of its width is due to c-quark decays, where the c-quark transition has been observed with Bþc →B0sπþ decays [2]. This leaves ∼10% for ¯bc →Wþ →¯qq annihilation amplitudes, which can be unambiguously probed in charmless final states. No charm- less Bþc decays have been reported to date, although searches show an indication at the level of 2.4 standard deviations (σ) [3]. RDðÞ0h ¼ fc fu × BðBþc →DðÞ0hþÞ; ð1Þ ð1Þ where h is π or K and BðBþc →DðÞ0hþÞ represents the corresponding branching fraction. The four observables are measured with a simultaneous fit to the D0πþ and D0Kþ invariant mass distributions. Theoretical estimates for BðBþc →J=ψπþÞ range from 6.0 × 10−4 [9] to 1.8 × 10−3 [10], which implies fc=fu values in the range 0.004–0.012 using the production ratio measured in Ref. [5] and the branching fraction BðBþ →J=ψKþÞ [11]. Estimates for BðBþc →D0KþÞ vary from 1.3 × 10−7 [6] to 6.6 × 10−5 [8], while estimates for BðBþc →D0πþÞ vary from 2.3 × 10−7 [6] to 2.3 × 10−6 [7]. Using Eq. (1), the expectation for RD0π is seen to cover the range 9 × 10−10 −3 × 10−8, while RD0K covers the range 5 × 10−10 −8 × 10−7. To test QCD factorization and explore the new physics potential of Bþc decays, rarer decays such as suppressed tree-level b →u transitions and b →s loop-mediated (penguin) decays can be studied, where the charm quantum number remains unchanged. The simplest decay is the color-allowed Bþc →DðÞ0πþ decay, illustrated in Fig. 1(a). The expected branching fraction for this decay is a factor jVub=Vcbj2 ≈0.007 lower than the favored b →c and color-allowed Bþc →J=ψπþ decay [4,5], placing this mode at the limit of sensitivity with current LHCb data. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Observation of B+ c D0K++ decays DOI: https://doi.org/10.1103/PhysRevLett.118.111803 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-143104 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-143104 Journal Article Published Version Originally published at: Bernet, R; Müller, K; Serra, N; Steinkamp, O; Straumann, U; Vollhardt, A; et al (2017). Observation of B+ c D0K++ decays. Physical Review Letters, 118(11):111803. DOI: https://doi.org/10.1103/PhysRevLett.118.111803 Originally published at: Bernet, R; Müller, K; Serra, N; Steinkamp, O; Straumann, U; Vollhardt, A; et al (2017). Observation of B+ c D0K++ decays. Physical Review Letters, 118(11):111803. DOI: https://doi.org/10.1103/PhysRevLett.118.111803 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) Full author list given at the end of the article. Observation of Bþc →D0Kþ Decays R. Aaij et al. (LHCb Collaboration) j (LHCb Collaboration) ( ) (Received 7 January 2017; revised manuscript received 17 February 2017; published 15 March 2017) Received 7 January 2017; revised manuscript received 17 February 2017; published 15 March 2017) (Received 7 January 2017; revised manuscript received 17 February 2017; published 15 March 2017) Using proton-proton collision data corresponding to an integrated luminosity of 3.0 fb−1, recorded by the LHCb detector at center-of-mass energies of 7 and 8 TeV, the Bþc →D0Kþ decay is observed with a statistical significance of 5.1 standard deviations. By normalizing to Bþ →¯D0πþ decays, a measurement of the branching fraction multiplied by the production rates for Bþc relative to Bþ mesons in the LHCb acceptance is obtained, RD0K ¼ ðfc=fuÞ × BðBþc →D0KþÞ ¼ ð9.3þ2.8 −2.5 0.6Þ × 10−7, where the first uncertainty is statistical and the second is systematic. This decay is expected to proceed predominantly through weak annihilation and penguin amplitudes, and is the first Bþc decay of this nature to be observed. DOI: 10.1103/PhysRevLett.118.111803 DOI: 10.1103/PhysRevLett.118.111803 A two-dimensional optimization is performed to determine the second stage BDT requirements for the two-body and four-body modes, where the signal S is compared to the number of background events B in data using a figure of merit S=ð ﬃﬃﬃﬃ B p þ 3=2Þ [25]. The value of B is determined within 50 MeV=c2 of the known Bþc mass. No PID information is used in the BDT training, so that the efficiency for B →D0Kþ and B →D0πþ decays is similar. The use of BDTs to select signal decays was validated by comparing the efficiency of the BDT requirements for Bþ →¯D0πþ decays in data and simulation, where close agreement was found across a wide range of BDT cuts. The purity of the selection is further improved by requiring all kaons and pions in the D0 decay to be identified with a PID selection that has an efficiency of about 85% per particle. RDðÞ0h ¼ N ðBþc →DðÞ0hþÞ N ðBþ →¯D0πþÞ × BðBþ →¯D0πþÞ × ξ; ð2Þ ð2Þ where N ðBþc →DðÞ0hþÞ represents the Bþc →DðÞ0hþ yield, N ðBþ →¯D0πþÞ represents the yield of Bþ →¯D0πþ normalization decays, BðBþ →¯D0πþÞ is the normalization mode branching fraction [11], and ξ is the ratio of efficiencies for reconstructing and selecting Bþ and Bþc mesons decaying to these final states. The LHCb detector is a single-arm forward spectrometer covering the pseudorapidity range 2 < η < 5, described in detail in Refs. [12,13]. The detector allows the reconstruction of both charged and neutral particles. For this analysis, the ring-imaging Cherenkov (RICH) detectors [14], distinguishing pions, kaons, and protons, are particu- larly important. Simulated events are produced using the software described in Refs. [15–22]. After reconstruction of the D0 meson candidate, the same selection is applied to the Bþc and Bþ candidates. The invariant mass of the D0 candidate must be within 25 MeV=c2 of its known value [11]. The other hadron originating from the B decay must have transverse momen- tum (pT) in the range 0.5–10.0 GeV=c and momentum (p) in the range 5–100 GeV=c, ensuring that the track is within the kinematic coverage of the RICH detectors that provide particle identification (PID) information. A kinematic fit is performed to each decay chain [23], with vertex constraints applied to both the B and D vertices, and the D0 candidate mass constrained to its known value. DOI: 10.1103/PhysRevLett.118.111803 However, this expectation may be enhanced by penguin and weak annihilation amplitudes, which will be more pronounced in the Bþc →DðÞ0Kþ mode [see Fig. 1(b) and 1(c)]. This motivates a search for the Bþc →DðÞ0Kþ and Bþc → DðÞ0πþ decays, particularly as the branching fraction estimates in the literature vary considerably [6–8]. This Letter reports a search for Bþc →D0πþ and Bþc → D0Kþ decays in pp collision data corresponding to FIG. 1. Tree (a), penguin (b), and weak annihilation (c) diagrams for the decays studied. In each case, the meson appearing before the comma denotes the favored decay. 111803-1 © 2017 CERN, for the LHCb Collaboration 111803-1 0031-9007=17=118(11)=111803(9) week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) combinations from data with invariant mass in the range 5900–7200 MeV=c2. For the first BDT, background can- didates with a D0 invariant mass more than 30 MeV=c2 away from the known D0 mass are used. In the second BDT, background candidates with a D0 invariant mass within 25 MeV=c2 of the known D0 mass are used. A loose cut on the classifier response of the first BDT is applied before training the second one. This focuses the second BDT training on backgrounds enriched with fully reconstructed D0 mesons. integrated luminosities of 1.0 and 2.0 fb−1 taken by the LHCb experiment at center-of-mass energies of 7 and 8 TeV, respectively, where the D0 meson is reconstructed in the Cabibbo-favored final states D0 →K−πþ or D0 →K−πþπ−πþ (inclusion of charge-conjugate proc- esses is implied throughout). Partially reconstructed Bþc →ðD0 →D0fπ0; γgÞhþ decays, where the neutral particle indicated in braces is not considered in the invariant mass calculation, are treated as additional signal channels. The number of Bþc decays is normalized by comparison to the number of Bþ →½ ¯D0 →Kþπ−ðπþπ−Þπþ decays. A fit to the invariant mass distribution of DðÞ0hþ candidates in the range 5800–6900 MeV=c2 enables a measurement of The inputs to all BDTs include properties of each particle (p, pT, and the IP significance) and additional properties of the B and D0 candidates (decay time, flight distance, decay vertex quality, radial distance between the decay vertex and the PV, and the angle between the reconstructed momentum vector and the line connecting the production and decay vertices). DOI: 10.1103/PhysRevLett.118.111803 The red solid curve illustrates Bþc →D0Kþ decays, the red dashed curve illustrates Bþc →D0Kþ decays, the green dashed curve represents Bþc →D0πþ decays, the gray shaded region represents partially reconstructed background decays, the cyan dashed line represents the combinatorial background, and the total PDF is displayed as a blue solid line. The small drop visible in the total Bþc →DðÞ0πþ PDF around the Bþc mass arises from the fact that the fit finds a small negative value for the Bþc →D0πþ yield. Partially reconstructed decays form a background at invariant masses lower than that of the signal peak. This background is described by a combination of parametric PDFs, with yield and shape parameters that are allowed to vary. A linear function describes the combinatorial back- ground. The yield of Bþ →¯D0Kþ decays, where the kaon is misidentified as a pion, is fixed using a simultaneous fit to correctly identified Bþ →¯D0Kþ events. Using a data- driven analysis of approximately 20 million Dþ decays reconstructed as Dþ →D0πþ, D0 →K−πþ, the proba- bility of kaon misidentification is determined to be 32%. The invariant mass fits to Bþ →ð ¯D0 →Kþπ−Þπþ and Bþ →ð ¯D0 →Kþπ−πþπ−Þπþ decays determine a total observed yield N ðBþ →¯D0πþÞ ¼ 309462 550. two particles are missed, with shape parameters taken from simulated Bþ →D0πþπ0 decays and scaled to account for the different momenta of the decay products in Bþc and Bþ decays. y ð Þ To measure N ðBþc →DðÞ0hþÞ, a simultaneous invariant mass fit to the Bþc →D0πþ and Bþc →D0Kþ samples is performed in the region 5800–6900 MeV=c2. Two-body and four-body D -decay candidates are included, where a Gaussian PDF describes the fully reconstructed Bþc signals. The mean of this Gaussian is fixed to the known Bþc mass [11]. The width of the Bþc →D0πþ PDF is taken from a fit to suppressed Bþ →ð ¯D0 →πþK−Þπþ decays, scaled up by a factor 1.3 to account for the difference in momenta of the decay products in Bþc →D0πþ and Bþ →¯D0πþ decays. The width of the Bþc →D0Kþ peak is related to that of Bþc →D0πþ decays by the ratio of the widths of the Bþ →¯D0Kþ and Bþ →¯D0πþ peaks found in the normali- zation mode fits. DOI: 10.1103/PhysRevLett.118.111803 The Bþ (Bþc ) meson candidates with an invariant mass in the interval 5080–5900 MeV=c2 (5800–6900 MeV=c2) and with a proper decay time above 0.2 ps are retained. Each B candidate is associated with the primary vertex (PV) to which it has the smallest impact parameter (IP), defined as the distance of closest approach of the candidate’s trajec- tory to a given PV. p Simulated signal samples are used to evaluate the relative efficiency for selecting Bþc and Bþ decays. The efficiency ratio is ξ ¼ ϵðBþÞ=ϵðBþc Þ, where ϵðBþÞ and ϵðBþc Þ re- present the combined efficiencies of detector acceptance, trigger, reconstruction, and offline selection. As both Bþc and Bþ mesons are required to decay to the same final-state particles, differences between ϵðBþÞ and ϵðBþc Þ arise due to differences in their masses and lifetimes. The Bþc meson lifetime is ð0.507 0.009Þ ps, which is 3.2 times shorter than that of the Bþ meson [11]. This results in a lower Bþc efficiency relative to Bþ by a factor 2.4, due to the proper decay time cut. The Bþc meson is heavier than the Bþ, which reduces by a factor 1.3 the fraction of Bþc decays in which all final-state particles are within the detector acceptance. However, as the BDTs are trained specifically on Bþc simulated decays, the offline selection efficiency is lower for Bþ decays, contributing a relative efficiency of 0.94. Overall, the efficiency ratio is ξ ¼ 3.04 0.16 ð2.88 0.15Þ for the two-body (four-body) D0 decay. The uncertainties are systematic, arising from the use of Two boosted decision tree (BDT) discriminators [24] are used for further background suppression. They are trained using simulated Bþc →½D0 →K−πþðπþπ−Þhþ signal decays and a sample of wrong-sign Kþπ−ðπþπ−Þhþ 111803-2 111803-2 ] 2 c ) [MeV/ ± h 0 D ( m 5800 6000 6200 6400 6600 6800 5 10 15 20 25 ± π 0 D → ± c B LHCb ) 2 c Candidates / (40 MeV/ 5 10 15 20 25 ± K 0 D → ± c B LHCb FIG. 2. Results of the simultaneous fit to the D0Kþ (top plot) and D0πþ (bottom plot) invariant mass distributions in the Bþc mass region, including the D0 →K−πþ and D0 →K−πþπ−πþ final states. Inclusion of the charge conjugate decays is implied. DOI: 10.1103/PhysRevLett.118.111803 The red solid curve illustrates Bþc →D0Kþ decays, the red dashed curve illustrates Bþc →D0Kþ decays, the green dashed curve represents Bþc →D0πþ decays, the gray shaded region represents partially reconstructed background decays, the cyan dashed line represents the combinatorial background, and the total PDF is displayed as a blue solid line. The small drop visible in the total Bþc →DðÞ0πþ PDF around the Bþc mass arises from the fact that the fit finds a small negative value for the Bþc →D0πþ yield. V I E W L E T T E R S week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) ] 2 c ) [MeV/ ± h 0 D ( m 5800 6000 6200 6400 6600 6800 5 10 15 20 25 ± π 0 D → ± c B LHCb ) 2 c Candidates / (40 MeV/ 5 10 15 20 25 ± K 0 D → ± c B LHCb 17 MARCH 2017 finite simulated samples and possible mismodeling of the simulated Bþc lifetime and production kinematics. To measure N ðBþ →¯D0πþÞ, binned maximum like- lihood fits to the invariant mass distributions of selected Bþ candidates are performed, where separate fits are employed for the two-body and four-body ¯D0 modes. The total probability density function (PDF) is built from four contributions. The Bþ →¯D0πþ decays are modeled by the sum of two modified Gaussian functions with asym- metric power-law tails and an additional Gaussian function as used in Ref. [26], all of which share a common peak position. Misidentified Bþ →¯D0Kþ candidates have an incorrect mass assignment and form a distribution dis- placed downward in mass, with a tail extending to lower invariant masses. They are modeled by the sum of two modified Gaussian PDFs with low-mass power-law tails. All PDF parameters are allowed to vary, with the exception of the tail parameters which are fixed to the values found in simulation. FIG. 2. Results of the simultaneous fit to the D0Kþ (top plot) and D0πþ (bottom plot) invariant mass distributions in the Bþc mass region, including the D0 →K−πþ and D0 →K−πþπ−πþ final states. Inclusion of the charge conjugate decays is implied. DOI: 10.1103/PhysRevLett.118.111803 π 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb K 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb decay. The value of RD0K is at the high end of theoretical predictions [6–8] and an expectation based on the observed Bþc →J=ψπþ yield at LHCb [28]. From Refs. [5] and [11], RJ=ψπ ¼ ð7.0 0.3Þ × 10−6 is obtained. As fc=fu is common to both RJ=ψπ and RD0K, the ratio of branching fractions is measured to be BðBþc →D0KþÞ= BðBþc →J=ψπþÞ ¼ 0.13 0.04 0.01 0.01, where the first uncertainty is statistical, the second is systematic, and the third comes from RJ=ψπ. FIG. 3. CLs p-value distributions for the RD0h observables. The dashed line represents the expected CLs values, where the 1σ and 2σ contours are indicated by the green (dark) and yellow (light) shaded regions, respectively. Upper limits are determined by the points at which the observed CLsþb p-values (black points connected by straight lines) fall below 5% (red solid line). Also displayed are the corresponding CLs ¼ CLsþb=CLb values (blue points connected by straight dotted lines). The absence of the Bþc →D0πþ mode shows that the Bþc →D0Kþ amplitude is not dominated by the tree-level b →u transition shown in Fig. 1(a), but rather by the penguin 1(b) and/or weak annihilation 1(c) diagrams. This result constitutes the first observation of such amplitudes in the decay of a Bþc meson. We express our gratitude to our colleagues in the CERN accelerator departments for the excellent performance of the LHC. We thank the technical and administrative staff at the LHCb institutes. We acknowledge support from CERN and from the national agencies: CAPES, CNPq, FAPERJ and FINEP (Brazil); NSFC (China); CNRS/IN2P3 (France); BMBF, DFG and MPG (Germany); INFN (Italy); FOM and NWO (Netherlands); MNiSW and NCN (Poland); MEN/IFA (Romania); MinES and FASO (Russia); MinECo (Spain); SNSF and SER (Switzerland); NASU (Ukraine); STFC (United Kingdom); NSF (USA). We acknowledge the computing resources that are provided by CERN, IN2P3 (France), KIT and DESY (Germany), INFN (Italy), SURF (Netherlands), PIC (Spain), GridPP (United Kingdom), RRCKI and Yandex LLC (Russia), CSCS (Switzerland), IFIN-HH (Romania), CBPF (Brazil), PL-GRID (Poland) and OSC (USA). We are indebted to the communities behind the multiple open source software packages on which we depend. DOI: 10.1103/PhysRevLett.118.111803 Partially reconstructed Bþc →D0hþ signal decays are modeled using a combination of para- metric PDFs, with yield and shape parameters that are allowed to vary. These decays contribute at lower invariant masses than the fully reconstructed signal decays, as a result of not considering the natural particle in the invariant mass calculation. An additional background component at low invariant mass is included to describe Bþc decays where Misidentified Bþc →D0πþðKþÞ decays in the Bþc →D0KþðπþÞ sample are modeled using the same PDFs as the normalization fits, with widths and peak positions scaled for the decay momentum difference. These shapes are fixed in the fit. Signal decays are split into separate samples with correct and incorrect kaon identification, with a kaon misidentification rate of 7% and a corresponding pion identification efficiency of 91% fixed using the data-driven Dþ analysis described above. An exponential function describes the combinatorial back- ground, which is fitted independently in the Bþc →D0πþ and Bþc →D0Kþ samples. The combinatorial yields, signal yields, and partially reconstructed Bþc →D0hþfπ0g and Bþc →D0hþfπ0g background yields are all free to vary. The fit to data is shown in Fig. 2, where a Bþc →D0Kþ yield of 20 5 events is found. All other signal yields are consistent with zero. To test the significance of each signal yield, CLs hypothesis tests [27] are performed. Upper limits at 95% confidence level (C.L.) are determined by the point 111803-3 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) π 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb K 0 D R 0 0.5 1 1.5 6 − 10 × -value p 0 0.5 1 LHCb FIG. 3. CLs p-value distributions for the RD0h observables. The dashed line represents the expected CLs values, where the 1σ and 2σ contours are indicated by the green (dark) and yellow (light) shaded regions, respectively. Upper limits are determined by the points at which the observed CLsþb p-values (black points connected by straight lines) fall below 5% (red solid line). Also displayed are the corresponding CLs ¼ CLsþb=CLb values (blue points connected by straight dotted lines). [1] I. P. Gouz, V. V. Kiselev, A. K. Likhoded, V. I. Romanovsky, and O. P. Yushchenko, Prospects for the Bc studies at LHCb, Phys. At. Nucl. 67, 1559 (2004). y [2] R. Aaij et al. (LHCb Collaboration), Observation of the Decay Bþc →B0sπþ, Phys. Rev. Lett. 111, 181801 (2013). [3] R. Aaij et al. (LHCb Collaboration), Study of Bþc decays to the KþK−πþ final state and evidence for the decay Bþc →χc0πþ, Phys. Rev. D 94, 091102 (2016). [1] I. P. Gouz, V. V. Kiselev, A. K. Likhoded, V. I. Romanovsky, and O. P. Yushchenko, Prospects for the Bc studies at LHCb, Phys. At. Nucl. 67, 1559 (2004). [2] R. Aaij et al. (LHCb Collaboration), Observation of the Decay Bþc →B0sπþ, Phys. Rev. Lett. 111, 181801 (2013). [3] R. Aaij et al. (LHCb Collaboration), Study of Bþc decays to the KþK−πþ final state and evidence for the decay Bþc →χc0πþ, Phys. Rev. D 94, 091102 (2016). DOI: 10.1103/PhysRevLett.118.111803 This is the first observation of the Bþc →D0Kþ 111803-4 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) [18] D. J. Lange, The EvtGen particle decay simulation package, Nucl. Instrum. Methods Phys. Res., Sect. A 462, 152 (2001). [4] V. M. Abazov et al. (D0 Collaboration), Observation of the Bc Meson in the Exclusive Decay Bc →J=ψπ, Phys. Rev. Lett. 101, 012001 (2008). [5] R. Aaij et al. (LHCb Collaboration), Measurement of Bþc Production at ﬃﬃﬃs p ¼ 8 TeV, Phys. Rev. Lett. 114, 132001 (2015). [19] J. Allison et al. (Geant4 Collaboration), Geant4 develop- ments and applications, IEEE Trans. Nucl. Sci. 53, 270 (2006). ( ) [6] H. F. Fu, Y. Jiang, C. S. Kim, and G.-L. Wang, Probing non- leptonic two-body decays of Bc meson, J. High Energy Phys. 06 (2011) 15. [20] S. Agostinelli et al. (Geant4 Collaboration), Geant4: A simulation toolkit, Nucl. Instrum. Methods Phys. Res., Sect. A 506, 250 (2003). y ( ) [7] D.-S. Du and Z.-T. Wei, Space-like Penguin effects in Bc decays, Eur. Phys. J. C 5, 705 (1998). [21] M. Clemencic, G. Corti, S. Easo, C. R Jones, S. Miglioranzi, M. Pappagallo, and P. Robbe, The LHCb simulation application, Gauss: Design, evolution and experience, J. Phys. Conf. Ser. 331, 032023 (2011). [8] J. Zhang and X.-Q. Yu, Branching ratio and CP violation of Bc →DK decays in the perturbative QCD approach, Eur. Phys. J. C 63, 435 (2009). [22] C.-H. Chang, C. Driouichi, P. Eerola, and X.-G. Wu, BCVEGPY: An event generator for hadronic production of the Bc meson, Comput. Phys. Commun. 159, 192 (2004). [9] D. Ebert, R. N. Faustov, and V. O. Galkin, Weak decays of the Bc meson to charmonium and D mesons in the relativistic quark model, Phys. Rev. D 68, 094020 (2003). [23] W. D. Hulsbergen, Decay chain fitting with a Kalman filter, Nucl. Instrum. Methods Phys. Res., Sect. A 552, 566 (2005). [10] C.-H. Chang and Y.-Q. Chen, Decays of the Bc meson, Phys. Rev. D 49, 3399 (1994). [11] C. Patrignani et al. (Particle Data Group Collaboration), Review of particle physics, Chin. Phys. C 40, 100001 (2016). [24] B. P. Roe, H.-J. Yang, J. Zhu, Y. Liu, I. Stancu, and G. DOI: 10.1103/PhysRevLett.118.111803 Individual groups or members have received support from AvH Foundation (Germany), EPLANET, Marie Skłodowska-Curie Actions and ERC (European Union), Conseil Général de Haute- Savoie, Labex ENIGMASS and OCEVU, Région Auvergne (France), RFBR and Yandex LLC (Russia), GVA, XuntaGal and GENCAT (Spain), Herchel Smith Fund, The Royal Society, Royal Commission for the Exhibition of 1851 and the Leverhulme Trust (United Kingdom). at which the p-value falls below 5%. All free variables in the fit are considered as nuisance parameters in this procedure. The p-value distributions for each RD0h meas- urement are shown in Fig. 3. The Bþc →D0hþ modes demonstrate no excess, and the RD0h CLs confidence intervals are determined similarly to that of RD0π. The upper limits at 95% confidence level found for RD0π, RD0π, and RD0K are RD0π < 3.9 × 10−7; RD0π < 1.1 × 10−6; RD0K < 1.1 × 10−6: RD0π < 3.9 × 10−7; RD0π < 1.1 × 10−6; RD0K < 1.1 × 10−6: The systematic uncertainties affecting the measurements are found to be much smaller than the statistical uncer- tainty, and do not alter the above upper limits. In the case of RD0K, the observed signal is of much higher significance. To determine the full uncertainty for RD0K, the systematic uncertainties affecting the measure- ment are accounted for. A systematic uncertainty of 1.1 × 10−8 is incurred from the use of fixed terms in the invariant mass fit. According to Eq. (2), several terms with associated relative uncertainties scale the measured signal yield: ξ with 5.3% uncertainty, BðBþ →¯D0πþÞ with 3.1% uncertainty [11], and N ðBþ →¯D0πþÞ with 0.14% uncer- tainty. The total systematic uncertainty, given by the sum in quadrature, is 6.2%. To determine the significance of the Bþc →D0Kþ peak, a likelihood scan is performed. The resulting −Δ logðLÞ value for the RD0K ¼ 0 hypothesis corresponds to a statistical significance of ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ −2Δ logðLÞ p ¼ 5.1σ for the signal. The final result is RD0K ¼ ð9.3þ2.8 −2.5 0.6Þ × 10−7; [3] R. Aaij et al. (LHCb Collaboration), Study of Bþc decays to the KþK−πþ final state and evidence for the decay Bþc →χc0πþ, Phys. Rev. D 94, 091102 (2016). where the first uncertainty is statistical and the second is systematic. R. Aaij,40 B. Adeva,39 M. Adinolfi,48 Z. Ajaltouni,5 S. Akar,59 J. Albrecht,10 F. Alessio,40 M. Alexander,53 S. Ali,43 G. Alkhazov,31 P. Alvarez Cartelle,55 A. A. Alves Jr.,59 S. Amato,2 S. Amerio,23 Y. Amhis,7 L. An,3 L. Anderlini,18 G. Andreassi,41 M. Andreotti,17,g J. E. Andrews,60 R. B. Appleby,56 F. Archilli,43 P. d’Argent,12 J. Arnau Romeu,6 A. Artamonov,37 M. Artuso,61 E. Aslanides,6 G. Auriemma,26 M. Baalouch,5 I. Babuschkin,56 S. Bachmann,12 J. J. Back,50 A. Badalov,38 C. Baesso,62 S. Baker,55 V. Balagura,7,c W. Baldini,17 R. J. Barlow,56 C. Barschel,40 S. Barsuk,7 W. Barter,56 F. Baryshnikov,32 M. Baszczyk,27 V. Batozskaya,29 B. Batsukh,61 V. Battista,41 A. Bay,41 L. Beaucourt,4 J. Beddow,53 F. Bedeschi,24 I. Bediaga,1 L. J. Bel,43 V. Bellee,41 N. Belloli,21,i K. Belous,37 I. Belyaev,32 E. Ben-Haim,8 G. Bencivenni,19 S. Benson,43 A. Berezhnoy,33 R. Bernet,42 A. Bertolin,23 C. Betancourt,42 F. Betti,15 M.-O. Bettler,40 M. van Beuzekom,43 Ia. Bezshyiko,42 S. Bifani,47 P. Billoir,8 T. Bird,56 A. Birnkraut,10 A. Bitadze,56 A. Bizzeti,18,u T. Blake,50 F. Blanc,41 J. Blouw,11,† S. Blusk,61 V. Bocci,26 T. Boettcher,58 A. Bondar,36,w N. Bondar,31,40 W. Bonivento,16 I. Bordyuzhin,32 A. Borgheresi,21,i S. Borghi,56 M. Borisyak,35 M. Borsato,39 F. Bossu,7 M. Boubdir,9 T. J. V. Bowcock,54 E. Bowen,42 C. Bozzi,17,40 S. Braun,12 M. Britsch,12 T. Britton,61 J. Brodzicka,56 E. Buchanan,48 C. Burr,56 A. Bursche,2 J. Buytaert,40 S. Cadeddu,16 R. Calabrese,17,g M. Calvi,21,i M. Calvo Gomez,38,m A. Camboni,38 P. Campana,19 D. H. Campora Perez,40 L. Capriotti,56 A. Carbone,15,e G. Carboni,25,j R. Cardinale,20,h A. Cardini,16 P. Carniti,21,i L. Carson,52 K. Carvalho Akiba,2 G. Casse,54 L. Cassina,21,i L. Castillo Garcia,41 M. Cattaneo,40 G. Cavallero,20 R. Cenci,24,t D. Chamont,7 M. Charles,8 Ph. Charpentier,40 G. Chatzikonstantinidis,47 M. Chefdeville,4 S. Chen,56 S. -F. Cheung,57 V. Chobanova,39 DOI: 10.1103/PhysRevLett.118.111803 McGregor, Boosted decision trees as an alternative to artificial neural networks for particle identification, Nucl. Instrum. Methods Phys. Res., Sect. A 543, 577 (2005). [12] A. A. Alves Jr. et al. (LHCb Collaboration), The LHCb detector at the LHC, J. Instrum. 3, S08005 (2008). [25] G. Punzi, in Statistical Problems in Particle Physics, Astrophysics, and Cosmology, edited by L. Lyons, R. Mount, and R. Reitmeyer (Stanford Linear Accelerator Center, Stanford, California, 2003), p. 79. [13] R. Aaij et al. (LHCb Collaboration), LHCb detector performance, Int. J. Mod. Phys. A 30, 1530022 (2015). [14] M. Adinolfi et al., Performance of the LHCb RICH detector at the LHC, Eur. Phys. J. C 73, 2431 (2013). [26] R. Aaij et al. (LHCb Collaboration), Measurement of CP observables in B →DK and B →Dπ with two- and four-body D meson decays, Phys. Lett. B 760, 117 (2016). [15] T. Sjöstrand, S. Mrenna, and P. Skands, PYTHIA 6.4 physics and manual, J. High Energy Phys. 05 (2006) 026. [16] T. Sjöstrand, S. Mrenna, and P. Skands, A brief introduction to PYTHIA 8.1, Comput. Phys. Commun. 178, 852 (2008). [27] L. Moneta et al., The RooStats Project, Proc. Sci., ACAT20102010 (2010) 057. [17] I. Belyaev et al., Handling of the generation of primary events in Gauss, the LHCb simulation framework, J. Phys. Conf. Ser. 331, 032047 (2011). [28] R. Aaij et al. (LHCb Collaboration), Measurement of the branching fraction ratio BðBþc →ψð2SÞπþÞ= BðBþc →J=ψπþÞ, Phys. Rev. D 92, 072007 (2015). R. Aaij,40 B. Adeva,39 M. Adinolfi,48 Z. Ajaltouni,5 S. Akar,59 J. Albrecht,10 F. Alessio,40 M. Alexander,53 S. Ali,43 G. Alkhazov,31 P. Alvarez Cartelle,55 A. A. Alves Jr.,59 S. Amato,2 S. Amerio,23 Y. Amhis,7 L. An,3 L. Anderlini,18 G. Andreassi,41 M. Andreotti,17,g J. E. Andrews,60 R. B. Appleby,56 F. Archilli,43 P. d’Argent,12 J. Arnau Romeu,6 A. Artamonov,37 M. Artuso,61 E. Aslanides,6 G. Auriemma,26 M. Baalouch,5 I. Babuschkin,56 S. Bachmann,12 J. J. Back,50 A. Badalov,38 C. Baesso,62 S. Baker,55 V. Balagura,7,c W. Baldini,17 R. J. Barlow,56 C. Barschel,40 S. Barsuk,7 W. Barter,56 F. Baryshnikov,32 M. Baszczyk,27 V. Batozskaya,29 B. Batsukh,61 V. Battista,41 A. Bay,41 L. Beaucourt,4 J. Beddow,53 F. Bedeschi,24 I. Bediaga,1 L. J. Bel,43 V. Bellee,41 N. Belloli,21,i K. Belous,37 I. Belyaev,32 E. Ben-Haim,8 G. Bencivenni,19 S. Benson,43 A. Berezhnoy,33 R. Bernet,42 A. Bertolin,23 C. Betancourt,42 F. Betti,15 M.-O. Bettler,40 M. van Beuzekom,43 Ia. Bezshyiko,42 S. Bifani,47 P. Billoir,8 T. Bird,56 A. Birnkraut,10 A. Bitadze,56 A. M. Chrzaszcz,42,27 X. Cid Vidal,39 G. Ciezarek,43 P. E. L. Clarke,52 M. Clemencic,40 H. V. Cliff,49 J. Closier,40 V. Coco,59 J. Cogan,6 E. Cogneras,5 V. Cogoni,16,40,f L. Cojocariu,30 G. Collazuol,23,o P. Collins,40 A. Comerma-Montells,12 A. Contu,40 A. Cook,48 G. Coombs,40 S. Coquereau,38 G. Corti,40 M. Corvo,17,g C. M. Costa Sobral,50 B. Couturier,40 G. A. Cowan,52 D. C. Craik,52 A. Crocombe,50 M. Cruz Torres,62 S. Cunliffe,55 R. Currie,55 C. D’Ambrosio,40 F. Da Cunha Marinho,2 E. Dall’Occo,43 J. Dalseno,48 P. N. Y. David,43 A. Davis,3 K. De Bruyn,6 S. De Capua,56 M. De Cian,12 J. M. De Miranda,1 L. De Paula,2 M. De Serio,14,d P. De Simone,19 C. T. Dean,53 D. Decamp,4 M. Deckenhoff,10 L. Del Buono,8 M. Demmer,10 A. Dendek,28 D. Derkach,35 O. Deschamps,5 F. Dettori,40 B. Dey,22 A. Di Canto,40 H. Dijkstra,40 F. Dordei,40 M. Dorigo,41 A. Dosil Suárez,39 A. Dovbnya,45 K. Dreimanis,54 L. Dufour,43 G. Dujany,56 K. Dungs,40 P. Durante,40 R. Dzhelyadin,37 A. Dziurda,40 A. Dzyuba,31 N. Déléage,4 S. Easo,51 M. Ebert,52 U. Egede,55 V. Egorychev,32 S. Eidelman,36,w S. Eisenhardt,52 U. Eitschberger,10 R. Ekelhof,10 L. Eklund,53 S. Ely,61 S. Esen,12 H. M. Evans,49 T. Evans,57 A. Falabella,15 N. Farley,47 S. Farry,54 R. Fay,54 D. Fazzini,21,i D. Ferguson,52 A. Fernandez Prieto,39 F. Ferrari,15,40 F. Ferreira Rodrigues,2 M. Ferro-Luzzi,40 S. Filippov,34 R. A. Fini,14 M. Fiore,17,g M. Fiorini,17,g M. Firlej,28 C. Fitzpatrick,41 T. Fiutowski,28 F. Fleuret,7,b K. Fohl,40 M. Fontana,16,40 F. Fontanelli,20,h D. C. Forshaw,61 R. Forty,40 V. Franco Lima,54 M. Frank,40 C. Frei,40 J. Fu,22,q W. Funk,40 E. Furfaro,25,j C. Färber,40 A. Gallas Torreira,39 D. Galli,15,e S. Gallorini,23 S. Gambetta,52 M. Gandelman,2 P. Gandini,57 Y. Gao,3 L. M. Garcia Martin,69 J. García Pardiñas,39 J. Garra Tico,49 L. Garrido,38 P. J. Garsed,49 D. Gascon,38 C. Gaspar,40 L. Gavardi,10 G. Gazzoni,5 D. Gerick,12 E. Gersabeck,12 M. Gersabeck,56 T. Gershon,50 Ph. Ghez,4 S. Gianì,41 V. Gibson,49 O. G. Girard,41 L. Giubega,30 K. Gizdov,52 V. V. Gligorov,8 D. Golubkov,32 A. Golutvin,55,40 A. Gomes,1,a I. V. Gorelov,33 C. Gotti,21,i R. Graciani Diaz,38 L. A. Granado Cardoso,40 E. Graugés,38 E. Graverini,42 G. Graziani,18 A. Grecu,30 P. Griffith,47 L. Grillo,21,40,i B. R. Gruberg Cazon,57 O. Grünberg,67 E. Gushchin,34 Yu. Guz,37 T. Gys,40 C. Göbel,62 T. Hadavizadeh,57 C. Hadjivasiliou,5 G. Haefeli,41 C. Haen,40 S. C. Haines,49 B. Hamilton,60 X. Han,12 S. Hansmann-Menzemer,12 N. Harnew,57 S. T. Harnew,48 J. Harrison,56 M. Hatch,40 J. He,63 T. Head,41 A. Heister,9 K. Hennessy,54 P. Henrard,5 L. Henry,8 E. van Herwijnen,40 M. Heß,67 A. Hicheur,2 D. Hill,57 C. Hombach,56 H. Hopchev,41 W. Hulsbergen,43 T. Humair,55 M. Hushchyn,35 D. Hutchcroft,54 M. Idzik,28 P. Ilten,58 R. Jacobsson,40 A. Jaeger,12 J. Jalocha,57 E. Jans,43 A. Jawahery,60 F. Jiang,3 M. John,57 D. Johnson,40 C. R. Jones,49 C. Joram,40 B. Jost,40 N. Jurik,57 S. Kandybei,45 M. Karacson,40 J. M. Kariuki,48 S. Karodia,53 M. Kecke,12 M. Kelsey,61 M. Kenzie,49 T. Ketel,44 E. Khairullin,35 B. Khanji,12 C. Khurewathanakul,41 T. Kirn,9 S. Klaver,56 K. Klimaszewski,29 S. Koliiev,46 M. Kolpin,12 I. Komarov,41 R. F. Koopman,44 P. Koppenburg,43 A. Kosmyntseva,32 A. Kozachuk,33 M. Kozeiha,5 L. Kravchuk,34 K. Kreplin,12 M. Kreps,50 P. Krokovny,36,w F. Kruse,10 W. Krzemien,29 W. Kucewicz,27,l M. Kucharczyk,27 V. Kudryavtsev,36,w A. K. Kuonen,41 K. Kurek,29 T. Kvaratskheliya,32,40 D. Lacarrere,40 G. Lafferty,56 A. Lai,16 G. Lanfranchi,19 C. Langenbruch,9 T. Latham,50 C. Lazzeroni,47 R. Le Gac,6 J. van Leerdam,43 A. Leflat,33,40 J. Lefrançois,7 R. Lefèvre,5 F. Lemaitre,40 E. Lemos Cid,39 O. Leroy,6 T. Lesiak,27 B. Leverington,12 T. Li,3 Y. Li,7 T. Likhomanenko,35,68 R. Lindner,40 C. Linn,40 F. Lionetto,42 X. Liu,3 D. Loh,50 I. Longstaff,53 J. H. Lopes,2 D. Lucchesi,23,o M. Lucio Martinez,39 H. Luo,52 A. Lupato,23 E. Luppi,17,g O. Lupton,40 A. Lusiani,24 X. Lyu,63 F. Machefert,7 F. Maciuc,30 O. Maev,31 K. Maguire,56 S. Malde,57 A. Malinin,68 T. Maltsev,36 G. Manca,16,f G. Mancinelli,6 P. Manning,61 J. Maratas,5,v J. F. Marchand,4 U. Marconi,15 C. Marin Benito,38 M. Marinangeli,41 P. Marino,24,t J. Marks,12 G. Martellotti,26 M. Martin,6 M. Martinelli,41 D. Martinez Santos,39 F. Martinez Vidal,69 D. Martins Tostes,2 L. M. Massacrier,7 A. Massafferri,1 R. Matev,40 A. Mathad,50 Z. Mathe,40 C. Matteuzzi,21 A. Mauri,42 E. Maurice,7,b B. Maurin,41 A. Mazurov,47 M. McCann,55,40 A. McNab,56 R. McNulty,13 B. Meadows,59 F. Meier,10 M. Meissner,12 D. Melnychuk,29 M. Merk,43 A. Merli,22,q E. Michielin,23 D. A. Milanes,66 M. -N. Minard,4 D. S. Mitzel,12 A. Mogini,8 J. Molina Rodriguez,1 I. A. Monroy,66 S. Monteil,5 M. Morandin,23 P. Morawski,28 A. Mordà,6 M. J. Morello,24,t O. Morgunova,68 J. Moron,28 A B M i 52 R M t i 61 F M h i 52 M M ld 43 M M i i 15 D Müll 56 J Müll 10 K Müll 42 V Müll 10 DOI: 10.1103/PhysRevLett.118.111803 Bizzeti,18,u T. Blake,50 F. Blanc,41 J. Blouw,11,† S. Blusk,61 V. Bocci,26 T. Boettcher,58 A. Bondar,36,w N. Bondar,31,40 W. Bonivento,16 I. Bordyuzhin,32 A. Borgheresi,21,i S. Borghi,56 M. Borisyak,35 M. Borsato,39 F. Bossu,7 M. Boubdir,9 T. J. V. Bowcock,54 E. Bowen,42 C. Bozzi,17,40 S. Braun,12 M. Britsch,12 T. Britton,61 J. Brodzicka,56 E. Buchanan,48 C. Burr,56 A. Bursche,2 J. Buytaert,40 S. Cadeddu,16 R. Calabrese,17,g M. Calvi,21,i M. Calvo Gomez,38,m A. Camboni,38 P. Campana,19 D. H. Campora Perez,40 L. Capriotti,56 A. Carbone,15,e G. Carboni,25,j R. Cardinale,20,h A. Cardini,16 P. Carniti,21,i L. Carson,52 K. Carvalho Akiba,2 G. Casse,54 L. Cassina,21,i L. Castillo Garcia,41 M. Cattaneo,40 G. Cavallero,20 R. Cenci,24,t D. Chamont,7 M. Charles,8 Ph. Charpentier,40 G. Chatzikonstantinidis,47 M. Chefdeville,4 S. Chen,56 S. -F. Cheung,57 V. Chobanova,39 111803-5 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) J. Cogan,6 E. Cogneras,5 V. Cogoni,16,40,f L. Cojocariu,30 G. Collazuol,23,o P. Collins,40 A. Comer L. De Paula,2 M. De Serio,14,d P. De Simone,19 C. T. Dean,53 D. Decamp,4 M. Deckenhoff,10 L. A. Dendek,28 D. Derkach,35 O. Deschamps,5 F. Dettori,40 B. Dey,22 A. Di Canto,40 H. Dijkstra, g y N. Farley,47 S. Farry,54 R. Fay,54 D. Fazzini,21,i D. Ferguson,52 A. Fernandez Prieto,39 F. Ferrari,15,40 F. Ferreira Rodrigues,2 M. Ferro-Luzzi,40 S. Filippov,34 R. A. Fini,14 M. Fiore,17,g M. Fiorini,17,g M. Firlej,28 C. Fit pp j p F. Fleuret,7,b K. Fohl,40 M. Fontana,16,40 F. Fontanelli,20,h D. C. Forshaw,61 R. Forty,40 V. Franco Lima,54 M. Frank,40 C. Frei,40 J. Fu,22,q W. Funk,40 E. Furfaro,25,j C. Färber,40 A. Gallas Torreira,39 D. Galli,15,e S. Gallorini,23 S. Gambetta,52 M. Lucio Martinez,39 H. Luo,52 A. Lupato,23 E. Luppi,17,g O. Lupton,40 A. Lusiani,24 X. Lyu,63 F. Machefert,7 F. Maciuc,30 J. F. Marchand,4 U. Marconi,15 C. Marin Benito,38 M. Marinangeli,41 P. Marino,24,t J. Marks,12 G. Martellotti,26 M. Martin,6 M. Martinelli,41 D. Martinez Santos,39 F. Martinez Vidal,69 D. Martins Tostes,2 L. M. Massacrier,7 A. Massafferri,1 R. Matev,40 A. Mathad,50 Z. Mathe,40 C. Matteuzzi,21 A. Mauri,42 E. Maurice,7,b B. Maurin,41 A. Mazurov,47 M. McCann,55,40 A. McNab,56 R. McNulty,13 B. Meadows,59 F. Meier,10 M. Meissner,12 D. Melnychuk,29 M. Merk,43 A M li22 q E Mi hi li 23 D A Mil 66 M N Mi d4 D S Mi l12 A M i i8 J M li R d i 1 M. McCann,55,40 A. McNab,56 R. DOI: 10.1103/PhysRevLett.118.111803 Vieites Diaz,39 H. Viemann,67 X. Vilasis-Cardona,38,m M. Vitti,49 V. Volkov,33 A. Vollhardt,42 B. Voneki,40 A. Vorobyev,31 V. Vorobyev,36,w C. Voß,9 J. A. de Vries,43 C. Vázquez Sierra,39 R. Waldi,67 C. Wallace,50 R. Wallace,13 J. Walsh,24 J. Wang,61 D. R. Ward,49 H. M. Wark,54 N. K. Watson,47 D. Websdale,55 A. Weiden,42 M. Whitehead,40 J. Wicht,50 G. Wilkinson,57,40 M. Wilkinson,61 M. Williams,40 M. P. Williams,47 M. Williams,58 T. Williams,47 F. F. Wilson,51 J. Wimberley,60 J. Wishahi,10 W. Wislicki,29 M. Witek,27 G. Wormser,7 S. A. Wotton,49 K. Wraight,53 K. Wyllie,40 Y. Xie,65 Z. Xing,61 Z. Xu,4 Z. Yang,3 Y. Yao,61 H. Yin,65 J. Yu,65 X. Yuan,36,w O. Yushchenko,37 K. A. Zarebski,47 M. Zavertyaev,11,c L. Zhang,3 Y. Zhang,7 Y. Zhang,63 A. Zhelezov,12 Y. Zheng,63 X. Zhu,3 V. Zhukov,33 and S. Zucchelli15 (LHCb Collaboration) 1Centro Brasileiro de Pesquisas Físicas (CBPF), Rio de Janeiro, Brazil 2Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3Center for High Energy Physics, Tsinghua University, Beijing, China 4LAPP, Université Savoie Mont-Blanc, CNRS/IN2P3, Annecy-Le-Vieux, France 5Clermont Université, Université Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 7LAL, Université Paris-Sud, CNRS/IN2P3, Orsay, France 8LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France 9I. Physikalisches Institut, RWTH Aachen University, Aachen, Germany 10Fakultät Physik, Technische Universität Dortmund, Dortmund, Germany 11Max-Planck-Institut für Kernphysik (MPIK), Heidelberg, Germany 12Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 13School of Physics, University College Dublin, Dublin, Ireland 14Sezione INFN di Bari, Bari, Italy 15Sezione INFN di Bologna, Bologna, Italy 16Sezione INFN di Cagliari, Cagliari, Italy DOI: 10.1103/PhysRevLett.118.111803 McNulty,13 B. Meadows,59 F. Meier,10 M. Meissner,12 D. Melnychuk,29 M. Merk,43 A. Merli,22,q E. Michielin,23 D. A. Milanes,66 M. -N. Minard,4 D. S. Mitzel,12 A. Mogini,8 J. Molina Rodriguez,1 I. A. Monroy,66 S. Monteil,5 M. Morandin,23 P. Morawski,28 A. Mordà,6 M. J. Morello,24,t O. Morgunova,68 J. Moron,28 , , , , , , , , , P. Naik,48 T. Nakada,41 R. Nandakumar,51 A. Nandi,57 I. Nasteva,2 M. Needham,52 N. Neri,22 S. Neubert,12 N. Neufeld,40 M. Neuner,12 T. D. Nguyen,41 C. Nguyen-Mau,41,n S. Nieswand,9 R. Niet,10 N. Nikitin,33 T. Nikodem,12 A. Nogay,68 A. Novoselov,37 D. P. O’Hanlon,50 A. Oblakowska-Mucha,28 V. Obraztsov,37 S. Ogilvy,19 R. Oldeman,16,f C. J. G. Onderwater,70 J. M. Otalora Goicochea,2 A. Otto,40 P. Owen,42 A. Oyanguren,69 P. R. Pais,41 A. Palano,14,d A. Novoselov, D. P. O Hanlon, A. Oblakowska Mucha, V. Obraztsov, S. Ogilvy, R. Oldeman, C. J. G. Onderwater,70 J. M. Otalora Goicochea,2 A. Otto,40 P. Owen,42 A. Oyanguren,69 P. R. Pais,41 A. Palano,14,d M. Palutan,19 A. Papanestis,51 M. Pappagallo,14,d L. L. Pappalardo,17,g W. Parker,60 C. Parkes,56 G. Passaleva,18 g g p S. Perazzini,40 P. Perret,5 L. Pescatore,47 K. Petridis,48 A. Petrolini,20,h A. Petrov,68 M. Petruzzo,22,q E. Picatoste Olloqui,38 111803-6 week ending 17 MARCH 2017 week ending 17 MARCH 2017 P H Y S I C A L R E V I E W L E T T E R S PRL 118, 111803 (2017) S. Poslavskii,37 C. Potterat,2 E. Price,48 J. D. Price,54 J. Prisciandaro,39,40 A. Pritchard,54 C. Prouve,48 V. Pugatch,46 A. Puig Navarro,42 G. Punzi,24,p W. Qian,50 R. Quagliani,7,48 B. Rachwal,27 J. H. Rademacker,48 M. Rama,24 C. Sanchez Mayordomo,69 B. Sanmartin Sedes,39 R. Santacesaria,26 C. Santamarina Rios,39 M. Santimaria,19 E. Santovetti,25,j A. Sarti,19,k C. Satriano,26,s A. Satta,25 D. M. Saunders,48 D. Savrina,32,33 S. Schael,9 M. Schellenberg,10 C. Sanchez Mayordomo,69 B. Sanmartin Sedes,39 R. Santacesaria,26 C. Santamarina Rios,39 M. Santimaria,19 E. Santovetti,25,j A. Sarti,19,k C. Satriano,26,s A. Satta,25 D. M. Saunders,48 D. Savrina,32,33 S. Schael,9 M. Schellenberg,10 M. Schiller,53 H. Schindler,40 M. Schlupp,10 M. Schmelling,11 T. Schmelzer,10 B. Schmidt,40 O. Schneider,41 A. Schopper,40 K. Schubert,10 M. Schubiger,41 M. -H. Schune,7 R. Schwemmer,40 B. Sciascia,19 A. Sciubba,26,k A. Semennikov,32 6 5 5 U. Uwer,12 C. Vacca,16,f V. Vagnoni,15,40 A. Valassi,40 S. Valat,40 G. Valenti,15 R. Vazquez Gomez,19 P. Vazquez Regueiro,39 S. Vecchi,17 M. van Veghel,43 J. J. Velthuis,48 M. Veltri,18,r G. Veneziano,57 A. Venkateswaran,61 M. Vernet,5 M. Vesterinen,12 J. V. Viana Barbosa,40 B. Viaud,7 D. Vieira,63 M. 1Centro Brasileiro de Pesquisas Físicas (CBPF), Rio de Janeiro, Brazil 2Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 3Center for High Energy Physics, Tsinghua University, Beijing, China 4LAPP, Université Savoie Mont-Blanc, CNRS/IN2P3, Annecy-Le-Vieux, France 5Clermont Université, Université Blaise Pascal, CNRS/IN2P3, LPC, Clermont-Ferrand, France 6CPPM, Aix-Marseille Université, CNRS/IN2P3, Marseille, France 7LAL, Université Paris-Sud, CNRS/IN2P3, Orsay, France 8LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France 9I. Physikalisches Institut, RWTH Aachen University, Aachen, Germany 10Fakultät Physik, Technische Universität Dortmund, Dortmund, Germany 11Max-Planck-Institut für Kernphysik (MPIK), Heidelberg, Germany 12Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 13School of Physics, University College Dublin, Dublin, Ireland 14Sezione INFN di Bari, Bari, Italy 15Sezione INFN di Bologna, Bologna, Italy 16Sezione INFN di Cagliari, Cagliari, Italy F. Polci,8 A. Poluektov,50,36 I. Polyakov,61 E. Polycarpo,2 G. J. Pomery,48 A. Popov,37 D. Popov,11,40 B. Popovici,30 S. Poslavskii,37 C. Potterat,2 E. Price,48 J. D. Price,54 J. Prisciandaro,39,40 A. Pritchard,54 C. Prouve,48 V. Pugatch,46 A. Puig Navarro,42 G. Punzi,24,p W. Qian,50 R. Quagliani,7,48 B. Rachwal,27 J. H. Rademacker,48 M. Rama,24 M. Ramos Pernas,39 M. S. Rangel,2 I. Raniuk,45 F. Ratnikov,35 G. Raven,44 F. Redi,55 S. Reichert,10 A. C. dos Reis,1 C. Remon Alepuz,69 V. Renaudin,7 S. Ricciardi,51 S. Richards,48 M. Rihl,40 K. Rinnert,54 V. Rives Molina,38 P. Robbe,7,40 A. B. Rodrigues,1 E. Rodrigues,59 J. A. Rodriguez Lopez,66 P. Rodriguez Perez,56,† A. Rogozhnikov,35 S. Roiser,40 A. Rollings,57 V. Romanovskiy,37 A. Romero Vidal,39 J. W. Ronayne,13 M. Rotondo,19 M. S. Rudolph,61 T. Ruf,40 P. Ruiz Valls,69 J. J. Saborido Silva,39 E. Sadykhov,32 N. Sagidova,31 B. Saitta,16,f V. Salustino Guimaraes,1 C. Sanchez Mayordomo,69 B. Sanmartin Sedes,39 R. Santacesaria,26 C. Santamarina Rios,39 M. Santimaria,19 E. Santovetti,25,j A. Sarti,19,k C. Satriano,26,s A. Satta,25 D. M. Saunders,48 D. Savrina,32,33 S. Schael,9 M. Schellenberg,10 M. Schiller,53 H. Schindler,40 M. Schlupp,10 M. Schmelling,11 T. Schmelzer,10 B. Schmidt,40 O. Schneider,41 A. Schopper,40 K. Schubert,10 M. Schubiger,41 M. -H. Schune,7 R. Schwemmer,40 B. Sciascia,19 A. Sciubba,26,k A. Semennikov,32 A. Sergi,47 N. Serra,42 J. Serrano,6 L. Sestini,23 P. Seyfert,21 M. Shapkin,37 I. Shapoval,45 Y. Shcheglov,31 T. Shears,54 L. Shekhtman,36,w V. Shevchenko,68 B. G. Siddi,17,40 R. Silva Coutinho,42 L. Silva de Oliveira,2 G. Simi,23,o S. Simone,14,d M. Sirendi,49 N. Skidmore,48 T. Skwarnicki,61 E. Smith,55 I. T. Smith,52 J. Smith,49 M. Smith,55 H. Snoek,43 l. Soares Lavra,1 M. D. Sokoloff,59 F. J. P. Soler,53 B. Souza De Paula,2 B. Spaan,10 P. Spradlin,53 S. Sridharan,40 F. Stagni,40 M. Stahl,12 S. Stahl,40 P. Stefko,41 S. Stefkova,55 O. Steinkamp,42 S. Stemmle,12 O. Stenyakin,37 H. Stevens,10 S. Stevenson,57 S. Stoica,30 S. Stone,61 B. Storaci,42 S. Stracka,24,p M. Straticiuc,30 U. Straumann,42 L. Sun,64 W. Sutcliffe,55 K. Swientek,28 V. Syropoulos,44 M. Szczekowski,29 T. Szumlak,28 S. T’Jampens,4 A. Tayduganov,6 T. Tekampe,10 G. Tellarini,17,g F. Teubert,40 E. Thomas,40 J. van Tilburg,43 M. J. Tilley,55 V. Tisserand,4 M. Tobin,41 S. Tolk,49 L. Tomassetti,17,g D. Tonelli,40 S. Topp-Joergensen,57 F. Toriello,61 E. Tournefier,4 S. Tourneur,41 K. Trabelsi,41 M. Traill,53 M. T. Tran,41 M. Tresch,42 A. Trisovic,40 A. Tsaregorodtsev,6 P. Tsopelas,43 A. Tully,49 N. Tuning,43 A. Ukleja,29 A. Ustyuzhanin,35 U. Uwer,12 C. Vacca,16,f V. Vagnoni,15,40 A. Valassi,40 S. Valat,40 G. Valenti,15 R. Vazquez Gomez,19 P. Vazquez Regueiro,39 S. Vecchi,17 M. van Veghel,43 J. J. Velthuis,48 M. Veltri,18,r G. Veneziano,57 A. Venkateswaran,61 M. Vernet,5 M. Vesterinen,12 J. V. Viana Barbosa,40 B. Viaud,7 D. Vieira,63 M. Vieites Diaz,39 H. Viemann,67 X. Vilasis-Cardona,38,m M. Vitti,49 V. Volkov,33 A. Vollhardt,42 B. Voneki,40 A. Vorobyev,31 V. Vorobyev,36,w C. Voß,9 J. A. de Vries,43 C. Vázquez Sierra,39 R. Waldi,67 C. Wallace,50 R. Wallace,13 J. Walsh,24 J. Wang,61 D. R. Ward,49 H. M. Wark,54 N. K. Watson,47 D. Websdale,55 A. Weiden,42 M. Whitehead,40 J. Wicht,50 G. Wilkinson,57,40 M. Wilkinson,61 M. Williams,40 M. P. Williams,47 M. Williams,58 T. Williams,47 F. F. Wilson,51 J. Wimberley,60 J. Wishahi,10 W. Wislicki,29 M. Witek,27 G. Wormser,7 S. A. Wotton,49 K. Wraight,53 K. Wyllie,40 Y. Xie,65 Z. Xing,61 Z. Xu,4 Z. Yang,3 Y. Yao,61 H. Yin,65 J. Yu,65 X. Yuan,36,w O. Yushchenko,37 K. A. Zarebski,47 M. Zavertyaev,11,c L. Zhang,3 Y. Zhang,7 Y. Zhang,63 A. Zhelezov,12 Y. Zheng,63 X. Zhu,3 V. Zhukov,33 and S. Zucchelli15 P H Y S I C A L R E V I E W L E T T E R S Wills Physics Laboratory, University of Bristol, Bristol, United Kingdom 49Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 50Department of Physics, University of Warwick, Coventry, United Kingdom 51STFC Rutherford Appleton Laboratory, Didcot, United Kingdom 52School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 53School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 54Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 55Imperial College London, London, United Kingdom 56School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 57Department of Physics, University of Oxford, Oxford, United Kingdom 58Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 59University of Cincinnati, Cincinnati, Ohio, USA 60University of Maryland, College Park, Maryland, USA 61Syracuse University, Syracuse, New York, USA 62Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, associated with Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 63University of Chinese Academy of Sciences, Beijing, China, associated with Center for High Energy Physics, Tsinghua University, Beijing, China 64School of Physics and Technology, Wuhan University, Wuhan, China, associated with Center for High Energy Physics, Tsinghua University, Beijing, China 65Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China, associated with Center for High Energy Physics, Tsinghua University, Beijing, China 66Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia, associated with LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, France 67Institut für Physik, Universität Rostock, Rostock, Germany, associated with Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 68National Research Centre Kurchatov Institute, Moscow, Russia, associated with Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia Imperial College London, London, United Kingdom 58Massachusetts Institute of Technology, Cambridge, Massachusetts, USA 59 59University of Cincinnati, Cincinnati, Ohio, USA 60 61Syracuse University, Syracuse, New York, USA 62Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rio de Janeiro, Brazil, 63University of Chinese Academy of Sciences, Beijing, China, f g gy y g y j g 64School of Physics and Technology, Wuhan University, Wuhan, China, 66Departamento de Fisica, Universidad Nacional de Colombia, Bogota, Colombia, associated with LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, Fran 67 associated with LPNHE, Université Pierre et Marie Curie, Université Paris Diderot, CNRS/IN2P3, Paris, Fra 67Institut für Physik Universität Rostock Rostock Germany with Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 68 68National Research Centre Kurchatov Institute, Moscow, Russia, associated with Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 111803-8 111803-7 111803-7 week ending 17 MARCH 2017 PRL 118, 111803 (2017) P H Y S I C A L R E V I E W L E T T E R S 17Sezione INFN di Ferrara, Ferrara, Italy 18Sezione INFN di Firenze, Firenze, Italy 19Laboratori Nazionali dell’INFN di Frascati, Frascati, Italy 20Sezione INFN di Genova, Genova, Italy 21Sezione INFN di Milano Bicocca, Milano, Italy 22Sezione INFN di Milano, Milano, Italy 23Sezione INFN di Padova, Padova, Italy 24Sezione INFN di Pisa, Pisa, Italy 25Sezione INFN di Roma Tor Vergata, Roma, Italy 26Sezione INFN di Roma La Sapienza, Roma, Italy 27Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland 28AGH - University of Science and Technology, Faculty of Physics and Applied Computer Science, Kraków, Poland 29National Center for Nuclear Research (NCBJ), Warsaw, Poland 30Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest-Magurele, Romania 31Petersburg Nuclear Physics Institute (PNPI), Gatchina, Russia 32Institute of Theoretical and Experimental Physics (ITEP), Moscow, Russia 33Institute of Nuclear Physics, Moscow State University (SINP MSU), Moscow, Russia 34Institute for Nuclear Research of the Russian Academy of Sciences (INR RAN), Moscow, Russia 35Yandex School of Data Analysis, Moscow, Russia 36Budker Institute of Nuclear Physics (SB RAS), Novosibirsk, Russia 37Institute for High Energy Physics (IHEP), Protvino, Russia 38ICCUB, Universitat de Barcelona, Barcelona, Spain 39Universidad de Santiago de Compostela, Santiago de Compostela, Spain 40European Organization for Nuclear Research (CERN), Geneva, Switzerland 41Institute of Physics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 42Physik-Institut, Universität Zürich, Zürich, Switzerland 43Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands 44Nikhef National Institute for Subatomic Physics and VU University Amsterdam, Amsterdam, Netherlands 45NSC Kharkiv Institute of Physics and Technology (NSC KIPT), Kharkiv, Ukraine 46Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine 47University of Birmingham, Birmingham, United Kingdom 48H.H. 111803-8 week ending 17 MARCH 2017 PRL 118, 111803 (2017) PRL 118, 111803 (2017) 69Instituto de Fisica Corpuscular, Centro Mixto Universidad de Valencia - CSIC, Valencia, Spain, associated with ICCUB, Universitat de Barcelona, Barcelona, Spain 70Van Swinderen Institute, University of Groningen, Groningen, Netherlands, associated with Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands †Deceased. aUniversidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. b bLaboratoire Leprince-Ringuet, Palaiseau, France. p g cP.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia d cP.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS) d dUniversità di Bari, Bari, Italy. eUniversità di Bologna, Bologna, Italy. f fUniversità di Cagliari, Cagliari, Italy. fUniversità di Cagliari, Cagliari, Italy. gUniversità di Ferrara, Ferrara, Italy. h gUniversità di Ferrara, Ferrara, Italy. h hUniversità di Genova, Genova, Italy. I IUniversità di Milano Bicocca, Milano, Italy. j IUniversità di Milano Bicocca, Milano, Italy. j jUniversità di Roma Tor Vergata, Roma, Italy. k jUniversità di Roma Tor Vergata, Roma, Italy. k kUniversità di Roma La Sapienza, Roma, Italy. l Università di Roma La Sapienza, Roma, Italy. lAGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. mLIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. p , , y lAGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. mLIFAELS L S ll U i it t R Ll ll B l S i lAGH - University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków, Poland. mLIFAELS L S ll U i i R Ll ll B l S i lAGH - University of Science and Technology, Faculty of Computer Science, Electronics a mLIFAELS, La Salle, Universitat Ramon Llull, Barcelona, Spain. LIFAELS, La Salle, Universitat Ramon Llull, Barc nHanoi University of Science, Hanoi, Viet Nam. nHanoi University of Science, Hanoi, Viet Nam. oUniversità di Padova, Padova, Italy. pUniversità di Pisa, Pisa, Italy. pUniversità di Pisa, Pisa, Italy. qUniversità degli Studi di Milano, Milano, Italy. r qUniversità degli Studi di Milano, Milano, Italy. rUniversità di Urbino, Urbino, Italy. rUniversità di Urbino, Urbino, Italy. sUniversità della Basilicata, Potenza, Italy. sUniversità della Basilicata, Potenza, Italy. tScuola Normale Superiore, Pisa, Italy. tScuola Normale Superiore, Pisa, Italy. uUniversità di Modena e Reggio Emilia, Modena, Italy. uUniversità di Modena e Reggio Emilia, Modena, Italy. vIligan Institute of Technology (IIT), Iligan, Philippines. vIligan Institute of Technology (IIT), Iligan, Philippines. wNovosibirsk State University, Novosibirsk, Russia. wNovosibirsk State University, Novosibirsk, Russia. 111803-9
https://openalex.org/W3093995193	https://pure.eur.nl/files/48423966/Holvast-and-Lindeman-2020-inquiry_into_the_blurring_boundaries_between_professionals_and_paraprofessionals.pdf	English	null	An inquiry into the blurring boundaries between professionals and paraprofessionals in Dutch courts and the public prosecution service	International journal of law in context	2,020	cc-by	15,347	Abstract The autonomous position of legal professionals is no longer self-evident. Professionals are under increased pressure to reform. This phenomenon is not only true for legal professionals. A broader trend – which is recognised for the medical profession, academic profession and alike – is that paraprofessionals are gain- ing a more prominent position. In this paper, we focus on the developments in the Dutch public justice system. We conduct a case-study on the role of paraprofessionals in courts and in the public prosecution service – two understudied legal institutions in this regard. By drawing on empirical data unfolding the working routine of the judiciary and the prosecution service, we find two paradigms that define the think- ing about professionalism: a traditional ‘pure professional’ paradigm and a new, more hybrid paradigm that includes (policy-based) managerial thinking. The latter appears to be enhanced by a New Public Management (NPM) approach within these institutions. Although we observe resistance among (para) professionals towards professional changes and ambiguity in the relationships between professionals and paraprofessionals, we also observe that managerialism has changed the work processes and the division of labour between professionals and paraprofessionals. Keywords: professionalism; managerialism; paraprofessionals; judiciary; prosecution office International Journal of Law in Context (2020), 16, 371–389 doi:10.1017/S1744552320000270 An inquiry into the blurring boundaries between professionals and paraprofessionals in Dutch courts and the public prosecution service N.L. Holvast1* and J.M.W. Lindeman2 1Erasmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, The Netherlands Corresponding author E mail: holvast@law eur nl 1Erasmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, The Netherlands Corresponding author E mail: holvast@law eur nl versity Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, nds 1Erasmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht School of Law, The Netherlands Corresponding author E-mail: holvast@law eur nl asmus University Rotterdam, Erasmus School of Law, The Netherlands and 2Utrecht University, Utrecht Scho e Netherlands Corresponding author. E-mail: holvast@law.eur.nl *Corresponding author. E-mail: holvast@law.eur.nl © The Author(s), 2020. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 1The Dutch body of judges that provides the administration of justice (de Rechtspraak) is in an international context com- monly referred to as ‘the judiciary’. The body of public prosecutors (openbaar ministerie) is often referred to as ‘the public prosecution service’. © The Author(s), 2020. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. prosecution service’. 1 Introduction Dutch judges and prosecutors – in the Netherlands, both appointed as ‘judicial civil servants’ – are traditionally portrayed as ‘pure professionals’, as described by Freidson (1994; 2001) and Abbott (1988)1: professionals who establish professional control of work and occupational protection (Noordegraaf, 2007). The adjudication of justice has long been regarded as the outcome of the labour of professional workers (judges and prosecutors). However, over the years, it is increasingly being per- ceived as a service provided by state institutions (the judiciary and the public prosecution service). Similar to many other professional domains (e.g. health care and education), ‘managerialism’ or ‘New Public Management’ (NPM) has had a considerable impact within the administration of justice (Noordegraaf, 2015; Langbroek and Westenberg, 2018; Visser et al., 2019). We notice that this has resulted in a shift from ‘traditional’ professionalism to ‘new’, more hybrid professionalism – which introduces NPM logics into the professionalism-paradigm (see Section 2.1). This shift and the ensuing organisational changes challenge the traditional division of power within these professional institutions (see similarly on the medical profession Bach et al., 2008; Kirkpatrick 1The Dutch body of judges that provides the administration of justice (de Rechtspraak) is in an international context com- monly referred to as ‘the judiciary’. The body of public prosecutors (openbaar ministerie) is often referred to as ‘the public prosecution service’. © The Author(s), 2020. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. NL Holvast and JMW Lindeman 372 et al., 2011). One way in which the power of professionals is challenged is by promoting stratification and by introducing different types, or levels, of professional occupations within the institutions (see Noordegraaf, 2007, pp. 762–763). These processes result in blurring boundaries between ‘full’ profes- sional and varying levels of ‘semi-’, ‘quasi-’ or ‘para-’ professional occupations (for magistrates’ assis- tants in the UK, see e.g. Raine and Willson, 1997; for legal practitioners, see Moorhead et al., 2003; see also Sommerlad et al., forthcoming). 1 Introduction Although it could be argued that both ‘full’ and ‘semi’ profes- sionals have (different) levels of professionalism, in this paper, we refer to the judges and prosecutors as ‘professionals’ and to all others involved – who may meet some, but not all, of the requirements to be a judge or a prosecution officer – as ‘paraprofessionals’.2 The latter group is more diverse in nature, consisting of judicial assistants, staff lawyers and a variety of prosecution assistants. Scholarly observations on the professional shift within the legal domain have predominantly focused on its effect on legal practitioners working at (large) law firms and not on legal professionals working within the administration of justice at state institutions. In this paper, we describe the changes within state institutions in more detail, with specific attention on the changes in the positions of para- professionals. We investigate how new professionalism has changed the formal position of paraprofes- sionals, focusing on the Dutch Judiciary and public prosecution service (PPS) as a case-study. We describe how professionals deal with the changes in what ‘professionalism’ entails and the conse- quences this has for the interaction between professionals and paraprofessionals. 2The latter group’s status is strongly connected to or derived from the ‘full’ professional. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 2.1 Professionalism and NPM Freidson (1970) and Abbott (1988) were among the first authors to introduce the concept of profes- sionalism, which can be contrasted with the concepts of a free market or a bureaucratic logic of orga- nising work. Professional occupations require specific skills and knowledge, which are achieved by both formal education and training as well as informal socialisation into the profession. Lawyers have traditionally been regarded as typical professionals. In their work, they serve public interests, but partly within a commercial setting (the latter being particularly true for legal practitioners; see also the influential work by Abel, 2003). y Recently, however, various authors have noticed a shift in the conditions under which professionals conduct their work. New organisational and managerial principles were introduced into the profes- sions. Evetts (2011) labels this as a transformation from ‘occupational’ professionalism to ‘organisa- tional’ or ‘new’ professionalism. This transformation results in ‘a shift from notions of partnership, collegiality, discretion and trust to increasing levels of managerialism, bureaucracy, standardization, assessment and performance review’ (Evetts, 2011, p. 406). Noordegraaf (2007) similarly refers to this new type of professionalism as ‘controlled professionalism’, considering that the organisations start to gain control over the professionals to some extent. In line with this reasoning, Noordegraaf (2007; 2015) also introduced the concept of ‘hybrid professionalism’. In this concept, organisational and managerial requirements (such as efficiency, budget control and productivity) go together with professional standards (such as quality standards, expertise, autonomy and independence), enhancing rather than weakening each other. g Particularly focusing on the legal profession, Sommerlad (1995) too recognises that the traditional legal profession is under attack by a new paradigm of managerialism. In his inaugural lecture, Moorhead (2014) argues that the legal profession today is precarious and challenged by new legal service providers. Similarly, Kritzer (1999) recognises a movement wherein legal professionalism has changed and ‘formal’ professionals have lost their exclusivity. As a result, ‘services previously provided only by members of formal professions can now be delivered by specialized general professionals or nonprofes- sionals’ (Kritzer, 1999, p. 713). Muzio and Ackroyd (2005) observe that external pressures have mainly resulted in increased internal stratification within law firms. In this context, the term International Journal of Law in Context 373 ‘deprofessionalisation’ has been used. However, we believe (with Paterson, 1996) that this movement can better be recognised as creating an altered form of professionalism rather than resulting in deprofessio- nalisation. 2.1 Professionalism and NPM Professional values still occupy a central place in the execution of the profession but are under pressure by other values and are therefore transformed and relocated within the professional institutions. p y p While the aforementioned literature mainly discusses the position of legal practitioners, there is a fair amount of literature that observes that a worldwide movement to managerialise public-sector insti- tutions has also affected the administration of justice at state institutions (see Freiberg, 2005; Heydebrand and Seron, 1990; Raine and Willson, 1997; Mak, 2008; Hondeghem et al., 2016; Lienhard et al., 2012; Visser et al., 2019). Managerial influences on the administration of justice are, for instance, observed in the US (Heydebrand and Seron, 1990), Australia (Freiberg, 2005; Spigelman, 2001) as well as in various European countries (e.g. for the UK, see Raine and Willson, 1997; McLaughlin et al., 2001; for France, see Vigour, 2015; Mak, 2008; for Switzerland, see Lienhard et al., 2012; for the Netherlands, see Visser et al., 2019; Langbroek and Westenberg, 2018; Mak, 2008). This movement, under the name New Public Management (NPM), stems from organisa- tional theories and focuses on public accountability and the use of best practices from the private sec- tor to improve the quality of services provided by public-service organisations (see e.g. Hood, 1991). Private-sector principles, such as efficiency, effectiveness, transparency and accountability, have become guiding concepts and are widely adhered to in courts and public prosecution offices. This is accompanied by the introduction of private-sector techniques, such as incorporating systems to ‘measure’ court performance and processes and incentives to increase the efficiency of courts (Contini and Carnevali, 2010). While the NPM approach could be regarded as a welcome initiative to help reduce the backlog of cases and improve the quality of justice (Contini and Carnevali, 2010; Jean and Pauliat, 2006), it gen- erates resistance from the professionals working within the system (Vigour, 2006; 2015; Holvast and Doornbos, 2015). This is partly related to the fact that the introduction of NPM principles is often accompanied by – or at least associated with – cuts in the budgets of the organisations. However, pro- fessionals hold more general objections to the movement. Judges particularly fear that NPM principles will impede traditional professional and legal values, such as judicial independence, impartiality and – most importantly – autonomy (Langbroek, 2001; see in relation to legal-aid practice Sommerlad, 1995). 2.1 Professionalism and NPM In fact, it is a much wider grievance that managerialism hinders the autonomous position of profes- sionals. Similar concerns are raised within the health sector and (higher) education (Bezes et al., 2012). g Professional workers are likely to oppose these managerialism influences, for example by creating sets of self-binding conditions for entering the profession and for performing the professional duties. These self-binding conditions (are proclaimed to) accommodate the provision of high-quality services. At the same time, and some authors argue that this is their core purpose, the conditions also function as a means to maintain a professional monopoly of the services offered by the profession (Larson, 1977). Professions will therefore be resilient to changes that challenge the existing system of profes- sional self-organisation and self-control (see e.g. Freidson, 2001). They find it problematic to accept that paraprofessionals can and should perform parts of the professional duties, as it means giving up part of the professional monopoly of providing legal services. However, it has become increasingly clear that this is a lost battle, as the market mechanism forces legal professionals to give up part of this monopoly to become more affordable (Susskind, 2017). This introduces new forms of professionalism and results in paraprofessional positions becoming more professionalised, a notable example being paralegals in law firms (Noordegraaf, 2011; Lively, 2001). ttps://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ownloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 3 The Dutch context: changing position of paraprofessionals in the judiciary and the PPS In the 1990s and 2000s, the Dutch administration of justice has adopted various NPM features (see more in Visser et al., 2019). The introduction of these features affected the formal position of para- professionals in the Dutch judiciary and the PPS in different ways. In both institutions, we observed that paraprofessionals have received a more central position in the work processes. However, the changes in the position of the paraprofessionals in the PPS are more far-reaching and more explicit than in the judiciary. We start this section by providing a brief description of the organisation of both institutions. Subsequently, we successively summarise the changes that occurred regarding the position of paraprofessionals in the judiciary and the PPS. 2.2 Methodology This paper combines a macro-level analysis of regulation and policy on the role of paraprofessionals with micro-level insights from empirical data collected through participant observations and inter- views within the Dutch judiciary and the PPS. For the regulation and policy analysis, we studied the literature on organisational developments within both institutions and conducted a review of NL Holvast and JMW Lindeman 374 policy documents published by the Council for the Judiciary (Raad voor de Rechtspraak), the Board of Prosecutors General (College van Procureurs-Generaal) and the Dutch Association for the Judiciary (Nederlandse Vereniging voor Rechtspraak, the professional body and trade union for judges and pub- lic prosecutors). The previous documents report only marginally about the role and duties of assistants and they provide little insight into the daily practice in the workplace. In order to gain more in-depth informa- tion, empirical data were needed. The empirical data derive from two research projects3: one on the work of public prosecutors in the Dutch PPS and another project on the involvement of judicial assis- tants in judicial decision-making at Dutch district courts. In the first project, Lindeman studied how public prosecutors define and fulfil their duties. He conducted fieldwork for the duration of twelve months within two district offices and one nationwide office of the PPS. He followed the prosecutors in their daily routine and observed their interactions with the PPS staff, police officers, judges, etc. He was able to observe how prosecutors interact with prosecution assistants and to what extent they follow the assistants’ judgments. During his fieldwork, he observed and spoke with numerous prosecution assistants. He also conducted fifteen interviews with prosecutors.4 In the second project, Holvast examined the involvement of judicial assistants in the judicial decision-making in two Dutch district courts (in the criminal-law and administrative-law divisions). She conducted fieldwork for the dur- ation of eight months. Holvast participated in the daily activities of the courts and followed the decision-making process in 137 cases, heard during twenty-seven hearings. She analysed the exchange of views orally and via memos prior to the hearing, observed the hearings and was able to attend and observe the deliberation sessions. She also examined the process of drafting and finalising the judg- ment by judges and assistants. Holvast interviewed over eighty court officials, mainly judges and judi- cial assistants.5 3These two PhD projects by Holvast and Lindeman were published in 2017. 4See more in Lindeman (2017, chapter 2). 5See more in Holvast (2017, chapter 2). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 3These two PhD projects by Holvast and Lindeman were published in 2017. 4See more in Lindeman (2017, chapter 2). 5See more in Holvast (2017, chapter 2). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cam 3.1 Managerial changes in the Dutch judicial and public prosecution organisations This magisterial position is most absolute for judges (as adjudicators) and somewhat less for prosecutors, who have to comply with criminal- policy regulations (Van de Bunt, 1985; Lindeman, 2017). The special position of judges and prosecutors has always been important for the organisational struc- ture of their respective institutions. Traditionally, the judiciary and the PPS were managed by the judges and prosecutors. At all courts, the periodical ‘court meeting’ (gerechtsvergadering), consisting of all judges, used to be an important decision-making body. These local bodies still have some decision-making power, but a more managerial structure was introduced in 2002, with the newly established national Council for the Judiciary at the top. At the court level, an additional management layer consisting of judge team lea- ders was introduced and the courts’ boards were required to include one non-judge board member. Another important change was the introduction of output financing. These changes resulted in a some- what more managerial organisation, in which collective work processes are – to some extent – restricting the autonomy of judges and moving more towards a ‘controlled professionalism’ as described by Noordegraaf (2015). Nonetheless, the autonomous position of judges and their authority in organising their work are still key elements within the judicial organisation. Hence, the NPM values have not replaced the traditional rule-of-law values, but these values co-exist (Mak, 2008; Langbroek and Westenberg, 2018). The organisational structure of the PPS has changed more radically. Significant NPM influences can be recognised in these developments. As within the judiciary, these influences also restrict the autonomy of prosecutors. In order to understand the impact of the changes, a short introduction on the organisation of the PPS is necessary (Zouridis, 2016). The PPS has authority over the police in criminal matters and decides whether to prosecute or dis- miss a case (along the lines of the so-called expediency principle). In the 1970s and 1980s, the Dutch government increasingly regarded criminal policy as a means to proactively fight crime and the PPS was supposed to be the ‘spider in the web’ in this battle. However, in the 1990s, it became clear that the PPS was not up to this task. After long deliberations, legislation implementing rigorous changes in the management structure of the PPS came into force in 1999. 3.1 Managerial changes in the Dutch judicial and public prosecution organisations While we portray judges and prosecution officers as professionals, and thereby compare them to other professionals such as doctors, professors and attorneys, it is important to note that judges and prose- cutors have a special position in society. In the Netherlands, both officials are appointed by law as a so-called ‘judicial civil servant’ (rechterlijk ambtenaar, Art. 1 of the Judicial Organization Act), pro- viding them with a special legal status codified in the Act on the Legal Status of Judicial Civil Servants. This differentiates them from other civil servants and gives them, in some ways, the status of personification of the administration of justice (one of the three branches of state power) (Langbroek and Westenberg, 2018). Especially for judges, this position also comes with various safe- guards regarding their independence, impartiality and integrity: judges are appointed for life (Art. 116 of the Dutch Constitution) and special rules for dismissal apply to ‘judicial civil servants’ International Journal of Law in Context 375 (Bovend’Eert, 2000). These safeguards apply to public prosecutors to a somewhat lesser extent: they are not appointed for life and the Minister of Justice can give directions to the Public Prosecutor’s Office (Van de Bunt and Van Gelder, 2012). Judicial assistants are defined in the Judicial Organization Act as ‘civil servants who work at courts’. Prosecution assistants are regular civil servants and are implicitly referred to as ‘other civil servants working at the prosecutor’s office’.6 The regular terms of appointment and dismissal for Dutch civil servants apply to both types of assistants. The formal status of ‘judicial civil servants’ also resonates in the way judges and prosecutors view their position and how they act. In the Netherlands, this is often referred to as their ‘magisterialness’ (in Dutch magistratelijkheid, which refers to a mindset of making independent decisions as a guardian of the rule of law, thereby considering the interests of all parties involved). For prosecutors, magister- ialness means that they are not merely a party in criminal proceedings seeking to ‘win’ a case: they are expected to oversee a process of establishing the truth that is compatible with the rule of law (Lindeman, 2017, pp. 24–26, 295). Developing and maintaining the attitude of a magistrate is part of the professional training of judges and prosecutors. 6Judicial Organization Act, Art. 126. 7The Judicial Revision Act merged nineteen district offices into ten. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at ww.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge J g , 7The Judicial Revision Act merged nineteen district offices into ten. 3.2 Paraprofessionals in the judiciary The paraprofessional, in the capacity of court assistant (griffier), has a long history in the Dutch judiciary, existing since the early 1800s (De Groot-van Leeuwen, 1991). Other than preparing the court record, which has been a key responsibility since their existence, duties of these assistants have changed drastically over time. From the 1960s to the 1980s, a substantial portion of the assistants consisted of internally trained, originally administrative, staff members. From the 1990s onwards, and particularly after the enact- ment of new legislation, their position was gradually professionalised. The largest change occurred after the introduction of the new management structure with the enactment of the Dutch Judiciary Organization and Management Act (Wet Organisatie en bestuur gerechten) and the Act on the Council for the Judiciary (Wet Raad voor de Rechtspraak), in 2005. The numbers of judicial assistants working at the courts vary somewhat per court type and division but, at district courts and Courts of Appeal, the judicial assistants slightly outnumber the judges working at the courts (see Holvast, 2017, p. 49).8 g y j g g p To implement the output-based financing structure that was introduced in 2005, the judiciary cre- ated a model that specifies how much time (measured in minutes) a judge and a judicial assistant are estimated to work on a specific type of case: the so-called Lamicie-model (see Van der Torre et al., 2007). The model determines what compensation courts subsequently receive for handling the cases, taking into account the supposed time spent on handling those cases by the judicial officers (Van der Torre et al., 2007). The figures are based on time writing surveys and are supposed to reflect the real division of work within the courts. The model revealed the practice that, in most types of cases, judicial assistants spend considerably more time working on the cases than judges. Shortly after the creation of this model, the internal training programme for judicial assistants deteriorated and the minimum requirement for holding a diploma from an institute of higher professional education was introduced (Abram et al., 2011). The latter was primarily a codification of an already existing prac- tice. In fact, many newly hired assistants had (and still have) finished even higher levels of education (an LLM from a Dutch law school). 3.1 Managerial changes in the Dutch judicial and public prosecution organisations A ‘top-down’ hierarchy was introduced, formalising the (until then informal) leadership by the Board of Prosecutors General, but still allowing the Minister of Justice to issue instructions to that board. Also, the practice to shift a part of the pro- secutors’ work to subordinates had a statutory basis that allowed a much broader application. Policy regulations issued by the Board of Prosecutors General became increasingly important instruments. These regulations constrain the discretionary role of individual prosecutors, as they hold instructions regarding the decision to prosecute. g g p Since the formal reorganisation in 1999, the PPS has almost continuously been engaged in revising and adjusting its organisation, for example by increasing the scale of the offices.7 Since the 2008 crisis, NL Holvast and JMW Lindeman 376 budget cuts have been a rule rather than an exception, urging the PPS to work as efficiently as possible, without losing sight of the primary objectives. As of 1 January 2019, the PPS also introduced output financing for a significant part of the organisation. These developments within the PPS display a strong pattern of centralisation of authority and a mandatory accountability within a strict hierarchy, to a certain extent even involving accountability to the Minister of Justice. g y These organisational changes in the institutions also had consequences for the position of parapro- fessionals. We will discuss this position in the next sections. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 8Unfortunately, no historic comparison can be provided; these numbers only recently became available. 3.2 Paraprofessionals in the judiciary The function of judicial assistants in district courts and civil and criminal Courts of Appeal have, since 2007, been divided into five categories: junior judicial staff members, judicial staff members, senior judicial staff members, staff lawyers and senior staff lawyers. The function profiles provide insight into the duties of judicial assistants. These profiles state that (both junior and senior) judicial assistants assist judges in preparing for hearings, during the hearings and in the processing and conceiving of judgments. Staff lawyers are usually specialists of a subfield of law. They are required to possess an LLM from a Dutch law school. Staff lawyers may provide advice on complex legal issues and cases with high social or economic impact. Additionally, they can con- tribute to the creation of new case-law and serve as sounding boards for judges and judicial assistants. The descriptions of duties in the profiles, however, remain abstract and can be interpreted in multiple ways. The function profiles do not indicate the manner in which judicial assistants are supposed to assist prior to the hearings or how to give advice. The profiles also do not mention the assistants’ role in the deliberation room; the research by Holvast (2017) revealed the informal norm that judicial assistants do participate in judicial deliberations. It can be extracted from the aforementioned documents that judicial assistants have attained a more central position within the process of adjudication. Several professional duties that were originally International Journal of Law in Context 377 reserved for judges are now partly conducted by judicial assistants. The transformation of the judicial-assistant position that occurred over time appears to follow from NPM-driven organisational changes, such as the output-based financing structure and efforts to improve the efficiency of work pro- cesses. The required qualifications for the function have become stricter and an assistant’s current role exceeds the more administrative tasks that the role primarily consisted of before. Most judges and assis- tants who worked at the courts during these transitions confirm that the judicial-assistant position has tacitly become more demanding. Examples thereof are the acceleration of the duties to draft judgments and the assistants’ active participation in deliberation sessions. The fact that the position of judicial assistant nowadays requires more legal knowledge and skills is most recently used as leverage in the negotiations of the collective labour agreements of judicial assistants. 3.3.1 Formalised mandate for prosecutor assistants The idea to allow prosecutors to delegate additional duties to assistants developed as early as the 1970s. That is not to say that delegating these duties was immediately a widespread practice. At first, prose- cutors were reluctant to allow the paraprofessionals to perform some of their duties (Van de Bunt, 1985). After all, these assistants did not possess a law degree and, as such, acquired their knowledge ‘on the job’. At the same time, the case-load gave the prosecutors no choice but to allow assistants to at least prepare their cases for them. This practice was facilitated by the prosecution guidelines, support- ing the assistants in their decision-making process. This deployment of assistants increased over the years, without a clear legal basis: the public prosecutor would still formally make the decisions. This changed in 1999 when the legal amendments mentioned above provided a formal mandate to assis- tants within the PPS to perform certain duties normally reserved for public prosecutors.9 Thus, while, in the judiciary, assistants work under the direct authority of a judge, prosecutors’ assistants have been mandated to make individual decisions for the past two decades. As such, in the 1990s and 2000s, NPM principles were more and more incorporated into the PPS organisation. On the one hand, prosecution assistants increasingly were university graduates. On the other hand, the PPS also started employing new types of assistants with lower education levels. Between 2000 and 2015, a broad range of assistants with varying levels of education were employed by the PPS: administrative judicial assistants (senior secondary vocational education)10; junior pros- ecution assistants (higher professional education)11; prosecution assistants (higher professional educa- tion or university education)12; and senior prosecution assistants and (senior) policy officers (mostly university education).13 Nonetheless, even the assistants with the lowest rank (administrative judicial assistants) were mandated to process and decide on (the most simple) cases. 3.2 Paraprofessionals in the judiciary However, as of today, many judicial assistants have temporary contracts and most assistants’ salaries are based on a scale for graduates from Universities of Applied Sciences, even though the vast majority have graduated from regular universities. 9Judicial Organisation Act, Art. 126. 10Administratief Juridisch Medewerkers who received middelbare beroepsopleiding (MBO). 11Junior Parketsecretaris (hoger beroepsonderwijs, HBO). 12Parketsecretarissen, HBO or universitair onderwijs. 13Senior Parketsecretarissen and (senior) beleidsmedewerkers, Universitair geschoold. l d d f htt // b id / IP dd 80 101 62 141 03 M 2021 t 11 00 31 bj t t th C b id C t f il bl t https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 9Judicial Organisation Act, Art. 126. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cam 3.3.2 The role of paraprofessionals in FOO and HIC The work in Public Prosecutor’s Offices can roughly be divided in two work streams: frequently occur- ring offences (FOO) and high impact crimes/organised crimes (HIC). There are significant differences between the positions of the assistants in both divisions. During the period 2000–2015, prosecution assistants were used extensively to process FOO. This was facilitated by an ever-increasing, intricate structure of policy regulations (guidelines and NL Holvast and JMW Lindeman 378 instructions) and ICT systems, which allowed assistants to work independently while still being mon- itored (Van de Bunt and Van Gelder, 2012; Lindeman, 2017).14 Almost 50 per cent of the cases regis- tered by the PPS were dealt with out of court.15 A vast majority of these cases were processed by assistants. This allowed a so-called bifurcation: prosecutors dealt with more serious and/or compli- cated cases, while assistants processed the run-of-the-mill cases. Assistants decided whether cases went to trial or were handled out of court. They decided which offence a suspect would be prosecuted for and (in the case of out-of-court proceedings) on the type and measure of the sentence. Lindeman (2017) observed that there was not much scrutiny of the assis- tants’ work. The FOO departments of the PPS district office virtually operated on a ‘stand-alone’ basis. Prosecutors would only be involved if a case was taken to court because a suspect did not agree to the proposed out-of-court sanction or if an assistant decided to summon the suspect. A highly critical report on the efficiency of the criminal justice chain (Algemene Rekenkamer, 2012) led to the implementation of a new procedure regarding FOO: the so-called ZSM procedure (Jacobs and Van Kampen, 2014).16 In a significant departure from the practice described above, the aim is now that all cases are preliminary decided upon by a prosecutor in an early stage. The prosecu- tor gives an indication as to the processing (a trial or an out-of-court proceeding). Subsequently, a prosecution assistant will execute this decision or, if necessary, prepare the case for trial. The use of prosecution assistants without legal training has diminished. A new function, the ‘assistant-prosecutor’, has been introduced. (Senior) prosecution assistants with a law degree can enter a traineeship to become an assistant-prosecutor. An assistant-prosecutor is always on duty together with a regular prosecutor at the ZSM desk. 3.3.2 The role of paraprofessionals in FOO and HIC Those cases that will be routed towards a trial will, ideally, be processed by this assistant-prosecutor, who will also represent the PPS at the trial. The assistant-prosecutor will also represent the PPS at trial in other simple cases, mostly heard by a single judge. In doing so, the assistant-prosecutor takes over a significant part of the workload tradition- ally assigned to regular prosecutors. Initial research by the Dutch Association for the Judiciary and follow-up research by the Board of Prosecutors General reveal that there are differences between the districts as to the scope of the competence and autonomy of assistant-prosecutors. It is not always clear whether the various manifestations of the assistant-prosecutor are in line with the national policies. While prosecution assistants have increasingly been assigned more demanding duties, their salary grade has remained almost unchanged and they rarely have permanent contracts. Even becoming an assistant-prosecutor is by no means a guarantee to enter the traineeship to become a fully qualified public prosecutor. In the departments where more serious and/or complicated criminal cases are dealt with and where prosecutors oversee the criminal investigations carried out by the police, also known as the HIC departments, (senior) prosecution assistants play a significant role. The role of prosecution assistants within these more complicated criminal cases is more commensurate with the role of judicial assis- tants. The legislation on their mandate does not allow the prosecution assistants to make independent decisions on the use of investigation methods (e.g. wire taps or surveillance orders) and/or coercive measures. However, the assistants do often offer significant assistance to the prosecutors. 14This ICT system called the Decision Support System, Beslissingsondersteuningssysteem – BOS, was used from the start of the reorganisation in 1999 until 2015. 15This figure has been quite consistent over the past decades. 16ZSM stands for ‘zo snel mogelijk’ which translates as ‘as soon as possible’. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 14This ICT system called the Decision Support System, Beslissingsondersteuningssysteem – BOS, was used from the start of the reorganisation in 1999 until 2015. 15This figure has been quite consistent over the past decades. 16ZSM stands for ‘zo snel mogelijk’ which translates as ‘as soon as possible’. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 14This ICT system called the Decision Support System, Beslissingsondersteuningssysteem – BOS, was used fro he reorganisation in 1999 until 2015. 15 4.1 Introduction While further substantiating the professional and paraprofessional collaboration in the workplace, the (para)professionals are guided by shared professional norms and ideals regarding the appropriate pos- ition of paraprofessionals within the process of decision-making. Our fieldwork in the judiciary and PPS reveals that, among professionals and paraprofessionals, there is not one dominant paradigm that clarifies their behaviour. Two different – and on various aspects competing – paradigms co-exist and define the role of paraprofessionals. These professional paradigms are related to the developments in professionalism described in Section 2.1. On the one hand, and that is the first paradigm, (para)professionals conform to a trad- itional (occupational or ‘pure’) definition of professionalism, in which the ‘full’ professional is regarded as holding the single authority over the professional decision-making. In this paradigm, it is regarded as inappropriate for the ‘full’ professionals to rely too strongly on the work of paraprofes- sionals. The professionals claim full jurisdiction over the process of adjudication/prosecution and the position of paraprofessionals is – in some occasions – marginalised. On the other hand, a second para- digm exists, which encompasses that professionals and paraprofessionals accept that their relationships have changed and that managerial indicators regarding productivity and policy influence their work, and they acknowledge that paraprofessionals can – to some extent – be regarded as professionals themselves. Within this paradigm, all officers at the court/PPS are viewed as partners in reaching the goal of delivering justice in an efficient and timely manner. Some (para)professionals have ideas that predominantly fit into one specific paradigm. More often, professionals’ and paraprofessionals’ behaviour appears to fit in both paradigms, opting for different sets of norms in different situations. In the following sections, we provide a detailed account of how we recognised these paradigms in the statements made and actions taken by professionals and paraprofessionals. 3.4 Concluding remarks In the past decades, the (formal) division of duties between professionals and paraprofessionals has been altered. More specifically: further allocation of duties to paraprofessionals has occurred. Within both institutions, these changes can, at least in part, be attributed to the NPM movement. At the PPS, some of the decision-making practices have increasingly been mandated to assistants, International Journal of Law in Context 379 which transferred them into some sort of quasi-prosecutors, culminating in the establishment of assistant-prosecutors. In the judiciary, all decision-making duties are still formally in the hands of the judges, with no official mandates for decision-making by judicial assistants. In both institutions, the official recognition of more demanding paraprofessional functions is still limited: neither the financial compensation nor other forms of appreciation have shown meaningful improvement. Additionally, the official policy provides general guidelines, but leaves a significant mar- gin of discretion as to how the professionals and paraprofessionals should give substance to their collaboration, leaving room for differences in execution. This makes it interesting to investigate which – largely informally defined – standards/norms regarding the division of labour are existent at the courts and PPS, and how these are linked to different concepts of professionalism. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor According to some respondents, this can be explained by the often more timid nature of assistants, related to the fact that they chose to fulfil a subordinate role with no decision-making responsibilities. Observations within the PPS were somewhat different in this regard: especially within the HIC teams, some assis- tants played dominant roles in team meetings. Also, socialising between prosecutors and assistants was customary, which, in the 1980s, would have been unthinkable (Van de Bunt, 1985). Despite prosecutor and assistant positions growing towards each other, periodical ‘prosecutors-only’ meetings were still being held (Lindeman, 2017). While the social boundaries between the groups of professionals and paraprofessionals have faded in the past decades and the interaction has become more informal (on the PPS in the 1980s, see Van de Bunt, 1985; on the judiciary, see Bevers, 2004, pp. 8–9), these boundaries have not disappeared com- pletely. A judge (A1 – resp. 42) explains that, when she started working in the judiciary around the turn of the century, it was explained to her that judges were not supposed to socialise too much with assistants, as that was unworthy of their rank. Within some court divisions, it was still common in 2012–2013 for judges and assistants to have lunch in separate groups. This demonstrates that – to some extent – a social distance still exists. Judicial assistants would, on average, also speak considerably less than judges during general staff meetings that were held in different courts. According to some respondents, this can be explained by the often more timid nature of assistants, related to the fact that they chose to fulfil a subordinate role with no decision-making responsibilities. Observations within the PPS were somewhat different in this regard: especially within the HIC teams, some assis- tants played dominant roles in team meetings. Also, socialising between prosecutors and assistants was customary, which, in the 1980s, would have been unthinkable (Van de Bunt, 1985). Despite prosecutor and assistant positions growing towards each other, periodical ‘prosecutors-only’ meetings were still being held (Lindeman, 2017). The previous paragraphs clearly indicate that both judges and prosecutors believe that there is a certain domain of authority exclusive to their profession. In the judiciary as well as in the PPS, we observed significant differences between individual judges/prosecutors as to the exact dimensions of that domain. 17But see Section 4.3.1. 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor On various occasions, we observed a scepticism towards a movement that would assign more far- reaching adjudicative or prosecutive duties to paraprofessionals. Several times an inherent hierarchy in the relationship between professionals and paraprofessionals was emphasised by respondents. They related this to the fact that the judge/prosecutor is the one appointed by law to adjudicate/pros- ecute. This claim of authority is more obvious with regard to judges (see Section 3.1) and this was indeed a stronger sentiment within the judiciary. However, prosecutors also expressed this claim of authority. The ‘full’ professionals thereby displayed behaviour of marking the boundaries of their pro- fession and being resilient to changes in work processes that might challenge these boundaries – which can be regarded as typical behaviour for ‘pure professionals’. It is interesting that not just professionals, but also several paraprofessionals defend these strict boundaries. 380 NL Holvast and JMW Lindeman Some authors suggest that the sense of responsibility for judgments (and in this context also deci- sions to use coercive measures, prosecute and/or demand a sentence) might be lost when paraprofes- sionals are involved (Fiss, 1983; Hol, 2001). Despite our finding that, indeed, paraprofessionals are involved to a greater extent in the decision-making process, a decline in a sense of responsibility was not generally observed in our research.17 One judge, for example, stated: ‘I make the decision. It is my responsibility, and I have to carry it. And if we can’t decide, I am the one who lies awake at night, not the judicial assistant. He will hear the next day what we’re going to do.’ (A1 – resp. 18) Judges’ responsibilities appear to rest heavily on their shoulders. Relying too strongly on the pro- ducts of judicial assistants is, from this perspective, excluded. Judges draw a clear line at the use of memos produced by judicial assistants. All judges proclaimed that it is inappropriate to prepare for a hearing by exclusively reading the memo prepared by an assistant and not opening the case files. One judge’s (A1 – resp. 38) opinion was more extreme. He said he did not read the memo at all, as he wanted to enter the hearing unbiased. However, this was not the leading opin- ion within the courts. Judges’ responsibilities appear to rest heavily on their shoulders. Relying too strongly on the pro- ducts of judicial assistants is, from this perspective, excluded. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor Judges draw a clear line at the use of memos produced by judicial assistants. All judges proclaimed that it is inappropriate to prepare for a hearing by exclusively reading the memo prepared by an assistant and not opening the case files. One judge’s (A1 – resp. 38) opinion was more extreme. He said he did not read the memo at all, as he wanted to enter the hearing unbiased. However, this was not the leading opin- ion within the courts. In the HIC division of the PPS, a similar claim of authority was displayed. Prosecutors claimed that assistants hardly ever made decisions without prior consent by the prosecutor, especially when it con- cerned decisions on the application of the expediency principle: ‘It’s an equal debate, but at the end of the day, the decision has your name on it, and you will be the one having to discuss the decision with parties concerned. No, I don’t think that decisions leave this office without the involvement of a prosecutor.’ (A2 – resp. 10) ‘It’s an equal debate, but at the end of the day, the decision has your name on it, and you will be the one having to discuss the decision with parties concerned. No, I don’t think that decisions leave this office without the involvement of a prosecutor.’ (A2 – resp. 10) While the social boundaries between the groups of professionals and paraprofessionals have faded in the past decades and the interaction has become more informal (on the PPS in the 1980s, see Van de Bunt, 1985; on the judiciary, see Bevers, 2004, pp. 8–9), these boundaries have not disappeared com- pletely. A judge (A1 – resp. 42) explains that, when she started working in the judiciary around the turn of the century, it was explained to her that judges were not supposed to socialise too much with assistants, as that was unworthy of their rank. Within some court divisions, it was still common in 2012–2013 for judges and assistants to have lunch in separate groups. This demonstrates that – to some extent – a social distance still exists. Judicial assistants would, on average, also speak considerably less than judges during general staff meetings that were held in different courts. 4.3 The ‘new’ professionals paradigm: judges/prosecution officers and assistants as partners in reaching professional goals 4.3.1 (Para)professionals taking into account ‘new professional’ values in their professional collaborations While we observed claims made regarding the hierarchy in the relationship between professionals and paraprofessionals, the equality in and similarities of the positions were just as frequently mentioned. Many judges underlined that they acknowledged judicial assistants for being colleagues with whom they closely collaborate in the process of adjudication and interchange intellectual ideas regarding the merits of cases. Assistants are often also regarded as equal partners by the HIC prosecutors and are welcomed to discuss almost all decisions with the prosecutor. Lindeman (2017) heard a pros- ecutor make the off-the-cuff claim that assistants could perform all the prosecutor’s tasks, except for the trial (A2, p. 105). A judge similarly said: ‘A judge is actually a sort of assistant plus: he should be able to do everything that an assistant can and chair the hearing’ (A1 – resp. 77). Judges and prosecutors regularly work with the same assistants, which can attribute to a certain team spirit. The fact that the majority of recently appointed judicial/prosecution assistants obtained a law degree appears to be important for judges and prosecutors to acknowledge them as discussion partners: ‘I always think: we employ lawyers for a reason …. We should make use of that’ (A1 – resp. 42). Various respondents also highlight the sounding-board function of the assistants and they recognise that the discussion with the assistants affects their considerations regarding cases. One judge explains how she, somewhat to her own surprise, really missed this type of contribution when – due to unforeseen circumstances – she was not accompanied by a knowledgeable assistant: ‘I realized that I really missed a discussion partner then. … I really missed someone to write the judgments. That, too. … But I mainly noticed that what I really missed was the exchange of thoughts. I didn’t expect that to be such an issue.’ (A1 – resp. 42) The discussion-partner role of judicial assistants is also exposed by the informal norm (largely given substance since the 1990s) that judicial assistants are asked to display their views on the case during deliberation sessions.18 Some judges regard this as a duty, emphasising their equal position in the dis- cussion of cases. Others consider this more to be a privilege or a social gesture, which they imagine assistants will appreciate. 18Although, in the interviews, judges widely endorse this informal work practice, they do not always act accordingly during the deliberation sessions (perhaps due to ‘pure’ professional ideas). In just over half of the deliberations observed by Holvast, the assistants were consistently offered the opportunity to speak first. 4.2 The ‘pure professional’ paradigm: emphasising the superior status and authority of the judge/ prosecutor The appearance at the public hearing was generally regarded as one of the key components of the profession of judge or prosecutor, which clearly distinguished these professionals from the paraprofes- sionals. Several assistants mentioned that they actually prefer a position out of the spotlight and believed themselves to be more suitable for such a role. At the courts, a marginal change occurred ttps://doi.org/10.1017/S1744552320000270 .org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at International Journal of Law in Context 381 in which, in administrative-law cases, some judges would allow assistants to ask questions to the par- ties (which in reality rarely happened). At the PPS, a notable shift in this regard is the introduction of the assistant-prosecutor (see Section 3.3). Still, a large portion of the judges and prosecutors believed that performing at the hearing was typ- ical for their profession. This also demarcates their professional position to the general public. These professionals reject the possibility of having assistants involved. When the researcher, for instance, asked a judge about her view on having judicial assistants ask questions, she responded: ‘No, that can- not happen, because the assistant is not present to ask questions during the hearing. If that happens during a hearing where parties are present, then it is wrong. Period’ (A1 – resp. 50). In summary, we observed (para)professionals who (still) adhere to traditional professional values, as described in the original literature on professionalism when it concerns certain parts of their work (see Larson, 1977; Abbott, 1988; Freidson, 1994). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 4.3 The ‘new’ professionals paradigm: judges/prosecution officers and assistants as partners in reaching professional goals In the administrative-law sections of the courts, it is common practice in single-judge cases for the judge and judicial assistant to meet after the hearing to discuss the cases. It is remarkable that these meetings are referred to as ‘deliberation sessions’, while formally the 18Although, in the interviews, judges widely endorse this informal work practice, they do not always act accordingly during the deliberation sessions (perhaps due to ‘pure’ professional ideas). In just over half of the deliberations observed by Holvast, the assistants were consistently offered the opportunity to speak first. NL Holvast and JMW Lindeman 382 judge takes the decision and, thus, actual deliberation is not a necessity. Most of these sessions appeared to occur on equal grounds, with both participants being actively involved in exchanging dif- ferent arguments regarding a case. A similar practice can be found in the various forms of peer ses- sions that prosecutors convene on a regular basis. For example, ‘sentence assessments’19 regularly take place. A prosecutor presents his/her case to other prosecutors with the objective of assessing the demand (regarding the sentence) that fits the crime best. The assistant who worked on the case with the prosecutor is also invited and actively participates in the meeting. Furthermore, judicial and prosecution assistants have another important means of sharing their views on the case: the preparatory memos that they make for judges and prosecution officers to pre- pare for the hearings (see on the importance of these memos in Dutch police judge cases also Van Oorschot, 2014). Some of these memos are mainly summaries of the case files, yet other memos clearly, and deliberately, reveal the views of the assistants on the demands that the prosecutor should present or what the judges’ decision should be. In some administrative-law cases, judicial assistants would write memos in a draft-judgment format, thereby to an extent already anticipating the legal rea- soning. In complicated cases, the prosecution assistant who assisted in conducting and overseeing the investigation and who prepared the case will attend the hearing before the trial such as to be able to assist the prosecutor when last-minute questions arise. Hence, while judges and prosecutors are by law required to act and decide in specific situations, the preparations of an assistant are by many consid- ered indispensable for these actions and decisions. 19Strafmaatoverleggen. 20Dutch law requires these warrants for many investigation methods, such as surveillance orders, requisition of informa- tion (e.g. CCTV footage) and wire taps. 21However, because both our research projects provide an analysis of the institutions at one specific point in time, we do not claim to have proof for a causal relation and/or a gradual development in this regard. It is even likely that both sets of values have always to some extent played a part in defining work relations. 22Lindeman’s (2017) observations on the FOO divisions are representative of the organisation of the processing of FOO in the timeframe prior to the ZSM procedure (roughly from 2000 until 2015). https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available a 19Strafmaatoverleggen. 20Dutch law requires these warrants for many investigation methods, such as surveillance orders, requisition of informa- tion (e.g. CCTV footage) and wire taps. 21However, because both our research projects provide an analysis of the institutions at one specific point in time, we do not claim to have proof for a causal relation and/or a gradual development in this regard. It is even likely that both sets of values have always to some extent played a part in defining work relations. 22Li d ’ (2017) b ti th FOO di i i t ti f th i ti f th i f FOO i 4.3 The ‘new’ professionals paradigm: judges/prosecution officers and assistants as partners in reaching professional goals This indicates that these professionals acknowledge that their work is to some extent ‘controlled’ by decisions about work processes – and the role of para- professionals therein – made on an organisational level (see Noordegraaf, 2015). At the HIC departments, when the police need a warrant for a specific investigative method,20 assistants will draw up the warrant (and determine whether there are sufficient grounds that meet the legal requirements). Assistants draft orders for pre-trial detention. Assistants prepare meetings with the police in ongoing investigations, and they even represent the prosecutor in such meetings. Some prosecutors (but certainly not all) sometimes temporarily yield some of their authority and allow assistants to decide on the arrest of persons ‘not caught in the act’, even when there is no legit- imation for such a mandate. The prosecutor will insist on being briefed on such decisions straightaway (Lindeman, 2017). Prosecution assistants also play important roles in the policy-related duties of public prosecutors. They prepare and attend selection consultations with the police, in which the case-load of HIC cases will be prioritised. They also prepare and attend tri-party consultations in which the city mayor, the chief of police and the public prosecutor discuss priorities regarding enforcement of crime and public order within districts. Hence, in this second paradigm, we observe (para)professionals taking into account ‘new profes- sional’ values in their professional collaborations. Thus, we observe that (para)professionals are aware of various values of which the importance was stressed in the NPM movement.21 y p y p g 22Lindeman’s (2017) observations on the FOO divisions are representative of the organisation of the processing of FOO in the timeframe prior to the ZSM procedure (roughly from 2000 until 2015). ww.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge 19Strafmaatoverleggen. 4.3.2 New professionalism in the extreme: the PPS 2000–201522 In the FOO divisions of the PSS, we observed an example of an extreme form of the ‘new professionals’ paradigm in which professionals do not so much work together to reach professional goals, but rather work on reaching these goals separately. y p y p g 22Lindeman’s (2017) observations on the FOO divisions are representative of the organisation of the processing of FOO in the timeframe prior to the ZSM procedure (roughly from 2000 until 2015). International Journal of Law in Context 383 During Lindeman’s fieldwork, assistants in the FOO division would work mostly independently from prosecutors and were formally mandated to independently decide cases. Prosecutors had no individual say as to which assistant processed which cases: management and policy regulations had taken over that part of the process and prosecutors often only came into play once a case ended up before the court. Prosecutors did not have the time or the means to actively interfere in those cases at an earlier stage. The decisions were made at great distance from the prosecutor. Therefore, the prosecutors did not claim authority over the cases: ‘Misdemeanors; I do have them [cases prepared by prosecution assistants] at my hearings, but they do not feel like my cases’ (A2 – resp. 7). At the same time, the prosecutors were not content with this division of duties because they regularly noticed mistakes and/or omissions in cases that would end up in court hearings that they had to prepare. Prosecutors commented on the undesirability of this situation, while at the same time emphasising that it was out of their hands. As a consequence of omissions in the files, they sometimes would ask for an acquittal or a stay of proceedings. One prosecutor (A2 – resp. 7) admitted to being aware that, due to a lack of scrutiny by the responsible assistants, people were paying fines for behaviour that was not necessarily liable for punishment. Still he admitted that he did not make the effort to raise awareness on this topic with the responsible assistants, nor with other prosecutors, nor the leadership of their district office. Another prosecutor (A2 – resp. 1) referred to similar issues as a ‘business risk’, while at the same time admitting that he believed it to be frustrating and that it could affect the credibility of the PPS. 4.3.2 New professionalism in the extreme: the PPS 2000–201522 Thus, the prosecutors did feel that they should have authority over the process, but perceived that – in reality – it was out of their control. They considered this to be primarily a problem for the institution rather than for them as professionals. It is in this regard that we observe how the organisational control strongly affects the division of duties between professionals and paraprofessionals, which leads to a decline in the sense of responsibility (discussed earlier in Section 4.2 above) that we did not observe as strongly in other divisions. In most situations described under this paradigm of ‘new professionals’, we see professionals acting as ‘hybrid professionals’ who combine pursuing professional standards with managerial requirements (see Noordegraaf, 2007). However, the situation at the FOO divisions appears to be one in which the professionals try to meet professional standards but the managerial set-up hinders them from meeting both requirements. This situation has somewhat changed since the introduction of the ZSM procedure (discussed in Section 3.3). One of the aims of this alteration was to increase the commitment of pro- secutors to FOO cases. In practice, however, a lot of cases are still processed by assistants, who have now been promoted to ‘assistant-prosecutors’. p p In the following section, we discuss some consequences of two co-existing, and in some respects conflicting, paradigms regarding the role of paraprofessionals. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available 5.1.1 Different levels of delegation of authority in complex and routine cases To anyone with an understanding of the diversity of cases that are handled by the PPS and the judi- ciary, it will not be surprising that different types of cases demand a different paraprofessional con- tribution. Among other aspects, this depends on the type of legislation involved, the procedure for handling the case and the content of the case. In our studies, we found a difference especially between routine, or run-of-the-mill, cases and complex cases.23 In complex cases – dealing with complex legal issues that require a modified process for handling the case, adjusted to the specifics of the case – the collaboration of professionals and paraprofessionals is quite diverse and to a large extent dependent on the specific needs of the professionals. Professionals still have significant control over the work processes and the collaboration is hardly directly affected by NPM requirements such as productivity and efficiency. Different professionals think and act differ- ently regarding which, and how many, content-related tasks are allocated to paraprofessionals (see also Section 4). How much authority judges and prosecutors are prepared to relinquish also depends on the legal knowledge and quality they attribute to the assistants and the trust they have in them. The idea that substantial expertise and specialisation are more important for the quality of one’s work than one’s formal position (being professional or paraprofessional) is consistent with research conducted on the quality of legal practitioners and paralegals (see Moorhead et al., 2003). There are few organisational guidelines regarding the collaboration between professionals and para- professionals in complex cases. In several of such cases, we observed collaboration on an equal basis, with paraprofessionals actively participating in the discussion regarding the merits of a case. However, in other cases, we observed professionals strongly taking the lead and allowing only a small role to be played by paraprofessionals. This was particularly observed at the courts where complex cases are regularly handled by panels of three judges. In that setting, an additional contribution to the judicial discussion by a judicial assistant was, at times, not needed.24 In routine cases – dealing with simple legal issues that can be handled in a rather uniform manner – the procedure for handling the cases is more established and regulated by organisational standards. 5 Analysis of practices in the workplace This section analyses the daily practices that we observed. We demonstrate how the two paradigms that shape the thinking about professional–paraprofessional relations played out at the level of the work- place. When analysing the judiciary and the PPS, first of all, we observe one major collective finding, which is related to the (para)professionals’ juggle between the different paradigms (see similarly Schott et al., 2016). Namely, we observed great variation on a workplace level in the manner in which profes- sionals and paraprofessionals divide tasks and give substance to their collaboration. We thereby observe that the type of collaboration and the level of delegation of authority differ greatly, depending on the type of cases, being either routine cases or complex cases. This – sometimes inexplicable – vari- ation also results in an ambiguity in the relationship between professionals and paraprofessionals. Second, we find that organisational aspects do – sometimes greatly – influence the nature and degree of collaboration and delegation. This influence is particularly visible in the PPS and is more subtly observed in the judiciary. NL Holvast and JMW Lindeman 384 23This is a simplified dichotomy of reality, as different grades of complexity exist. 24This situation has not been observed much at the prosecution’s offices, as it is not very common that multiple prose- cutors deal with one case. See also Lindeman (2017). 5.1 Great variation and ambiguity in the professional–paraprofessional collaboratio On the one end of the spectrum, we observe occasions on which the professional hardly utilises the paraprofessional, other than for purely administrative and secretarial tasks. This occurs mainly in the judiciary. Some judges tend to check all the products of judicial assistants extensively, especially when they consider a judicial assistant to be less meticulous. Other judges simply make little use of the memos and drafts produced by assistants. On the other end of the spectrum, we observe occasions on which the professional utilises the para- professional to the extent that they leave a large part of the initial decision-making up to the parapro- fessional and the professional mainly functions as a controller at the end of the process. The most extreme example of this form of collaborating has been described above regarding the FOO division of the PPS, where prosecutors have given up part of their professional territory. This substantial variation is partly related to the variety in needs with regard to assistance in dif- ferent types of cases; each case requires different legal research to be conducted as well as different clerical assistance. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at 23This is a simplified dichotomy of reality, as different grades of complexity exist. 24This situation has not been observed much at the prosecution’s offices, as it is not very common th cutors deal with one case. See also Lindeman (2017). ww.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ed from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge 5.1.2 Ambiguity on the relations between professionals and paraprofessionals Nonetheless, even in cases with very comparable characteristics, the type and amount of involvement of paraprofessionals vary greatly. We believe that this variance is related to the autonomy that the pro- fessionals still are given by their institutions in deciding how to collaborate with paraprofessionals.25 How the professionals give substance to their discretion appears to be dependent on the paradigms they adhere to. The unpredictability of the division of duties also results in a fair amount of ambiguity in the relationship between these officials. In the judiciary, where there is relatively little official policy that defines the role of paraprofessionals, it is unclear which informal norms should be leading when giving substance to the role of paraprofessionals in practice. In the PPS, we observed that prosecutors often were not aware of the specific requirements regarding education and/or experience for assistants: ‘This is not something that was handed out to me when I started working here. I know about it in general, but if you ask me in detail, I won’t know. It’s not an issue that we normally encounter’ (A2 – resp. 8). Furthermore, prosecutors were not always aware of the formal limits to the mandate that most assistants were working under. Although most (para)professionals have certain, sometimes strong, ideas about the appropriate type and amount of division of labour between professionals and parapro- fessionals, they are uncertain about whether and how to propagate these ideas. As a result, judges and prosecutors only rarely talk to paraprofessionals to share their expectations about their work or provide them with feedback.26 Judicial assistants are, for instance, unsure about what is expected of them during deliberation sessions. They are regularly invited to share their views on cases at the start of the discussions in the deliberations room, but are they also expected to get involved in the discussion that follows from this? When judicial assistants have written draft judgments, judges often make adjustments to the drafts and the assistants are asked to revise them. However, the reasons for making the adjustments are hardly ever explained to the assistants. When judges were asked why they rarely shared their views on the quality and usefulness of the work of judi- cial assistants, they explained that this was partly because they were uncertain about the work stan- dards that judicial assistants had to meet. 25Paraprofessionals’ views also play a role in this, but a proactive paraprofessional has to first be accommodated to act as such by the professional; see also Holvast (2017, pp. 161–162). 26The absence of a culture of sharing feedback in the judiciary was also repeatedly observed by a committee that conducted inspections of the courts. See Commissie visitatie gerechten, Rapport visitatie gerechten (2014), pp. 51–55; Rapport Visitatie Gerechten (2018), p. 20. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, availa 5.1.1 Different levels of delegation of authority in complex and routine cases Usually, these cases – which are the bulk of the cases at district courts and the PPS – do not require a lot of deliberations, as the outcome is clear-cut. A relatively large proportion of the paraprofessionals’ duties in these cases therefore consists of administrative duties. However, in this regard, we observe different official views from the institutions regarding the extent to which judicial or prosecutorial duties can be mandated to paraprofessionals. As described above, in the FOO division, paraprofes- sionals are mandated to make prosecution decisions and, more recently, to even function as a International Journal of Law in Context 385 prosecutor in court. While prosecutors are at times sceptical about this delegation of authority, they accept this as something that is out of their control. In the judiciary, where the sentiment that the judge ought to have the final say is much stronger, no such official delegation of authority is observed. Even in routine cases, the judge always is the one who officially decides the cases, even though these cases are at times completely prearranged by judicial assistants. This final responsibility is considered key to many judges. One judge notes: ‘He [the suspect] has the right, which is also captured in the constitution, to get the judge offered to him by law. … So he has a right to see me. Not a judicial assistant, but me!’ (A1 – resp. 38). In short, we observe a rather varied, and in that sense also comparable, situation in courts and the prosecution office regarding complex cases. Regarding routine cases, we observe that – especially the official – delegation of duties goes much further within the prosecution office. 5.2 Organisation and official policy determine the work processes We have already given various examples of situations in which the official policy of the institutions influences the organisation of the work in the workplace. In Lindeman’s study, it became clear that policy (and regulation) played an extensive role in the developments that resulted in the establishment of particular working processes. In fact, the role of policy and regulation was so extensive that prose- cutors felt that they no longer had individual autonomy over the processing of cases. They were not familiar with the assistants who were mandated to decide on the cases and there was no procedure in place to provide them with feedback. This led to a disconnection between the prosecutors and the paraprofessionals. Prosecutors no longer felt that the cases dealt with in the FOO division were ‘their’ cases, even when they ended up having to handle the case on trial. It seems that values trad- itionally associated with the professional (individual prosecutors) have been appropriated by the insti- tution and in turn have been attributed (within a strict framework of policy and regulations) to paraprofessionals. Thus, in this concrete situation, deprofessionalisation of the ‘full’ professional seems to take place. This is accompanied by an increasing professionalisation of the paraprofessional. The PPS has partly reversed these developments with the introduction of the ZSM procedure. However, the introduction of assistant-prosecutors reveals that prosecutors have undeniably lost ter- ritory. The introduction of this new function has met some criticism. It has been touted (by a judge) as being merely a trick with a change of labels, leading to an inflation of the position of prosecutors (Kristen et al., 2017). At the same time, prosecutors, who felt disconnected with these cases anyway, do not always mourn this ‘lost territory’. The previous developments have not been observed at the HIC divisions of the PPS and at the judi- ciary. The professional values and managerial requirements are in those (parts of the) institutions mainly communicating vessels. Still, it cannot be denied that organisational aspects and official policy have also been determinative for the manner in which the professional and paraprofessionals conduct their work. Due to the high workload that is assigned to prosecutors and judges (see Visser et al., 2019, pp. 45–47) and due to centrally introduced work processes introduced to increase efficiency, judges and prosecutors are not able to perform all their professional duties themselves and must rely heavily on assistants. 5.1.2 Ambiguity on the relations between professionals and paraprofessionals Judges were especially unsure about whether they could expect that judicial assistants should function as a discussion partner. Public prosecutors on the one hand appear more at ease about working with assistants. At the same time, they may feel that some assistants have been forced upon them: it is the organisation that decides the scope of the assistants’ mandate. However, even when prosecutors are largely supportive of employing assistants, they do ask themselves what a suitable division of labour would be and what conditions would be necessary to achieve this ideal situation. bridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 m https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core ter 386 NL Holvast and JMW Lindeman In both institutions, this results in a situation in which the conditions for fruitful professional– paraprofessional interaction remain largely unexpressed. Various paraprofessionals mention the uncertainty of this status quo. They are unsure whether they are expected to play an active role in the decision-making process, by challenging the arguments produced by the professionals, or whether they are expected to follow the instructions of professionals without asking critical questions. 5.2 Organisation and official policy determine the work processes This also results in situations in which the professionals are more or less forced to sur- render part of their professional territory. Part of the work that defines the magisterial character of judges and prosecutors (e.g. forming an unbiased opinion without the risk of any prejudice when deciding on a case) has been appropriated by their institutions and attributed to paraprofessionals. While there is room left for discussion about the optimal division of duties, the effects of managerial measurements have made it difficult for judges and prosecutors to arrange significant changes in order to reclaim territory in situations that they believe need this. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cam 6 Conclusion In this paper, we described how, in the past decades, the traditionally professional-oriented organisa- tion of the Dutch judiciary and the PPS changed, under influences such as the NPM movement. In our analysis of official documents, we find that these changes also altered the position of the judges, pro- secutors and their assistants. Similarly to many other public-sector institutions as well as to legal prac- tice, the employment of assistants has increased significantly in the judiciary and the PPS. There also has been a considerable amount of professionalisation among these assistants, to the point at which they can be referred to as paraprofessionals. Our empirical research reveals that these paraprofessionals International Journal of Law in Context 387 play a significant role in the day-to-day work at the judiciary and PPS. Especially at the PPS, parapro- fessionals have been given relatively large autonomy, by way of a mandate construction. However, individual judges, prosecutors and paraprofessionals have different attitudes towards the appropriate division of duties. In these attitudes, we recognise attributes from the traditionally leading ‘pure pro- fessionalism’ (which emphasises the superior status and autonomy of the professional) as well as the ‘new professionalism’ (which emphasises the collaborative aspect of the professional and paraprofes- sional in reaching professional goals). We have defined these as two competing paradigms. In practice, we observe that these paradigms are visible in different forms and variations in both institutions. The views and practices of professionals in the judiciary fit more dominantly within the first paradigm and the views and practices within the PPS (particularly the divisions dealing with FOO) fit more dom- inantly within the second paradigm. It has become clear that, while professionals have continued to occupy an important position within their institutions, the managerialism of the organisational structures of both institutions has had a considerable impact on the division of duties between professionals and paraprofessionals. The new professional perspective, however, has not replaced the traditional perspective on the alloca- tion of duties, but has set a new, more hybrid perspective in action. The co-existence of these two para- digms and the fact that both institutions have little regulation that concretely defines the territory of the (para)professionals result in a wide variation in collaboration between professionals and parapro- fessionals in practice as well as in ambiguity regarding the position of paraprofessionals. 6 Conclusion Such a diver- sified landscape in daily practices in the workplace is not unique for our research setting. It can be distinguished in other professional settings as well (see e.g. Maynard-Moody and Musheno, 2003). In fact, many (legal) professionals are likely to struggle to define their relationship to paraprofes- sionals. Nonetheless, empirical evidence of such a struggle within the public justice system has been scarce up until now. We believe that some discretion in defining the territory of professionals and the collaboration between professionals and paraprofessionals is essential, as different situations may require different professional and paraprofessional attention. At the same time, not discussing the ideals behind differ- ent practices can result in incomprehensible or arbitrary differences in the allocation of duties in the workplace. It can even result in a vacuum in which no one feels responsible anymore. A possible answer to the, sometimes inexplicable, variation in work practices and the uncertainty about the appropriate division of labour among (para)professionals might be the creation of professional stan- dards for the employment of paraprofessionals. In order to have an actual effect on the daily work practices, we believe that it is key that not just managers, but also professionals and paraprofessionals, are involved in the creation of these standards. Additionally, if significant duties in the adjudication process are delegated to paraprofessionals, this should also be reflected in the safeguards relating to their function and in the financial compensation. In recent publications, Noordegraaf (2015) argued that the perspective of professionalism ‘beyond hybridity’ may counter these issues. Professionals will have to take up responsibilities regarding the organisation of their work, so that they are not only treating cases based on parameters set out by managers (Noordegraaf, 2015; Noordegraaf and Siderius, 2016). ‘Organising professionalism’ means that professional workers also share a responsibility for organising the structures around their work, such as acquiring and maintaining expertise, co-operating with others (e.g. in teams), prioritis- ing and selecting the work (bearing in mind strategic and budgetary considerations), detecting and preventing risks and failures, and accounting for their actions. We believe that this might work not only for the relationship of professionals and managers, but also for professionals and paraprofessionals. Conflicts of Interest. None Conflicts of Interest. None Acknowledgements. The authors would like to thank the participants of the section seminar of the Department STeM at the Erasmus School of Law as well as Peter Mascini and the anonymous reviewers for their valuable comments on earlier drafts of this paper. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, avai References Fiss OM (1983) The bureaucratization of the judiciary. The Yale Law Journal 92, 1442–1467. Freiberg A (2005) Managerialism in Australian criminal justice: RIP for KPIs? Monash University Law Revi Freidson E (1970) Profession of Medicine: A Study of the Sociology of Applied Knowledge. New York: Harper & Row. idson E (1994) Professionalism Reborn: Theory, Prophecy, and Policy. Chicago: University of Chicago Press. Freidson E (2001) Professionalism, the Third Logic: On the Practice of Knowledge. Chicago: University of Chicago Press. Heydebrand W and Seron C (1990) Rationalizing Justice: The Political Economy of Federal District Courts. University of New York Press. Hol AM (2001) De rechterlijke macht in spagaat: Over rechterlijke samenwerking en bestuurljke grenzen. In and Schoep GK (eds), Rechterlijke samenwerking. Deventer: Gouda Quint, pp. 91–100. 1) De rechterlijke macht in spagaat: Over rechterlijke samenwerking en bestuurljke grenzen. In Cleiren CPM GK (eds), Rechterlijke samenwerking. Deventer: Gouda Quint, pp. 91–100. Holvast NL (2017) In the Shadow of the Judge: The Involvement of Judicial Assistants in Dutch District Courts. The Hague Eleven International Publishing. g Holvast NL and Doornbos N (2015) Exit, voice, and loyalty within the judiciary: judges’ responses to new managerialism in the Netherlands’. Utrecht Law Review 11, 49–63. Hondeghem A, Rousseaux X and Schoenaers F (eds) (2016) Modernisation of the Criminal Justice Chain and the Judicial S t N I i ht T t C p ti d H C pit l S it l d S i I t ti l P bli hi Hondeghem A, Rousseaux X and Schoenaers F (eds) (2016) Modernisation of the Criminal Justice Chain and the Judicial System: New Insights on Trust, Cooperation and Human Capital. Switzerland: Springer International Publishing. m A, Rousseaux X and Schoenaers F (eds) (2016) Modernisation of the Criminal Justice Chain and the Jud New Insights on Trust, Cooperation and Human Capital. Switzerland: Springer International Publishing. Hood C (1991) A public management for all seasons? Public Administration 69, 3–19. Jacobs P and Van Kampen P (2014) Dutch ‘ZSM settlements’ in the face of procedural justice: the sooner the better? Utrecht Law Review 10, 73–85. Jean J-P and Pauliat H (2006) An evaluation of the quality of justice in Europe and its developments in France. Utrecht Law Review 2, 44–60. Kirkpatrick I, Dent M and Kragh Jespersen P (2011) The contested terrain of hospital management: Professional projects and healthcare reforms in Denmark. Current Sociology 59, 489–506. Kristen FGH et al. Conflicts of Interest. None Acknowledgements. The authors would like to thank the participants of the section seminar of the Department STeM at the Erasmus School of Law as well as Peter Mascini and the anonymous reviewers for their valuable comments on earlier drafts of this paper. NL Holvast and JMW Lindeman 388 References Abbott A (1988) The System of Professions: An Essay on the Division of Expert Labor. Chicago: University of Chicago Press. Ab l RL ( ) E li h L b M k d S Th P li i f P f i li O f d O f d U i i P Abram T et al. (2011) Partners in Crime: de juridisch medewerker in de strafrechtspraak, een professional na Den Haag: Programma Strafsector. Abram T et al. (2011) Partners in Crime: de juridisch medewerker in de strafrechtspraak, een professional naast de rechter. Den Haag: Programma Strafsector. Algemene Rekenkamer (2012) Prestaties in de strafrechtketen. Den Haag: Sdu. Bach S, Kessler I and Heron P (2008) Role redesign in a modernised NHS: the case of health care assistants. Human Resource Management Journal 18, 171–187. Bach S, Kessler I and Heron P (2008) Role redesign in a modernised NHS: the case of health care assistants. Human Resource Management Journal 18, 171–187. g Bevers PJJM (2004) Samenwerken binnen de rechterlijke organisatie. Den Haag: Boom Juridische Uitgevers. Bevers PJJM (2004) Samenwerken binnen de rechterlijke organisatie. Den Haag: Boom Juridische Uitgevers rs PJJM (2004) Samenwerken binnen de rechterlijke or Bezes P et al. (2012) New public management and professionals in the public sector: what new patterns beyon Sociologie du travail 54, 1–52. Bezes P et al. (2012) New public management and professionals in the public sector: what new patterns beyond opposition? Sociologie du travail 54, 1–52. PPT (2000) Benoeming en ontslag van rechters. Deventer: W.E.J. Tjeenk Willink. Bovend’Eert PPT (2000) Benoeming en ontslag van rechters. Deventer: W.E.J. Tjeenk Willink. Contini F and Carnevali D (2010) The quality of justice in Europe: conflicts, dialogue and politics. Research Institute on Judicial Systems Italian National Research Council. Available at https://www.sisp.it/files/papers/2010/francesco-contini- davide-carnevali-888.pdf (accessed 30 September 2020). Contini F and Carnevali D (2010) The quality of justice in Europe: conflicts, dialogue and politics. Research Institute on Judicial Systems Italian National Research Council. Available at https://www.sisp.it/files/papers/2010/francesco-contini- davide-carnevali-888.pdf (accessed 30 September 2020). p ( p ) De Groot-van Leeuwen LE (1991) De rechterlijke macht in Nederland: Samenstelling en denkbeelden van de zittende en staande magistratuur. Arnhem: Gouda Quint. p p De Groot-van Leeuwen LE (1991) De rechterlijke macht in Nederland: Samenstelling en denkbeelden van de zittende en staande magistratuur. Arnhem: Gouda Quint. g Evetts J (2011) A new professionalism? Challenges and opportunities. Current Sociology 59, 406–422. References (2017) Evaluatie van de Wet herziening gerechtelijke kaart – Samenwerking in de strafrechtketen. Den Haag: WODC/Universiteit Utrecht. Kritzer HM (1999) The professions are dead, long live the professions: legal practice in a postprofessional world. Law & Society Review 33, 713–759. Langbroek PM (2001) Quality Management and Autonomy for Court Organisations, EGPA paper 2001, Study Group on Management and Delivery of Justice, p. 6. Langbroek PM (2001) Quality Management and Autonomy for Court Organisations, EGPA paper 2001, Study Group on Management and Delivery of Justice, p. 6. Langbroek PM and Westenberg MRM (2018) Court Administration and Quality Work in Judiciaries in Four European M and Westenberg MRM (2018) Court Administration and Quality Work in Judiciaries in Four European Empirical Exploration and Constitutional Implications. Bern: Stämpfli Verlag. Langbroek PM and Westenberg MRM (2018) Court Administration and Quality Work in Judicia Countries: Empirical Exploration and Constitutional Implications. Bern: Stämpfli Verlag. oek PM and Westenberg MRM (2018) Court Administration and Quality Work in ntries: Empirical Exploration and Constitutional Implications. Bern: Stämpfli Verlag. Larson MS (1977) The Rise of Professionalism: A Sociological Analysis. Berkeley, CA: University of California Press. Lienhard A, Kettiger D and Winkler D (2012) Status of court management in Switzerland. International Journal for Court Administration 4, 41–67. Lindeman JMW (2017) Officieren van justitie in de 21e eeuw: Een verslag van participerend observatieonderzoek naar kopvatting en taakinvulling van officieren van justitie. Den Haag: Boom Juridische Uitgevers. Lively KJ (2001) Occupational claims to professionalism: the case of paralegals. Symbolic Interaction 24, 343 Mak E (2008) De rechtspraak in balans: Een onderzoek naar de rol van klassiek-rechtsstatelijke beginselen en ‘new public management’-beginselen in het kader van de rechterlijke organisatie in Nederland, Frankrijk en Duitsland. Oisterwijk: Wolf Legal Publishers. g Maynard-Moody S and Musheno M (2003) Cops, Teachers, Counselors: Stories from the Front Lines of Public Service. Ann Arbor: University of Michigan Press. International Journal of Law in Context 389 McLaughlin E, Muncie J and Hughes G (2001) The permanent revolution: New Labour, new public management and the modernization of criminal justice. Criminal Justice 1, 301–318. Moorhead R (2014) Professionalism: some empirical and behavioural perspectives on lawyers. Current Lega 447–481. Moorhead R, Paterson A and Sherr A (2003) Contesting professionalism: legal aid and nonlawyers in England and Wales. Law & Society Review 37, 756–808. y Muzio D and Ackroyd S (2005) On the consequences of defensive professionalism: recent changes in the legal labour pro- cess. Cite this article: Holvast NL, Lindeman JMW (2020). An inquiry into the blurring boundaries between professionals and paraprofessionals in Dutch courts and the public prosecution service. International Journal of Law in Context 16, 371–389. https://doi.org/10.1017/S1744552320000270 References Journal of Law and Society 32, 615–642. f y Noordegraaf M (2007) From ‘pure’ to ‘hybrid’ professionalism. present-day professionalism in ambiguous public domains. Administration & Society 48, 761–785. Noordegraaf M (2011) Risky business: how professionals and professional fields (must) deal with organizational issues. Organization Studies 32, 1349–1371. Noordegraaf M (2015) Hybrid professionalism and beyond: (new) forms of public professionalism in changing organiza- tional and societal contexts. Journal of Profession and Organization 2, 187–206. Noordegraaf M and Siderius K (2016) Perspectieven op publieke professionaliteit: van pr organiserende professionaliteit. Tijdschrift voor Management & Organisatie 70, 4–19. Noordegraaf M and Siderius K (2016) Perspectieven op publieke professionaliteit: van professionals (in organisaties) naar organiserende professionaliteit. Tijdschrift voor Management & Organisatie 70, 4–19. g p j f g g Paterson A (1996) Professionalism and the legal services market. International Jou rofessionalism and the legal services market. International Journal of the Legal Profession 3, 137–168. Paterson A (1996) Professionalism and the legal services market. International Journal of the Legal Professio Raine JW and Willson MJ (1997) Beyond managerialism in criminal justice. The Howard Journal 36, 80–95 Schott C, Van Kleef D and Noordegraaf M (2016) Confused professionals? Capacities to cope with pressures on professional work. Public Management Review 18, 1–28. g Sommerlad H (1995) Managerialism, and the legal profession: a new professional paradigm. International Journal of the Legal Profession 2, 159–186. g f Sommerlad H et al. (Forthcoming) Casualisation and proletarianisation. In Abel RL and Sommerlad H, with Hammerslev O d S h l U ( d ) L i 21st C S i i C i Th i Bl b H P bli hi Sommerlad H et al. (Forthcoming) Casualisation and proletarianisation. In Abel RL and Sommerlad H, with Hammerslev O and Schultz U (eds), Lawyers in 21st Century Societies: Comparative Theories. Bloomsbury: Hart Publishing. et al. (Forthcoming) Casualisation and proletarianisation. In Abel RL and Sommerlad H, with Hammerslev O z U (eds) Lawyers in 21st Century Societies: Comparative Theories Bloomsbury: Hart Publishing erlad H et al. (Forthcoming) Casualisation and proletarianisation. In Abel RL and Sommerlad H, with Hamm Schultz U (eds), Lawyers in 21st Century Societies: Comparative Theories. Bloomsbury: Hart Publishing. Spigelman JJ (2001) The ‘New Public Management’ and the courts. Australian Law Journal 75, 748–760. Susskind R (2017) Tomorrow’s Lawyers: An Introduction to Your Future, 2nd edn. https://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 Downloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at References Oxford: Oxford University Pre Van de Bunt HG (1985) Officieren van justitie: verslag van een participerend observatieonderzoek. Zwoll n de Bunt HG (1985) Officieren van justitie: verslag van een participerend observatieonderzoek. Zwolle: Tjee d B HG d V G ld JL (2012) Th D h i i C i d J i 41 117 140 ff j g p p j Van de Bunt HG and Van Gelder JL (2012) The Dutch prosecution service. Crime and Justice 41, 117–140. Van der Torre A et al. (2007) Rechtspraak: Productiviteit in perspectief. Den Haag: Sociaal en Cultureel Rechtspraak. Van Oorschot I (2014) Seeing the case clearly: file-work, material mediation, and visualizing practices in a D court. Symbolic Interaction 37, 439–457. Vigour C (2006) Justice: l’introduction d’une rationalité managériale comme euphémisation des enjeux politiques. Droit et société 63–64, 425–455. Vigour C (2015) Professions in policy and knowledge transfer: adaptations of lean management, and jurisdictional conflict in a reform of the French public service. International Journal of Sociology 45, 112–132. Visser M, Schouteten R and Dikkers J (2019) Controlling the courts: New Public Management and the Dutch judiciary. Justice System Journal 40, 39–53. Zouridis S (2016) From justice archipelago to security and justice chain: strategy-organisation configurations in the Dutch criminal justice system. In Hondeghem A, Rousseaux X and Schoenaers F (eds), Modernisation of the Criminal Justice Chain and the Judicial System: New Insights on Trust, Cooperation and Human Capital. Cham: Springer International Publishing, pp. 79–93. Cite this article: Holvast NL, Lindeman JMW (2020). An inquiry into the blurring boundaries between professionals and paraprofessionals in Dutch courts and the public prosecution service. International Journal of Law in Context 16, 371–389. https://doi.org/10.1017/S1744552320000270 Cite this article: Holvast NL, Lindeman JMW (2020). An inquiry into the blurring boundaries between professionals and paraprofessionals in Dutch courts and the public prosecution service. International Journal of Law in Context 16, 371–389. https://doi.org/10.1017/S1744552320000270 tps://www.cambridge.org/core/terms. https://doi.org/10.1017/S1744552320000270 ownloaded from https://www.cambridge.org/core. IP address: 80.101.62.141, on 03 Mar 2021 at 11:00:31, subject to the Cambridge Core terms of use, available at
https://openalex.org/W2754134997	https://revistas.ucr.ac.cr/index.php/rbt/article/download/26975/30648	English	null	Land use and biotic integrity in shallow streams of the Hondo River basin, Yucatan Peninsula, Mexico	Revista de Biología Tropical	2,017	cc-by	6,244	Received 21-III-2017. Corrected 18-VII-2017. Accepted 16-VIII-2017. Abstract: Aquatic environments face a variety of threats in the Hondo River basin, Southeastern Yucatán Peninsula. Some of these impacts, like pollution by pesticides, may depend on land use and cover. Our objec tive was to assess the effect of land use/cover using a previously published index of biotic integrity (IBI), based on fishes and designed for shallow streams in the Hondo River basin. Our hypothesis was that land uses that cause deforestation and pollution, such as urbanization, cattle breeding, or sugar cane fields, would be reflected in low IBI values, at least at some spatial scales. The 23 sites originally used in 2008-2009 to estimate by elec trofishing the relative abundance and other characteristics of selected fish species and guilds to construct the IBI, were revisited in February 2010, to validate by direct inspection the type of land use/cover suggested by landscape information in digital databases. We analyzed the effect of seven types of land use/cover (agriculture, pasture, human settlements, water bodies, wetlands, forest, and secondary vegetation) on the IBI values, at four spatial scales, i.e., the percent of every land use/cover at progressively larger circles (125, 250, 500, and 1 000 m diameter) centered on the water body where the IBI value was measured. Correlations were established among the percent land/use cover by scale around 23 sites, and with their corresponding IBI values. Then, Student’s t tests were calculated to examine significant differences in land use/cover between groups of localities above and below the median IBI value, and Mann-Whitney’s U tests were applied to compare IBI values between localities with and without a given landscape cover. Agriculture, human settlements, and secondary vegetation correlated negatively with the IBI; forests positively. Differences were significant (p<0.05) for forest (higher in sites with higher IBI values) and human settlements (lower in sites with higher IBI). Of all the landscape categories located in the Hondo River basin, with the exception of pasture, those of anthropogenic origin tended to be detrimental to aquatic biotic integrity. Rev. Biol. Trop. 65 (4): 1448-1458. Epub 2017 December 01. Key words: human impact, fishes, environmental risk, landscape dynamics, index of biotic integrity, aquatic diversity. Land use and biotic integrity in shallow streams of the Hondo River basin, Yucatán Peninsula, Mexico Rodrigo I. Pacheco-Díaz, Juan J. Schmitter-Soto, Birgit Schmook, Gerald A. Islebe & Holger Weissenberger El Colegio de la Frontera Sur, Apdo. Postal 424, MX-77000 Chetumal, Mexico; rpacheco@live.com.mx, jschmitt@ecosur.mx, bschmook@ecosur.mx, gislebe@ecosur.mx, holgerweissen@ecosur.mx Correspondence Rodrigo I. Pacheco-Díaz, Juan J. Schmitter-Soto, Birgit Schmook, Gerald A. Islebe & Holger Weissenberger El Colegio de la Frontera Sur, Apdo. Postal 424, MX-77000 Chetumal, Mexico; rpacheco@live.com.mx, jschmitt@ecosur.mx, bschmook@ecosur.mx, gislebe@ecosur.mx, holgerweissen@ecosur.mx Correspondence Received 21-III-2017. Corrected 18-VII-2017. Accepted 16-VIII-2017. MATERIAL AND METHODS The study area is located in the Southeast ern Yucatán Peninsula, in the Mexican part of the Hondo River basin (Fig. 1). The limits of the Hondo River basin are not well defined, but we estimated an extension of 8 174 km² in the Mexican side. The highest tributaries flow from a plateau at ca. 250 m above sea level, with no clear watershed to divide them from adjacent basins. The river’s main course follows a geo logical fault and, as its tributaries, flows slowly, with a slope always less than 5°. Typical soils are regosols, vertisols, and rendzines (Arriaga Cabrera et al., 2000).The climate is warm and sub-humid, with an average annual rainfall from 1 200 to 1 400 mm, and a mean tem perature of 24-28 °C (INEGI, 2009), although in recent years precipitation has fluctuated greatly (IPCC, 2013). The natural vegetation is mostly medium subdeciduous forest, domi nated by Manilkara zapota (L., 1759), Bucida buceras L., 1759, or Cryosophila stauracantha (Heynh., 1846), as well as mangrove and other aquatic vegetation (Miranda, 1958). ( ) Indices of biotic integrity (IBI) are useful tools for the estimation of current ecosystem condition, as they integrate information at mul tiple levels of biological organization and at different spatial scales (Karr, 1981). Therefore, IBI are complementary indicators to physical and chemical methods to estimate ecosystem health, although a serious drawback is the fact that they need to be tailored specifically almost for every basin, due to natural differences in biogeography and ecology. These indices, originally developed for streams, have also been applied to other aquatic environments, e.g. estuaries (Fisch, Branco, & Menezes, 2016). Besides fish, other organisms have been used as indicators, for example plants and invertebrates (Wilcox et al., 2002), diatoms (Fore, 2002), and even birds (Córdova-Ávalos, Alcántara-Carbajal, Guzmán-Plazola, Mendo za-Martínez, & González-Romero, 2009). In Mexico, IBI have been developed most ly for rivers and lakes (Schmitter-Soto, 2014). Schmitter-Soto, Ruiz-Cauich, Herrera-Pavón, and González-Solís (2011) developed a fish- based IBI for shallow streams in the Hondo River basin, based mainly on the relative abun dance of selected opportunistic species and sensitive ecological guilds. In this study, an IBI created for shallow streams in the Hondo basin (Schmitter-Soto et al., 2011), was analyzed as a function of landscape-level data. The IBI was developed from 23 sampling sites distributed on the Mexi can side of the Hondo River basin (Fig. 1A). Received 21-III-2017. Corrected 18-VII-2017. Accepted 16-VIII-2017. Along the Mexican-Belizean Hondo River basin, most human settlements used to subsist on forestry, agriculture, livestock, and tourism (although since the early 2000s most of them rely on government subsidies and remittances from the United States or from Cancún and Playa del Carmen: Castellanos, 2010). Some of these activities threaten freshwater systems due to creation of waste, such as organic matter and pesticides, among others. Agricul ture, transport, and urban residual water are the main sources of pollution (Buenfil-Rojas, Álvarez-Legorreta, & Cedeño-Vázquez, 2014; Sánchez, Álvarez-Legorreta, Pacheco, Carrillo, & González, 2016). Additionally, the presence of invasive species in the region, like tilapia Oreochromis mossambicus (Peters, 1852) and its hybrids, as well as armored catfish Ptery goplichthys pardalis (Castelnau, 1855) and the partial or total physical modification of the landscape at different scales threaten fresh water bodies and their biota (Schmitter-Soto Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1448 & Caro, 1997; Esselman, Schmitter-Soto, & Allan, 2013; Schmitter-Soto, Quintana, Valdéz- Moreno, Herrera-Pavón, & Esselman, 2015). & Caro, 1997; Esselman, Schmitter-Soto, & Allan, 2013; Schmitter-Soto, Quintana, Valdéz- Moreno, Herrera-Pavón, & Esselman, 2015). Central America (van Oosterhout & van der Velde, 2014), but no such analysis is yet avail able for Mexico. Thus, the aim of this study was to assess the effect of the land use/cover mosaic on the IBI based on fishes, as developed by Schmitter-Soto et al. (2011) for the Hondo River basin, Mexico. Our hypothesis was that land uses involving deforestation and pollution, such as urbanization, cattle breeding, or sugar cane fields, would correlate with low IBI val ues, at least at some spatial scales. ) The most important land use changes in the Hondo River basin are the conversion of natural vegetation to sugar cane plantations, pasture for cattle, physical changes of the water channel to create touristic facilities, and local tilapia production. This affects ripar ian systems, which possess high biodiversity and provide environmental services, food and recreational resources for the local population. Riparian ecosystems are also highly vulnerable to natural disturbances, such as floods or fires (Naiman et al., 2005). MATERIAL AND METHODS This index used twelve metrics based on fishes, with a maximum score of 60 and a minimum of zero; details are given by Schmitter-Soto et al. (2011), who also provided environmental data for every sampled locality used to design Some studies have incorporated landscape components to analyze their influence on IBI values, in North (e.g. Richards & Host, 1994; Snyder, Young, Villella, & Lemarié, 2003), South (Pinto, Araújo, & Hughes, 2006) and Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1449 g. 1. Study area in the Hondo River basin, with (A) sampled localities in the context of permanent and intermitte ter flows, and (B) identified land-uses (modified from Pacheco Díaz, 2011, and Schmitter-Soto et al., 2011) mbols: upper basin, open circles; middle basin, circles with a centered dot; lower basin, solid circles. Fig. 1. Study area in the Hondo River basin, with (A) sampled localities in the context of permanent and intermittent surface water flows, and (B) identified land-uses (modified from Pacheco Díaz, 2011, and Schmitter-Soto et al., 2011). Locality symbols: upper basin, open circles; middle basin, circles with a centered dot; lower basin, solid circles. (Schmitter-Soto et al., 2011) were visited in February 2010. At each site, a visual survey of the natural vegetation and land use (agricul ture, pasture for cattle, secondary vegetation, or test the index. No new components were added to the IBI. To collect field data for landscape vari ables, all locations used to construct the IBI Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1450 RESULTS among others) was conducted. A Garmin global positioning system device was used to georref erence the sites. Subsequently, the type of veg etation at each site was determined, using the vegetation classification for the Yucatán Pen insula by Miranda (1958). This field informa tion was used to corroborate available digital databases and maps (García & Secaira, 2006; INEGI, 2009). The data were processed with the geographic information system ArcView 3.2 (ESRI, 1999). Coverage and extension of agricultural and livestock activities, delimita tion of the basin, and land use were recorded, and a land use and land cover map was pro duced (Fig. 1B). MATERIAL AND METHODS For February 2010, we identified 17 land use and land cover types, which were grouped into seven categories or classes for analysis: agriculture, pasture, human settlements, water bodies, wetlands, forest, and secondary vegeta tion (Table 1; Fig. 1B). Considering altitudinal stratification, we found that the upper and middle basin was dominated by forest, followed by pasture; the other classes did not exceed 20 % of the remaining coverage. In the lower basin two classes dominated, namely forest and agricul ture, followed by secondary vegetation (Figs. 1B, 2). In the lower basin we found a larger area occupied by wetlands, water bodies, and human settlements. Pasture in the lower basin represented a smaller percentage than in the other two altitudinal sections (Fig. 2). Using any of the four different scales of analysis, pas ture had the highest percentage of land cover, ranging from 31 to 37 %; agricultural use had a range of 24 to 26 %; wetlands from 7 to 14 %, secondary vegetation from 8 to 12 %; forests from 6 to 10 %; human settlements from 6 to 9 %, and water bodies from 2 to 9 %. On the land use and land cover map, pro gressively larger circular buffers (125, 250, 500, and 1 000 m) were defined around each of the sampling sites in order to measure the coverage (in m2) at different scales for each land use/cover class at each site. A multiple Pearson correlation analysis was performed among the percentage covers for each land scape (land use/land cover) class and the local IBI values. In addition, Student’s t tests were calculated to look for significant differences in cover between high- and low-IBI groups of localities (above and below the median IBI value), and Mann-Whitney’s U tests were applied to compare IBI values between locali ties with vs. without a given landscape cover. Significance in all cases was set at p<0.05; we used the program Infostat, vers. 2008 (di Rien zo et al., 2008), and the website Vassarstats (Lowry, 2016). The correlation between the IBI for the Hondo River basin and the different land use/ cover classes was similar at all scales, always either negative or positive. Correlation was inverse between the index and agriculture (r = -0.17 to -0.32). Correlation between land use/cover types and IBI values (only Scale 4, 1 000 m, shown) agric forst past settl secve water wetln IBI -0.18 0.26 0.16 -0.45 -0.18 -0.27 0.40 agric 1 -0.34 -0.74 0.08 -0.22 -0.18 -0.08 forst 1 0.05 -0.39 -0.21 -0.12 -0.12 past 1 -0.33 -0.27 -0.24 -0.23 settl 1 0.37 0.55 -0.05 secve 1 0.44 0.20 water 1 0.29 Abbreviations: agric, agriculture; forst, forest; past, pasture; settl, human settlements; secve, secondary vegetation; wetln, wetlands. MATERIAL AND METHODS Human settlements also presented an inverse correlation to the IBI (r = -0.15 to -0.46), as did secondary vegetation Land use/cover classes in the Hondo River basin Classes Land use and land cover Cover (%) Forest High and medium subdeciduous forest 45 Pasture Pasture for cattle raising 17 Secondary vegetation Secondary vegetation 16 Agriculture Irrigation and temporal agriculture 12 Wetlands Mangrove, riparian vegetation and other hydrophytes 8 Human settlements Human settlements 1 Water bodies Lakes, cenotes, rivers, and streams 1 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1451 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1451 Fig. 2. Distribution of land use/cover in the different altitude layers in the Hondo River basin. DISCUSSION These pollutants remain suspended or precipitate, affecting most of the biota present, increasing deformities and mortality, and altering growth, reproduction, feeding, and survival of fish (Cooper, 1993). g ( p ) Many of the sampling sites, especially in the lower part of the basin, are also used for recreational purposes (rustic pools), which can be harmful to aquatic biota (Allan, 2004) because of ditching, building of dams and other water retaining structures (damaging not only to potamodrome species, but potentially frag menting all fish populations), clearing of ripar ian vegetation, and the addition of chemical substances (bleach and detergent) for the main tenance of the sites. In the upper basin, streams provide drinking water for farm animals (pigs and poultry). Canalization of the sites alters the evaporation balance, increases the frequency and magnitude of floods, contributes to the alteration of flow dynamics, and increases ero sion and transport of nutrients, sediments and pollutants (Walsh et al., 2005). Altered drain age systems, soil compaction, and modification of the mosaic of natural habitats, patches, and ecotones further affect the diversity of aquatic organisms (Allan et al., 1997). The effects of human settlements are similar to those caused by agriculture (van Sickle et al., 2004). Riparian vegetation is usually sparse in agricultural areas, and streams are modified given the spatial distribution and irrigation of fields, thus affecting fish communities, since the structure of riparian zones in turn affect the structure of aquatic habitats (Roth et al., 1996), especially because of a lack of vegetation in the stream banks, where many fish species find shelter as young. Nevertheless, Wang, Lyons, Kanehi, Bannerman, and Emmons (2000) com mented that urban uses are more harmful to environmental health than agriculture (because of the increased runoff, which alters the stream physically and chemically), and Snyder et al. (2003) even reported a positive relationship between the spread of agriculture and a fish- based IBI (although their finding might be due to including pasture and row-crops in the same land-use category). In the case of pasture for cattle farm ing, this study found positive values with respect to biotic integrity, a counter-intuitive result that refutes our initial hypothesis with respect to this particular land use. However, the same association was found by Fitzpatrick et al. (2001) in Wisconsin and by Pinto et al. (2006) in Brazil. DISCUSSION (r = -0.19 to -0.28). Positive correlations existed for pasture (r = 0.15 to 0.23), wetlands (r = 0.32 to 0.40), and forests (r = 0.20 to 0.23). Several land use/cover types were correlated among themselves, e.g. water bodies, secondary veg etation, and human settlements; agriculture and pasture were strongly inversely correlated, almost mutually exclusive (Table 2). In general, studies of land use on stream condition have found that, where forests domi nate (at least in temperate regions), IBI values tend to be higher (i.e., biotic integrity is better). In contrast, in localities with a high propor tion of agriculture (>40 %) and human settle ments, the IBI tends to be lower (Roth, Allan, & Erickson, 1996; Allan, Erickson, & Fay, 1997; Allan, 2004). However, only two differences between groups of localities were found to be statisti cally significant: Localities with a high IBI (>50) had a mean forest cover of 62.5 % vs. only 10.0 % for sites with lower IBI (t= 5.4). Localities with any human settlement cover had lower IBI values (mean 42.3) than those without human settlements (IBI mean 46.5; U= 29.5). In our study, agriculture had an inverse (although not significant) correlation to biotic integrity. In streams of Wisconsin (Richards & Host, 1994) and Michigan (Roth et al., 1996) the inverse correlation was significant Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1452 and strong, up to r = -0.70. Similarly, Wichert and Rapport (1998) found that fish commu nities significantly declined in streams in Southern Ontario as agriculture intensified. For streams in Illinois, Fitzpatrick, Scudder, Lenz, and Sullivan (2001) reported that IBI values tend to be low when agriculture cover is >10 % in the stream buffer zone (r = -0.76). In our study area, the main agricultural activity (close to 100 % of the agriculture cover in the lower basin) was sugar cane production, which causes a large deposition of sediment, fertiliz ers and herbicides in surrounding water bodies (Yu, DeLaune, Tao, & Beine, 2008). Carpenter et al. (1998) analyzed nutrient enrichment from crop fields and observed an increase in trophic levels, increasing production and bio mass of many organisms, resulting in changes in species composition, usually with lower diversity (evenness). et al., 2011). Detergents, oil derivatives, and domestic sewage are discharged into freshwa ter bodies in the Hondo basin. DISCUSSION These are the likely causes why pasture did not adversely affect aquatic integrity in the Hondo River basin. 1999), thus reducing wash-out. Other studies in pasture have shown an increase in micro organism communities that keep nutrient lev els stable (Bardgett, Mawdsley, Edwards, & Hobbs, 1999). Moreover, pasture does not need much input of fertilizers and herbicides relative to other crops in the region (e.g. sugar cane), and burning usually occurs only every two years. These are the likely causes why pasture did not adversely affect aquatic integrity in the Hondo River basin. Secondary vegetation, in spite of being a more “natural” land use, presented an inverse (although not significant) correlation to the aquatic biotic integrity. This can be explained by the fact that secondary vegetation results from the removal of native vegetation, partly by natural disasters like hurricanes, fires, and floods, but more importantly by human activi ties, such as land clearing for agriculture and the expansion of urban centers, among others (Gómez-Pompa, 1971). In the upper basin, secondary vegetation is the result of clearing for human settlements and pasture; in the lower basin, it can be attributed to agriculture, human settlements and pasture. Forests showed a positive correlation with aquatic biotic integrity, as reported by several authors (Omernik, Abernathy, & Male, 1981; Roth et al., 1996; Fitzpatrick et al., 2001). For ests favor aquatic ecosystems health because they act as traps for nutrients and toxins, retaining and limiting their leakage into water bodies. Forested areas also preserve chan nel morphology, stabilize flow, and provide organic matter, food and shelter for aquatic organisms. Wetlands showed a positive cor relation with respect to biotic integrity, as reported by other authors (Roth et al., 1996; Fitzpatrick et al., 2001). Vegetation in wetlands is important for freshwater ecosystems because it reduces excessive light and temperature, growth of algae and aquatic plants, loss of stability of the sediment, nutrient retention, changes in the quantity and quality of organic matter, and alteration of the trophic structure of the system (Findlay, Quinn, Hickey, Burrell, & Downes, 2001). The presence of lentic water bodies showed an inverse correlation with biotic integrity in streams. This is probably because most pollut ants are carried and deposited in these aquatic systems, with an enrichment in nutrients from external sources (Carpenter et al., 1998). DISCUSSION Both studies reported that an increase in pasture results in a decline of urban uses. Other studies indicated that pasture has a slightly positive effect on ecosystem health (Strayer et al., 2003). Biotic integrity was significantly lower where urban areas increased; this expected result is consistent with most findings else where (e.g. Pinto et al., 2006; van Oosterhout & van der Velde, 2014). The largest human settlement in the study area is the city of Che tumal, near the mouth of Hondo River, but other important urban areas lie mainly near the Hondo River main channel (Schmitter-Soto In our study area, pasture can act like natu ral vegetation: as grass covers the ground most of the year, soil erosion and runoff are avoided. Pasture reduces the concentration of nitrogen and other nutrients in the soil (Laurance et al., Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1453 Lower values of the Hondo River IBI are mainly correlated with the scarcity or absence of sensitive species (e.g. Hyphesso brycon compressus [Meek, 1904]), frequent excess of tolerant species (e.g. Rhamdia gua temalensis [Günther, 1864], Astyanax bacalar ensis Schmitter-Soto, 2017, Thorichthys meeki Brind, 1918), occasional presence of exotics (Oreochromis mossambicus [Peters, 1852], O. niloticus [L., 1758], Pterygoplichthys pardalis [Castelnau, 1855]), dominance of herbivores (e.g. Vieja melanura [Günther, 1862], perhaps due to the proliferation of filamentous algae at organic-enriched sites), or dominance of oppor tunistic species (e.g. Poecilia mexicana Stein dachner, 1863) (Schmitter-Soto et al., 2011). Jones III, Helfman, Harper, and Bolstad (1999) found lower fish abundance as riparian vegeta tion decreased, mainly because of the loss of benthic species, which were replaced by detri tivorous species, tolerant to adverse environ mental conditions and even invasive species. Likewise, associated vegetation has proven to be effective in the reduction of nitrogen and phosphorus, in both ground and surface water (Gergel, Turner, Miller, Melack, & Stanley, 2002). In Costa Rica, Lorion and Kennedy (2009) concluded that riparian vegetation was critical to biotic integrity in tropical streams. 1999), thus reducing wash-out. Other studies in pasture have shown an increase in micro organism communities that keep nutrient lev els stable (Bardgett, Mawdsley, Edwards, & Hobbs, 1999). Moreover, pasture does not need much input of fertilizers and herbicides relative to other crops in the region (e.g. sugar cane), and burning usually occurs only every two years. RESUMEN Uso del suelo e integridad biótica en arroyos someros de la cuenca del río Hondo, península de Yuca tán, México. Los ambientes acuáticos en la cuenca del río Hondo, sureste de la península de Yucatán, enfrentan diversas amenazas. Algunos de estos impactos, e.g. con taminación por plaguicidas, pueden depender del uso de suelo o cobertura. El objetivo fue evaluar el efecto del uso del suelo/cobertura sobre un índice biótico de integridad (IBI) basado en peces, publicado previamente, diseñado para arroyos someros en esta cuenca. La hipótesis era que los usos del suelo que involucran deforestación y conta minación, entre ellos urbanización, ganadería o cultivo de caña de azúcar, se verían reflejados en valores bajos del IBI, por lo menos a ciertas escalas espaciales. Los 23 sitios usados originalmente para estimar por electropesca la abundancia relativa y otras características de especies y gremios selectos de peces, para construir el IBI, fueron visitados de nuevo en febrero 2010 para validar por inspec ción directa el tipo de uso del suelo/cobertura sugerido por la información de paisaje en bases de datos digitales. Se analizó el efecto de siete tipos de uso del suelo/cobertura (agricultura, ganadería, asentamientos humanos, cuerpos de agua, humedales, bosque y vegetación secundaria) sobre los valores del IBI, a cuatro escalas espaciales, i.e., el por centaje de cada uso del suelo/cobertura en círculos progre sivamente mayores (125, 250, 500 y 1 000 m de diámetro) centrados en el cuerpo de agua donde se midió el valor del IBI. Los porcentajes de uso del suelo/cobertura por escala se correlacionaron entre sí y con los valores correspondien tes del IBI para explorar su asociación; luego, mediante pruebas t de Student se buscaron diferencias significativas en cobertura entre grupos de localidades por encima y por debajo del valor mediano del IBI, así como pruebas U de Mann-Whitney para comparar valores del IBI entre localidades con o sin una cobertura dada. La agricultura, asentamientos humanos y vegetación secundaria mostraron una relación negativa con el IBI, lo cual sugiere un impacto de las actividades humanas sobre la salud de los ecosiste mas acuáticos. Las diferencias fueron significativas para el bosque (mayor en sitios con mejor integridad acuática) y los asentamientos humanos (menor en sitios con mayor valor del IBI). RESUMEN De todas las clases paisajísticas en la cuenca del río Hondo, las de origen antrópico, excepto los pasti zales para ganadería, tendieron a ser perjudiciales para la integridad biótica acuática. Palabras clave: impacto humano peces riesgo ambiental It should be acknowledged that most of the correlations found between land use/cover classes and IBI values were not significant, mostly because of the small sample size. It is desirable to increase the number of sampled localities, so that there is a representation of the differences and relationships between index values and landscape components (Pinto et al., 2006). Nevertheless, our correlations are use ful as early warnings (a conclusion similar to Snyder’s et al., 2003). The most detrimental land use/cover was urban use, while the most positive ones were wetlands and forests. The lower basin, where agriculture and human settlements predomi nate, and where IBI indicates that aquatic environmental health is compromised, should receive attention more urgently. Cattle pas tures uses were expected to be detrimental, but were actually positive; secondary veg etation was expected to be positive, but this was not the case. The relationship between landscape and local aquatic integrity is not always intuitively predictable. This conclu sion has important implications for manage ment, so we recommend that due attention has to be paid to the interaction between these two levels of complexity for monitoring and restoration programs. DISCUSSION This condition reflects the presence of livestock, agriculture, and human settlements near water bodies, as many of these systems are devoid of riparian vegetation. The lack of riparian vegetation accelerates the rate and magnitude of pollution from organic matter and pesti cides carried by runoff, causing changes in the availability of nutrients because of the loss of processes of filtration provided by forests and riparian vegetation. Lentic systems function differently from lotic ones, because the mechanical process of degradation of pollutants is low or absent, Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1454 financed by project SEMARNAT-CONACYT 2006/01 (23674), and was part of the M.Sc. thesis of the first author. financed by project SEMARNAT-CONACYT 2006/01 (23674), and was part of the M.Sc. thesis of the first author. financed by project SEMARNAT-CONACYT 2006/01 (23674), and was part of the M.Sc. thesis of the first author. affecting the dilution of harmful substances; in addition, aeration is deficient, so most of the pollutants have a longer life (Schmitter- Soto et al., 2002). However, Pinto et al. (2006) found that the presence of lakes and ponds was positive (r = 0.71) for biotic integrity in Brazilian streams. REFERENCES Allan, J. D. (2004). Landscapes and riverscapes: the influence of land use on stream ecosystems. Annual Review of Ecology, Evolution, and Systematics, 35(2002), 257-284. http://doi.org/10.1146/annurev. ecolsys.35.120202.110122 Esselman, P. C., Schmitter-Soto, J. J., & Allan, J. D. (2013). Spatiotemporal dynamics of the spread of African tilapias (Pisces: Oreochromis spp.) into rivers of northeastern Mesoamerica. Biological Invasions, 15(7), 1471-1491. http://doi.org/10.1007/ s10530-012-0384-9 Allan, J. D., Erickson, D. L., & Fay, J. (1997). The influen ce of catchment land use on stream integrity across multiple spatial scales. Freshwater Biology, 37(1), 149-161. http://doi.org/10.1046/j.1365-2427.1997. d01-546.x Findlay, S., Quinn, J. M., Hickey, C. W., Burrell, G., & Downes, M. (2001). Effects of land use and riparian flowpath on delivery of dissolved organic carbon to streams. Limnology and Oceanography, 46(2), 345- 355. http://doi.org/10.4319/lo.2001.46.2.0345 Arriaga Cabrera, L., Espinosa-Rodríguez, J. M., Aguilar- Zúñiga, C., Martínez-Romero, E., Gómez- Mendoza, L., & Loa, E. (2000). Regiones terrestres prioritarias de México. Mexico City: Comisión Nacional para el Conocimiento y Uso de la Biodiversidad. Fisch, F., Branco, J. O., & Menezes, J. T. de. (2016). Ictiofauna como indicador de la integridad biótica de un ambiente estuarino. Acta Biológica Colombia na, 2121(11), 27-3827. http://doi.org/10.15446/abc. v21n1.46151 Bardgett, R. D., Mawdsley, J. L., Edwards, S., & Hobbs, P. J. (1999). Plant species and nitrogen effects on soil biological properties of temperate upland grasslands. Ecology, 13, 650-660. Fitzpatrick, F. A., Scudder, B. C., Lenz, B. N., & Sullivan, D. J. (2001). Effects of multiscale environmental characteristics on agricultural stream biota in eas tern Wisconsin. Journal of the American Water Resources Association, 37(6), 1489-1507. http://doi. org/10.1017/CBO9781107415324.004 Buenfil-Rojas, A. M., Álvarez-Legorreta, T., & Cedeño- Vázquez, J. R. (2014). Metals and metallothioneins in Morelet’s crocodile (Crocodylus moreletii) from a transboundary river between Mexico and Beli ze. Archives of Environmental Contamination and Toxicology, 68(2), 265-273. http://doi.org/10.1007/ s00244-014-0088-5 Fore, L. S. (2002). Response of diatom assemblages to human disturbance: development and testing of a multimetric index for the Mid-Atlantic Region (USA). In T. P. Simon (Ed.), Biological response signatures: indicator patterns using aquatic commu nities (pp. 445-480). Boca Raton, FL: CRC. Carpenter, S. R., Caraco, N. F., Correll, D. L., Howarth, R. W., Sharpley, A. N., & Smith, V. H. (1998). Nonpoint pollution of surface waters with phosphorus and nitro gen. Ecological Applications, 8(1998), 559-568. http:// doi.org/10.1890/1051-0761(1998)008[0559:NPOSW W]2.0.CO;2 García, G., & Secaira, F. (2006). ACKNOWLEDGMENTS Thanks to Susannah McCandless for reviewing our English, to the editors and anonymous reviewers for useful com ments on the manuscript, to Roberto Her rera for field support, and to Janneth Padilla for help with the figures. The Mexi can government issued collecting permit No. DGOPA.04715.240810.2914. This work was Palabras clave: impacto humano, peces, riesgo ambiental, variación del paisaje, índice biótico de integridad, diversi dad acuática. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1455 Environmental System Research Institute (ESRI). (1999). ArcView GIS. Version 3.2. New York: Environmental Systems Research Institute. Environmental System Research Institute (ESRI). (1999). ArcView GIS. Version 3.2. New York: Environmental Systems Research Institute. REFERENCES Una visión para el futuro: cartografía de las selvas maya, zoque y olmeca: plan ecorregional de las selvas maya, zoque y olmeca. San José, Costa Rica: TNC Infoterra, Conservation International. Castellanos, M. B. (2010). A return to servitude: Maya migration and the tourist trade in Cancún. Minnea polis: University of Minnesota Press. Gergel, S. E., Turner, M. G., Miller, J. R., Melack, J. M., & Stanley, E. H. (2002). Landscape indicators of human impacts to riverine systems. Aquatic Sciences, 64, 118-128. Cooper, C. M. (1993). Biological effects of agricultu rally derived surface water pollutants on aquatic systems-a review. Journal of Environmental Quality, 22(3), 402-408. Gómez-Pompa, A. (1971). Posible papel de la vegetación secundaria en la evolución de la flora tropical. Biotró pica, 3(2), 125-135. Córdova-Ávalos, A., Alcántara-Carbajal, J. L., Guzmán- Plazola, R., Mendoza-Martínez, G. D., & González- Romero, V. (2009). Desarrollo de un Índice de Integridad Biológica Avifaunístico para dos asocia ciones vegetales de la Reserva de la Biósfera Panta nos de Centla, Tabasco. Universidad y Ciencia, 25(1), 1-22. Retrieved from http://www.publicaciones.ujat. mx/publicaciones/uciencia/abril2009/1--340.pdf Instituto Nacional de Estadística Geografía e Informática (INEGI). (2009). Mapa digital de México. Retrie ved January 1, 2010, from http://gaia.inegi.org.mx/ mdm5/viewer.html Intergovernmental Panel on Climate Change (IPCC). (2013). The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. New York. Di Rienzo, J. A., Casanoves, F., Balzarini, M. G., Gonzalez, L., Tablada, M., & Robledo, C. W. (2008). InfoStat, versión 2008. Grupo InfoStat. Córdoba, Argentina: FCA, Universidad Nacional de Córdoba. Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1456 at multiple spatial scales. Landscape Ecology, 11(3), 141-156. http://doi.org/10.1007/BF02447513 Jones III, E. B. D., Helfman, G. S., Harper, J. O., & Bols tad, P. V. (1999). Effects of riparian forest removal on fish assemblages in southern Appalachian streams. Conservation Biology, 13(6), 1454-1465. Sánchez, A. J., Álvarez-Legorreta, T., Pacheco, J. G., Carri llo, L., & González, R. A. (2016). Calidad del agua subterránea: acuífero sur de Quintana Roo, México. Tecnología y Ciencias del Agua, 7(4), 75-96. Karr, J. R. (1981). Assessment of biotic integrity using fish communities. Fisheries, 6(6), 21-27. http://doi. org/10.1577/1548-8446(1981)006<0021:AOBIUF>2 .0.CO;2 Schmitter-Soto, J. J. (2014). Los índices bióticos de integridad en el monitoreo ambiental. In C. Gon zález-Zwarth, A. Vallarino, J. C. Pérez, & A. Low Pfeng (Eds.), Bioindicadores: guardianes de nues tro futuro ambiental (pp. 61-78). REFERENCES Mexico City: INECC, ECOSUR. Laurance, W. F., Fearnside, P. M., Laurance, S. G., Dela monica, P., Lovejoy, T. E., Rankin-De Merona, J. M., …, Gascon, C. (1999). Relationship between soils and Amazon forest biomass: a landscape-scale study. Forest Ecology and Management, 118(1-3), 127-138. http://doi.org/10.1016/S0378-1127(98)00494-0 Schmitter-Soto, J. J., & Caro, C. I. (1997). Distribution of tilapia, Oreochromis mossambicus (Perciformes: Cichlidae), and water body characteristics in Quin tana Roo, Mexico. Revista de Biología Tropical, 45(3), 1257-1261. Lorion, C. M., & Kennedy, B. P. (2009). Riparian forest buffers mitigate the effects of deforestation on fish assemblages in tropical headwater streams. Ecologi cal Applications, 19(2), 468-479. Schmitter-Soto, J. J., Comín, F. A., Escobar-Briones, E., Herrera-Silveira, J., Alcocer, J., Suárez-Morales, E., …, Steinich, B. (2002). Hydrogeochemical and biological characteristics of cenotes in the Yucatán Peninsula (SE Mexico). Hydrobiologia, 467, 215- 228. http://doi.org/10.1023/A:1014923217206 Lowry, R. (2016). VassarStats: website for statistical computation. Retrieved October 1, 2016, from http:// vassarstats.net/index.html Miranda, F. (1958). Estudios acerca de la vegetación. In E. Beltrán (Ed.), Los recursos naturales del sureste y su aprovechamiento (Vol. 2, pp. 215- 271). Mexico City: Instituto Mexicano de Recursos Naturales Renovables. Schmitter-Soto, J. J., Quintana, R., Valdéz-Moreno, M. E., Herrera-Pavón, R. L., & Esselman, P. C. (2015). Armoured catfish (Pterygoplichthys pardalis) in the Hondo River basin, Mexico-Belize. Mesoamericana, 19(3), 9-19. Naiman, R. J., Bechtold, S., Drake, D. C., Latterell, L. L., O’Keefe, T. C., & Balian, E. V. (2005). Orig ins, patterns, and importance of heterogeneity in riparian systems. In G. Lovett, C. G. Jones, M. G. Turner, & K. C. Weathers (Eds.), Ecosystem Function in Heterogeneous Landscapes (pp. 279- 309). New York: Springer. Schmitter-Soto, J. J., Ruiz-Cauich, L. E., Herrera-Pavón, R. L., & González-Solís, D. (2011). An Index of Bio tic Integrity for shallow streams of the Hondo River basin, Yucatán Peninsula. Science of the Total Envi ronment, 409(4), 844-852. http://doi.org/10.1016/j. scitotenv.2010.11.017 Omernik, J. M., Abernathy, A. R., & Male, L. M. (1981). Stream nutrient levels and proximity of agricultural and forest land to streams: some relationships. Jour nal of Soil and Water Conservation, 36(4), 227-231. Snyder, C. D., Young, J. A., Villella, R., & Lemarié, D. P. (2003). Influences of upland and riparian land use patterns on stream biotic integrity. Lands cape Ecology, 18, 647-664. http://doi.org/10.1023/ B:LAND.0000004178.41511.da Pacheco Díaz, R. I. (2011). Efectos del paisaje sobre un índice biótico de integridad en el río Hondo (M.Sc. Thesis). REFERENCES El Colegio de la Frontera Sur, Chetumal, Mexico. Strayer, D. L., Beighley, R. E., Thompson, L. C., Brooks, S., Nilsson, C., Pinay, G., & Naiman, R. J. (2003). Effects of land cover on stream ecosystems: Roles of empirical models and scaling issues. Ecosystems, 6(5), 407-423. http://doi.org/10.1007/s10021-002-0170-0 Pinto, B. T., Araújo, F. G. C., & Hughes, R. M. (2006). Effects of landscape and riparian condition on a fish index of biotic integrity in a large southeastern Brazil river. Hydrobiologia, 556(1), 69-83. http://doi. org/10.1007/s10750-005-9009-y van Oosterhout, M. P., & van der Velde, G. (2014). An advanced Index of Biotic Integrity for use in tropical shallow lowland streams in Costa Rica: Fish assemblages as indicators of stream ecosystem health. Ecological Indicators, 48, 687-698. http://doi. org/10.1016/j.ecolind.2014.09.029 Richards, C., & Host, G. E. (1994). Examining land influences on stream habitat and microinvertebrates: A GIS approach. Journal of the American Water Resources Association, 30(4), 729-738. van Sickle, J., Baker, A., Herlihy, P., Bayley, S., Gregory, P., Ashkenas, L., & Li, J. (2004). Projecting the biological condition of streams under alternative Roth, N. E., Allan, J. D., & Erickson, D. L. (1996). Lands cape influences on stream biotic integrity assessed Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1457 scenarios of human land use. Ecological Applica tions, 14(2), 368-380. rehabilitation of riparian systems in an agricultural drainage basin. Environmental Management, 22(3), 425-443. http://doi.org/10.1007/s002679900117 Walsh, C. J., Roy, A. H., Feminella, J. W., Cottingham, P. D., Groffman, P. M., & Morgan II, R. P. (2005). The urban stream syndrome: current knowledge and the search for a cure. Journal of the North American Benthological Society, 24(3), 706-723. http://doi. org/10.1899/04-028.1 Wilcox, D. A., Meeker, J. E., Hudson, P. L., Armita ge, B. J., Black, M. G., & Uzarski, D. G. (2002). Hydrologic variability and the application of Index of Biotic Integrity metrics to wetlands: A great lakes evaluation. Wetlands, 22(3), 588-615. http:// doi.org/10.1672/0277-5212(2002)022[0588:HVATA O]2.0.CO;2 Wang, L., Lyons, J. D., Kanehi, P., Bannerman, R., & Emmons, E. (2000). Watershed urbanization and changes in fish communities in southeastern Wis consin streams. Journal of the American Water Resources Association, 36(5), 1173-1189. http://doi. org/10.1111/j.1752-1688.2000.tb05719.x Yu, K., DeLaune, R. D., Tao, R., & Beine, R. L. (2008). Nonpoint source of nutrients and herbicides associa ted with sugarcane production and its impact on Loui siana coastal water quality. Journal of Environmental Quality, 37(6), 2275-2283. Wichert, G. A., & Rapport, D. REFERENCES J. (1998). Fish commu nity structure as a measure of degradation and Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 65 (4): 1448-1458, December 2017 1458
https://openalex.org/W2995012874	http://evu.encyclopedia.kyiv.ua/wp-content/uploads/2019/articles/do-pytannia-stvorennia-natsionalnoi-dovidkovoi-sluzhby-entsyklopedychno-osvitnoho-spriamuvannia.pdf	Ukrainian	null	On the modeling of a national encyclopedic-and-educational service	Enciklopedičnij vìsnik Ukraïni	2,019	cc-by	3,480	DOI: 10.37068/evu.11.3 DOI: 10.37068/evu.11.3 Енциклопедичний вісник України / The Encyclopedia Herald of Ukraine (2019) 11 © Інститут енциклопедичних досліджень НАН України / NASU Institute of Encyclopedic Research ISSN 2706-9990 (print) ISSN 2707-000X (online) у якій діє служба «Learndirect» («Навчання навпростець»). Ключові слова: енциклопедичне знання, енциклопедії, ціложиттєве на вчання, освіта для дорослих, «Навчання навпростець». On the modeling of a national encyclopedic-and-educational service Serhii Boltivets State Institute of Family and Youth Policy, Ministry of Youth and Sports of Ukraine, Kyiv, Ukraine Ключові слова: енциклопедичне знання, енциклопедії, ціложиттєве на вчання, освіта для дорослих, «Навчання навпростець». До питання створення національної довідкової служби енциклопедично-освітнього спрямування Сергій Болтівець Державний інститут сімейної та молодіжної політики Міністерства молоді та спорту України, Київ, Україна Резюме: Стаття висвітлює питання необхідності створення національної довідкової служби, що функціонувала б у вигляді безкоштовної телефонної лінії і, можливо, базувалася б на основі сучасних українських енциклопе дичних ресурсів. Поява такої служби обґрунтована у зв’язку з міжнародним соціальним рухом ціложиттєвого навчання (lifelong learning), яким в Україні опікується Всеукраїнське координаційне бюро Міжнародної громадсько- державної програми «Освіта дорослих України». Концепція ціложиттєвого навчання відстоює науково виважену ідею про здатність людини навчатися в будь-якому віці. Її визначено Радою Європи важливим компонентом євро пейської соціальної моделі. Відповідно, одним із завдань європейських дер жав є забезпечення прагнень громадян до розвитку, отримання ними освіти впродовж усього життя. Ідея створення національної довідкової служби в Україні ґрунтується на досвіді європейських країн, зокрема Великої Британії, у якій діє служба «Learndirect» («Навчання навпростець»). On the modeling of a national encyclopedic-and-educational service Serhii Boltivets State Institute of Family and Youth Policy, Ministry of Youth and Sports of Ukraine, Kyiv, Ukraine Serhii Boltivets State Institute of Family and Youth Policy, Ministry of Youth and Sports of Ukraine, Kyiv, Ukraine Сергій Болтівець Abstract: The article defends the necessity to establish a national help service titled Navchannia Navprostets (Learn Direct). It would be a not-for-profit telephone line based on modern encyclopedias of Ukraine. The creation of such a service is justified by the Lifelong Learning as an international social movement, which in Ukraine is supervised by the Ukrainian Coordination Bureau of the International Public-State Program Adult Education in Ukraine. The concept of lifelong learn ing is upheld by the scholarly explained idea of a human ability to learn at any age. It has been identified by the Council of Europe as an important component of the social model of Europe. Accordingly, one of the tasks of the European states is to ensure the citizens’ aspirations for development and to receive lifelong education. The idea of starting up the national help service in Ukraine is based on the experien ce of European countries, first of all, we mean a service in the UK (Learndirect). Keywords: encyclopedic knowledge, encyclopedias, lifelong learning, adult edu cation, Learndirect, International Adult Learners’ Week. Одним із завдань будь-якої державної (національної) енциклопедії є єднання дорослих громадян кожної країни у їхніх уявленнях про себе і світ. Енциклопедія – це найавторитетніше джерело знань про кожну краї ну, а тому вона належить до атрибутів державності так само, як гімн, герб і прапор. Вони вирізняють державу серед інших у світі, визначають її не повторність. Однак лише за енциклопедією можлива оцінка духовно-інте лектуальної і матеріальної вартості того, чим володіє і що здатна створити власними зусиллями кожна держава. 1999 року на мене поклали місію готувати й здійснити приєднання України як держави до всесвітнього руху країн-членів ООН у проведенні першого Міжнародного тижня освіти дорослих (International Adult Learners’ Week), що поєднав події, які відбуваються під час національних тижнів для можливості перейняти досвід інших країн у проведенні цього свята, сприя ти співробітництву організацій, що працюють в царині надання освіти до рослим громадянам. Офіційним початком Міжнародного тижня освіти до рослих стало засідання «Платформа для майбутнього» 8 вересня 2000 року в м. 1 Докладніше про Міжнародний тиждень освіти дорослих у Ганновері 2000 року див.: [Weil, Martinez]. On the modeling of a national encyclopedic-and-educational service Ганновері (Німеччина) під час Міжнародної виставки «Експо–2000», що була заключним засіданням дискусії «Побудова суспільства, яке на вчається – знання, інформація і людський розвиток» (Building Learning Societies – Knowledge, Information and Human Development) і водночас по чатком Міжнародного тижня освіти дорослих ООН і ЮНЕСКО1. Для відображення участі держави в проведенні першого національно го Тижня освіти дорослих в України мною був підготовлений і виданий у встановленому порядку наказ Міністерства освіти і науки України від 32 До питання створення національної довідкової . 08. 08. 2000 року № 370 «Про проведення Міжнародного тижня освіти дорослих». Цим наказом був затверджений склад організаційного коміте ту з проведення цієї події в додатку № 1, програма заходу в додатку № 2 і «Методичні рекомендації до програми проведення першого в Україні Міжнародного тижня освіти дорослих» у додатку № 3. 08. 08. 2000 року № 370 «Про проведення Міжнародного тижня освіти дорослих». Цим наказом був затверджений склад організаційного коміте ту з проведення цієї події в додатку № 1, програма заходу в додатку № 2 і «Методичні рекомендації до програми проведення першого в Україні Міжнародного тижня освіти дорослих» у додатку № 3. р р у у У підготовлених методичних рекомендаціях висвітлено зарубіжний досвід проведення національних тижнів навчання дорослих багатьох кра їн, та особливу увагу ми привернули до досвіду Великої Британії, яка і є ініціаторкою проведення тижнів навчання дорослих на роботі, починаючи від 1992 року. Так, у цій країні працює постійна телефонна лінія допомоги, яка має назву «Навчання навпростець» і діє за телефоном у Лондоні 080- 100-900 безкоштовно. Її основне завдання – надавати в конфіденційній формі інформацію і поради щодо освіти й працевлаштування. Досвідчені консультанти працюють весь рік від 9-ї до 20-ї години щодня з понеділка до п’ятниці та від 9-ї до 12-ї години щосуботи. Під час проведення тижня освіти дорослих лондонська лінія «Навчання навпростець» оснащуєть ся додатковими каналами й ресурсами для обробки збільшеної кількос ті запитів розширеної тематики. Для людей з вадами слуху забезпечують письмове спілкування за тим же безкоштовним номером. Кожній люди ні, якщо вона одержує допомогу як безробітна, надсилають інформацію про проведення тижня із запрошенням зателефонувати. Консультанти лі нії розшукають потрібні освітні курси і нададуть інші освітні послуги для будь-кого, хто зателефонував. On the modeling of a national encyclopedic-and-educational service У Великій Британії центральний організаційний комітет, що забезпе чує проведення національного тижня освіти дорослих, діє за такими на прямами: • забезпечення фінансування проекту; • здійснення планування; • пошук підтримки й залучення до участі в заході різноманітних професійних організацій, освітніх установ, державних і приватних підприємств; • розроблення маркетингової стратегії; • розроблення й виготовлення роздаткових матеріалів для організа цій-учасниць, зокрема інформаційних листів, плакатів, футболок, листівок тощо; • підтримка координаторів у різних місцевостях країни, забезпечення контактування між ними; • розвиток зв’язків із пресою на всіх рівнях; • розвиток комунікації зі всіма основними партнерами; • забезпечення політичної підтримки на всіх рівнях; 33 Сергій Болтівець • залучення нових організацій (музеї, супермаркети, в’язниці, спор тивні центри, бібліотеки) до співпраці у сфері навчання дорослих людей країни. • залучення нових організацій (музеї, супермаркети, в’язниці, спор тивні центри, бібліотеки) до співпраці у сфері навчання дорослих людей країни. • залучення нових організацій (музеї, супермаркети, в’язниці, спор тивні центри, бібліотеки) до співпраці у сфері навчання дорослих людей країни. Варто відзначити співмірність змістової спрямованості енциклопедич ного знання і міжнародних тижнів освіти дорослих ЮНЕСКО в Україні. Так, упродовж перших трьох років ці заходи мали універсальну спрямова ність, а, починаючи від 2003 року, почала виявлятись тематична спрямо ваність національних тижнів освіти дорослих (як і в інших країнах-членах ООН, які проводять відповідні заходи): 2003 рік: «Освіта дорослих для безпеки життя»; 2003 рік: «Освіта дорослих для безпеки життя»; 2004 рік: «Освіта дорослих для демократії»; 2005 рік: «Освіта дорослих для свободи совісті і віри»; 2006 рік: «Освіта дорослих для збереження роду, землі і води»; 2007 рік: у формі освітньої магістралі єднання Півночі й Півдн України під гаслом «Освіта дорослих для культури ціложиттєвог навчання»; 2004 рік: «Освіта дорослих для демократії»; 2005 рік: «Освіта дорослих для свободи совісті і віри»; 2006 рік: «Освіта дорослих для збереження роду, землі і води»; 2007 рік: у формі освітньої магістралі єднання Півночі й Півдня України під гаслом «Освіта дорослих для культури ціложиттєвого навчання»; 2008 рік: «Освіта дорослих для безпеки громадян і організацій об’єд наної Європи» за участі офіційних делегацій Республіки Грузія, Рес публіки Азербайджан, Республіки Вірменія, Республіки Польща, Ро сійської Федерації , Республіки Словаччина, Республіки Чехія у зв’язку з військовим нападом РФ на Республіку Грузія; 2009 рік: «Освіта дорослих для сучасної родини»; 2010 рік: «Освіта дорослих для розуміння й соціального партнер ства»; 2011 рік: «Ноосфера для освіти зрілої людини»; 2012 рік: «Освіта дорослих єднає всі покоління»; 2013 рік: «Життя є освіта, а теорія освіти є, власне, теорією життя. On the modeling of a national encyclopedic-and-educational service Тарас Шевченко»; 2014 рік: «Освіта татового життя»; 2015 рік: «Українська мова – це найперший вчинок свідомої культури порозуміння дорослих людей в Україні»; 2016 рік: «Татусь – мій навчитель»; 2016 рік: «Татусь – мій навчитель»; 2017 рік: «Реабілітаційна Україна для життя, здоров’я й безпеки в Європі»; 2018 рік: «Життя є освіта. Тарас Шевченко». 2018 рік: «Життя є освіта. Тарас Шевченко». Міжнародні тижні освіти дорослих в Україні втілюють центральну місію ЮНЕСКО – сприяти здійсненню права кожної людини на освіту протягом усього життя, інтеграції ціложиттєвого навчання і життя. Місія сучасних Міжнародні тижні освіти дорослих в Україні втілюють центральну місію ЮНЕСКО – сприяти здійсненню права кожної людини на освіту протягом усього життя, інтеграції ціложиттєвого навчання і життя. Місія сучасних 34 До питання створення національної довідкової енциклопедичних проектів, таких, наприклад, як «Енциклопедія Сучасної України» [Енциклопедія Сучасної...], можна сказати, є аналогічною. енциклопедичних проектів, таких, наприклад, як «Енциклопедія Сучасної України» [Енциклопедія Сучасної...], можна сказати, є аналогічною. р у Метою Українського тижня, як і національних тижнів згаданих країн, є інтеграція впродовж життя людини освіти в якнайширших рутинних кон текстах, таких як родина і суспільство, освіта, робота і дозвілля, заохочу вання дорослих, зрілих, літніх і старших людей до продовження власного навчання для якнайповнішої реалізації свого таланту і розкриття різнома нітних можливостей. Особлива увага суспільства в ході проведення цих за ходів привертається до реалізації таланту дорослих людей з обмеженими фізичними можливостями, розширення знань в царині культури здоров’я і безпеки життя, спільного освітнього зростання кількох поколінь – дідусів і бабусь, тат і мам, синів і дочок. У справі життєздатного об’єднання людства на основі ціннісних під став і цільових орієнтирів пріоритетом є ціложиттєва освіта (longlife learning) кожної людини. Ціложиттєвість виступає в сучасному культур но-освітньому контексті як ідея, принцип навчання, якість освітнього про цесу, умова становлення людини. Рада Європи визначила ціложиттєве навчання одним з основних компонентів європейської соціальної моделі. Таке навчання не обмежується лише сферою освіти. Воно є також критич ним чинником у сферах зайнятості й соціального забезпечення, економіч ного зростання і конкурентоспроможності. 2 Докладніше про «Learndirect» див.: [Learndirect...]. On the modeling of a national encyclopedic-and-educational service Для України вкрай важливо розвивати індивідуальні практики ціло життєвого навчання своїх громадян, відновлювати предковічні традиції народного саморуху до власної досконалості, загальносуспільного руху су цільної грамотності Козацької Доби та руху «Просвіт» 19–20 століть, зав дяки якому український народ після найважчих втрат відновлював свою життєздатність, сяйво культурної поваги і розквіт усіх наук, мистецтв, власного господарства і промисловості, багаторазово множив число тяму щих і здібних, талановитих і геніальних людей, чиї здобутки представлені в духовно-інтелектуальному піднесенні як власної країни, так і більшості інших країн світу. Протягом останнього двадцятиліття підготовки та проведення два дцяти тижнів для освіти дорослих у різних варіаціях обговорюється пи тання запровадження в Україні згаданої британської моделі «Навчання навпростець» («Learndirect»2). Розмова щодо цього з керівництвом од нієї з найбільших компаній мобільного зв’язку «Київстар» завершилася безрезультатно, хоча її президент Ігор Литовченко стверджує про те, що «Київстар» – соціально відповідальна компанія, дбає про те, аби техноло гії, які стають частиною життя українців, забезпечували якнайбільше ко ристі українським сім’ям і суспільству [Литовченко, с. 5]. 35 Сергій Болтівець Звернення з цього питання до публічного акціонерного товариства «Укртелеком», яке «надає повний спектр телекомунікаційних послуг в усіх регіонах країни ... і є лідером ринку швидкісного фіксованого доступу до мережі Інтернет та займає провідні позиції в галузі фіксованої телефо нії» [Соціальна відповідальність...], також не увінчалося успіхом. Яким же може бути раціональне використання інформаційних баз на зразок «Енциклопедії Сучасної України», крім їхнього прямого при значення: служити неупередженим джерелом об’єктивних знань? Яким, окрім іншого, має бути соціальний ефект від енциклопедичного знання? Досягнення соціального ефекту, як і його політичного, економічного, вій ськового та інших вимірів, можливе лише шляхом ширення енциклопе дичних знань, наприклад, через національну довідкову службу, про ство рення якої йдеться у цій статті. На наш погляд, національне значення індивідуально важливих знань кожного громадянина України незалежно від віку та інших відмінностей є справою загальнонаціональної ваги, а отже, спеціальним законом мало би бути втілено у відкритті безкоштовної телефонної лінії, яка б доповни ла наявні в Україні служби екстреної допомоги (101 – службу порятунку Державної служби України з надзвичайних ситуацій, 102 – поліцію, 103 – швидку допомогу, 104 – аварійну службу газу, 109 – довідкову службу) національною довідковою службою «Навчання навпростець». У перспек тиві всі названі номери екстреної допомоги населенню, крім довідкової, будуть об’єднані в єдиний телефонний номер 112, а тому є ймовірність створення єдиного номера соціальної допомоги, який доповнює довідкову службу соціально важливими для громадян України енциклопедичними знаннями. On the modeling of a national encyclopedic-and-educational service Основними засадами національної енциклопедичної довідкової служ би «Навчання навпростець» (далі НЕДС НН) вбачаємо такі: 1. На базі певних енциклопедичних ресурсів Всеукраїнське коорди наційне бюро Міжнародної громадсько-державної програми «Освіта до рослих України», що діє за сприяння Інституту ціложиттєвого навчання ЮНЕСКО, може виконувати індивідуальні запити мільйонів українців на енциклопедичне знання. 2. На відміну від найпоширенішої електронної форми спілкування, те лефонна лінія НЕДС НН є прямим спілкуванням з різними категоріями населення, які цього потребують, що має значний психогігієнічний вимір попередження ризикованої і небезпечної поведінки, включаючи суїци дальні наміри додзвонювачів. НЕДС НН в цьому вимірі є психогігієніч ною службою країни, а, отже, потужним засобом її суспільної психогігієни для збереження людських життів. р 3. Енциклопедичне знання, як і будь-яке знання, буття якого мож ливе лише у формі актуального мислення людини, існує в ненастанному 3. Енциклопедичне знання, як і будь-яке знання, буття якого мож ливе лише у формі актуального мислення людини, існує в ненастанному 36 До питання створення національної довідкової . русі, збагачуючись у своєму загальносуспільному обігу. Виходячи з цієї методологеми, НЕДС НН є засобом нагромадження, уточнення та вдо сконалення енциклопедичного знання, у неймовірно значних наслідках чого ми невдовзі зможемо переконатись, адже усне спілкування з міль йонами громадян формуватиме нові, допоки невідомі виміри пізнаваль них зацікавлень нації, відтак – виміри її актуального інтелектуального розвитку. р у 4. Організаційне функціонування НЕДС НН – це чергування консуль тантів лінії з метою прийому індивідуальних заявок на загальновживане й рідкісне знання, аналіз та узагальнення яких є створенням пізнавального запиту нації в його індивідуальному різноманітті. 4. Організаційне функціонування НЕДС НН – це чергування консуль тантів лінії з метою прийому індивідуальних заявок на загальновживане й рідкісне знання, аналіз та узагальнення яких є створенням пізнавального запиту нації в його індивідуальному різноманітті. у у у р 5. Особливою цінністю НЕДС НН є створювані нею обриси пізна вального запиту української нації на неіснуюче знання, яке позначаєть ся загальновживаним терміном «наука» без усвідомлення того, що наука є суміжжям між знанням і незнанням. Цим НЕДС НН формує запит на перспективні дослідження в Україні та їх пріоритетні напрями. 5. Особливою цінністю НЕДС НН є створювані нею обриси пізна вального запиту української нації на неіснуюче знання, яке позначаєть ся загальновживаним терміном «наука» без усвідомлення того, що наука є суміжжям між знанням і незнанням. Цим НЕДС НН формує запит на перспективні дослідження в Україні та їх пріоритетні напрями. 6. On the modeling of a national encyclopedic-and-educational service Категорія дорослих, віднесених до цієї основної бази користувачів – додзвонювачів, запитувачів НЕДС НН – має включати також: • безробітних і членів їхніх сімей; • переселенців і членів їхніх сімей; • пенсіонерів; • пенсіонерів; • молодь у віці до 35 років; • мам і татусів у відпустках для догляду за дитиною до досягнення 3-річного віку; • мам і татусів у відпустках для догляду за дитиною до досягнення 3-річного віку; • учасників і демобілізованих учасників бойових дій; • звільнених з місць позбавлення волі до працевлаштування за осно вним місцем роботи. • звільнених з місць позбавлення волі до працевлаштування за осно вним місцем роботи. Решта дорослих, не віднесених до цієї основної бази користувачів – до дзвонювачів, запитувачів НЕДС НН, може стати платниками телефонних запитів так само, як платною є довідкова служба телефонів. Разом з цим з огляду на необхідність культивування соціальної відповідальності бізнесу держава має бути взірцем власної соціальної відповідальності перед своїми громадянами. Це означає, що пізнавальні запити усіх зацікавлених укра їнців, які звертаються до НЕДС НН доцільно фінансувати з Державного бюджету України в однаковій мірі. Запровадження в нашій державі досвіду телефонної служби Великої Британії «Навчання навпростець» у формі національної енциклопедичної довідкової служби розглядаємо взірцем соціальної відповідальності дер жави і бізнесу перед усіма своїми громадянами. Сучасні енциклопедич ні ресурси України є певною сформованою базою достовірних знань для усіх громадян України незалежно від їхнього віку та інших індивідуальних особливостей. Саме вони можуть бути підґрунтям для створення в Україні своєї національної служби «Навчання навпростець». On the modeling of a national encyclopedic-and-educational service Статті сучасних фахових енциклопедій створюють кваліфікова ні автори, які самим фактом їх написання засвідчують свою компетент ність у відповідних питаннях і проблемах. Логічно, що використання «Енциклопедії Сучасної України» та низки галузевих, регіональних ен циклопедій, виданих як академічними, так і позаакадемічними колекти вами, є джерелом пошуку відповідей на запитання й запити, що надхо дитимуть на телефонну лінію НЕДС НН. Поряд із цими джерелами, що вже набули статусу загальновживаного знання, виникатимуть суміжні за питання, проблеми і пізнавальні запити на знання, яке ми вже визначили як рідкісне. В усіх цих випадках для надання обґрунтованої відповіді на запити будуть залучатися відповідні фахівці, які володіють цими рідкіс ними знаннями або можуть їх здобути в терміни, прийнятні для відповіді додзвонювачам, наприклад, у триденний, тижневий, десятиденний або місячний строк. 7. Виконання пізнавальних запитів, що полягає у підготовці письмо вих відповідей як матеріалів для майбутніх енциклопедичних статей або уточнень, доповнень до вже наявних, та усному повідомленню їх запиту вачам (додзвонювачам) є оплачуваною роботою великої кількості залуче них фахівців. 7. Виконання пізнавальних запитів, що полягає у підготовці письмо вих відповідей як матеріалів для майбутніх енциклопедичних статей або уточнень, доповнень до вже наявних, та усному повідомленню їх запиту вачам (додзвонювачам) є оплачуваною роботою великої кількості залуче них фахівців. ф 8. Основною базою користувачів – додзвонювачів, запитувачів НЕДС НН, запити яких виконуються за кошти Державного бюджету України, є інваліди всіх нозологій та члени їхніх сімей, загальна чисель ність яких нині в Україні становить близько 6 мільйонів осіб і продовжує зростати у зв’язку із вторгненням збройних сил Російської Федерації на територію України, зубожінням більшості населення, недоступності з ба 8. Основною базою користувачів – додзвонювачів, запитувачів НЕДС НН, запити яких виконуються за кошти Державного бюджету України, є інваліди всіх нозологій та члени їхніх сімей, загальна чисель ність яких нині в Україні становить близько 6 мільйонів осіб і продовжує зростати у зв’язку із вторгненням збройних сил Російської Федерації на територію України, зубожінням більшості населення, недоступності з ба 37 Сергій Болтівець гатьох об’єктивних і суб’єктивних причин закладів загальної середньої, вищої, професійної та інших видів освіти, культури, охорони здоров’я, соціального захисту для повносправних дорослих та дорослих інвалідів і членів їхніх сімей. гатьох об’єктивних і суб’єктивних причин закладів загальної середньої, вищої, професійної та інших видів освіти, культури, охорони здоров’я, соціального захисту для повносправних дорослих та дорослих інвалідів і членів їхніх сімей. ЛІТЕРАТУРА Енциклопедія Сучасної України: Т. 1–21 / Редкол.: І. М. Дзюба, М. Г. Же лезняк та ін.; Інститут енциклопедичних досліджень НАН Украї ни. Київ, 2001–2019. Енциклопедія Сучасної України: Т. 1–21 / Редкол.: І. М. Дзюба, М. Г. Же лезняк та ін.; Інститут енциклопедичних досліджень НАН Украї ни. Київ, 2001–2019. Енциклопедія Сучасної України: Т. 1–21 / Редкол.: І. М. Дзюба, М. Г. Же лезняк та ін.; Інститут енциклопедичних досліджень НАН Украї ни. Київ, 2001–2019. Литовченко І. Вступне слово. Діти в Інтернеті: як навчити безпеці у віртуальному світі: посібник для батьків / Інститут психології ім. Г. С. Костюка НАПН України. Київ, 2010. Литовченко І. Вступне слово. Діти в Інтернеті: як навчити безпеці у віртуальному світі: посібник для батьків / Інститут психології ім. Г. С. Костюка НАПН України. Київ, 2010. 38 До питання створення національної довідкової .. Соціальна відповідальність компанії «Укртелеком». Укртелеком: офі ційний веб-сайт. URL: https://new.ukrtelecom.ua/about/sotsIalna_ vIdpovIdalnIst (дата звернення: 02.12.2019). Learndirect: Official website. 2000. URL: https://www.learndirect.com (дата звернення: 02.12.2019). Weil M., Martinez F. The Learning Festivals Guide. An Internationally- Produced Communication Tool In Support of the Launch of the International Adult Learners / Institute of Education Sciences of the US Department of Education, 2010. URL: https://www.academia. edu/391643 (дата звернення: 02.12.2019). REFERENCES Dziuba, I. M., Zhelezniak, M. H. et al. (Eds.). (2001-2019). Entsyklopediia Suchasnoi Ukrainy [The Encyclopedia of Modern Ukraine] (21 vols.). Kyiv: NASU Institute of Encyclopedic Research [in Ukrainian]. Learndirect: Official website. (2000). Retrieved December 02, 2019, from https://www.learndirect.com. Lytovchenko I. (2010). Vstupne slovo [Foreword]. In Dity v Interneti: yak navchyty bezpetsi u virtualnomu sviti: posibnyk dlia batkiv [Kids on the Internet: How to teach safety in the virtual world: A guide for parents]. Kyiv: NAPSU H.S. Kostiuk Institute of Psychology [in Ukrainian]. Martinez, F. & Weil, M. (2010). The Learning Festivals Guide. An Inter nationally-Produced Communication Tool in Support of the Launch of the International Adult Learners. Institute of Education Sciences of the US Department of Education. Retrieved December 02, 2019, from https://www.academia.edu/391643. Sotsialna vidpovidalnist kompanii "Ukrtelekom" [Social responsibility of Ukrtelecom JSC] (n. d.). Ukrtelekom: Official website. Retrieved December 02, 2019, from https://new.ukrtelecom.ua/about/sotsI alna_vIdpovIdalnIst [in Ukrainian]. 39
https://openalex.org/W2132688806	https://www.frontiersin.org/articles/10.3389/fphy.2014.00080/pdf	English	null	Simulation of main plasma parameters of a cylindrical asymmetric capacitively coupled plasma micro-thruster using computational fluid dynamics	Frontiers in physics	2,015	cc-by	7,905	1. INTRODUCTION plasma jet. Unlike larger diameter lower pressure plasma devices, such as the Helicon Double Layer Thruster (HDLT), that produce thrust through ion accerelation in an expanding plasma [11, 12], these smaller plasma systems rely on the neutral gas to produce the thrust, hence increasing the momentum of the neutral gas increases thrust. An example of a thruster designed on this prin- ciple is an asymmetric hollow cathode glow discharge, estimated to produce 1 mN of thrust for a 1 kW power input [7]. The development and use of micro-satellites, such as CubeSat’s, has created a need for new micro-propulsion systems that are small, lightweight and low power [1]. The thrust produced needs to be of the order of a few μN to mN, with high propellant efﬁciency to reduce the need for excessive propellant to be carried on board. Currently, cold gas thrusters are widely used as micro-propulsion devices, where an inert gas is expelled through a nozzle to produce thrust. However, as there is no gas heating within the system the thrust produced per kilogram of propellant is low. Heating the gas through electric or chemical means increases the propellant efﬁciency or speciﬁc impulse (Isp) of the system. Electric heating, used in electric propulsion devices, has advantages over traditional chemical propulsion, due to a higher speciﬁc impulse (Isp) [2] and the ability to operate with green propellants, reducing the dangers associated with toxic propellants [3]. Another thruster based on the concept of neutral gas heating from ion-neutral charge exchange collisions, currently under development at The Australian National University, is an elec- trothermal radio-frequency (rf) capacitively coupled plasma (CCP) discharge device, known as “Pocket Rocket” [13, 14]. Recent rovibrational spectroscopy experiments have conﬁrmed a neutral gas temperature around 1100 K in argon at 10 W for pressures between 0.5 Torr and 4.0 Torr [15] with heating occuring from collisions within the plasma volume and surface heating from ion bombardment [16]. Argon gas is the standard operating propellant and is used in this work. Nitrogen gas (N2) is occasionally used as the operating propellant for experimental spectroscopy diagnostics, however due to the higher number of internal degrees of freedom in a diatomic nitrogen molecule com- pared with atomic argon, the neutral gas temperature achieved is lower (∼430 K for 10 W power input 15), hence the effectiveness as a thruster is reduced. ORIGINAL RESEARCH ARTICLE bli h d 06 J 2015 ORIGINAL RESEARCH ARTICLE bli h d 06 J 2015 PHYSICS published: 06 January 2015 doi: 10.3389/fphy.2014.00080 Amelia Greig , Christine Charles* and Roderick W. Boswell Space Plasma, Power and Propulsion Laboratory, Research School of Physics and Engineering, The Australian National Univers opulsion Laboratory, Research School of Physics and Engineering, The Australian National University, Canberra, ACT, Australia Space Plasma, Power and Propulsion Laboratory, Research School of Physics and Engineerin Computational ﬂuid dynamics (CFD) simulations of a radio-frequency (13.56 MHz) electrothermal capacitively coupled plasma (CCP) micro-thruster have been performed using the commercial CFD-ACE+ package. Standard operating conditions of a 10 W, 1.5 Torr argon discharge were used to compare with previously obtained experimental results for validation. Results show that the driving force behind plasma production within the thruster is ion-induced secondary electrons ejected from the surface of the discharge tube, accelerated through the sheath to electron temperatures up to 33.5 eV. The secondary electron coefﬁcient was varied to determine the effect on the discharge, with results showing that full breakdown of the discharge did not occur for coefﬁcients less than or equal to 0.01. Edited by: Edited by: Jean-Pierre Boeuf, University of Toulouse CNRS, France Reviewed by: Gerjan Hagelaar, University of Toulouse CNRS, France Emilie Despiau-Pujo, LTM - University Grenoble Alpes/CNRS/CEA-Leti Minatec, France Jean-Pierre Boeuf, University of Toulouse CNRS, France Jean-Pierre Boeuf, University of Toulouse CNRS, France Correspondence: Correspondence: Christine Charles, Space Plasma, Power and Propulsion Laboratory, Research School of Physics and Engineering, The Australian National University, Building 60, Mills Road, Canberra, ACT 0200, Australia e-mail: christine.charles@anu.edu.au Keywords: plasma thruster, computational ﬂuid dynamics, radiofrequency plasmas, micro-discharge, electric propulsion 1. INTRODUCTION h d l Simulation of main plasma parameters of a cylindrical asymmetric capacitively coupled plasma micro-thruster using computational ﬂuid dynamics Simulation of main plasma parameters of a cylindrical asymmetric capacitively coupled plasma micro-thruster using computational ﬂuid dynamics Amelia Greig , Christine Charles and Roderick W. Boswell Space Plasma, Power and Propulsion Laboratory, Research School of Physics and Engineering, The Australian National University, Canberra, ACT, Australia Amelia Greig , Christine Charles* and Roderick W. Boswell Space Plasma, Power and Propulsion Laboratory, Research School of Physics and Engineering, The Australian National University, Canberra, ACT, Australia 1. INTRODUCTION Heavier noble elements such as krypton or xenon are also suitable propellant gases although more expensive. Current electric micro-thruster concepts in use or develop- ment include resistojets, arcjets, hollow cathode thrusters and a radio-frequency (rf) capillary discharge [1, 4–7]. Small diam- eter plasma discharges with high operating pressures are also being investigated for use as micro-thrusters, requiring low power densities to produce high density collisional plasmas in small vol- umes. Within a collisional plasma ion-neutral charge exchange collisions accelerate the background neutrals in a process termed “neutral pumping” [8–10], resulting in neutral gas heating and an increase in momentum of the neutral gas component of the January 2015 \| Volume 2 \| Article 80 \| 1 www.frontiersin.org www.frontiersin.org Simulation of a plasma micro-thruster Greig et al. FIGURE 1 \| The physical Pocket Rocket device. The physical Pocket Rocket device currently consists of a 18 mm long, 4.2 mm inside diameter alumina tube, with a pow- ered copper electrode placed at the midpoint and two grounded aluminum electrodes placed at either end of the tube, as shown in Figure 1. Operational rf power (13.56 MHz) in the order of a few tens of Watts is coupled to the CCP discharge within the tube. The use of rf power introduces some technical chal- lenges in regards to power system size, such as miniaturizing the matching network, which are currently being overcome [17]. The discharge gas is held at pressures around a few Torr in an upstream plenum chamber with grounded aluminum walls before pass- ing through the tube. Downstream, a glass expansion tube with 50 mm diameter connects the device to a vacuum chamber ﬁtted with a rotary pump, creating pressures in the vacuum chamber approximately two times lower than in the plenum chamber. The end plate of the device connecting to the glass tube (thick black line at start of expansion tube in Figure 1) is also grounded alu- minum, creating a large asymmetry between the powered and grounded electrode areas resulting in a self-bias developing on the alumina (dielectric) tube. The plasma discharge produced has an ionization degree around 0.44% [15] at 1.5 Torr and 10 W, giving peak plasma densities in the center of the tube around 2 × 1012 cm−3 [14]. FIGURE 1 \| The physical Pocket Rocket device. 1. INTRODUCTION The governing equations for the heavy particle ﬂow parameters are mass conservation and the Navier-Stokes equation, given in Equations 1 and 2 respectively, where ρ is density, ⃗V is velocity, μ is viscosity (taken as constant 2.09 × 10−5 kgm−3 for argon) and SMx represents momentum added from any deﬁned source, such as the inlet. The pressure, p, is the sum of the static partial pressures for all heavy species, calculated from the ideal gas law and the electron pressure (pe), calculated as pe = nekTe, where ne is electron density, k is Boltzmann’s constant and Te is the electron temperature. Electric probe measurements of plasma parameters in Pocket Rocket are difﬁcult as the diameter of the discharge tube is not much larger than the diameter of the probe tip. Optical measure- ments are useful as they are non-invasive and measurements of parameters such as radical concentrations [18], electron density proﬁles [14] and gas temperature [15, 19] can be made. Computer based modeling such as particle-in-cell (PIC) and computational ﬂuid dynamics (CFD) simulations provide alternate methods to experiments for analyzing plasma based discharges. Fully kinetic PIC simulations of rf CCP discharges have previously been performed for one [20–23], two [24, 25] and three [26] spatial dimensions. However, higher dimension PIC simulations become very computationally expensive. Computational ﬂuid dynamics (CFD) simulations provide a ﬂuid model alterna- tive to PIC simulations, where ﬂuid based equations are solved over a gridded geometry using numerical methods. Successful CFD simulations utilizing commercial packages, such as CFD- ACE+ (Computational Fluid Dynamics Advaced Computing Environment), have been performed for weakly ionized and dual frequency CCPs [27–29]. Here the CFD-ACE+ package is used for a 2D ﬂuid based simulation giving the main plasma parame- ters (electron density and temperature, plasma potential, DC bias, etc.) of the Pocket Rocket device, for comparison with previously obtained experimental results. 1. INTRODUCTION δρ δt + ∇.(ρ ⃗V) = 0 (1) δ(ρ ⃗V) δt + ∇.(ρ ⃗Vu) = −∇.p + ∇.(μ∇u) + SM (2) (1) δ(ρ ⃗V) δt + ∇.(ρ ⃗Vu) = −∇.p + ∇.(μ∇u) + SM (2) (2) A transport equation of the form shown in Equation 3 is solved for each species including ions, where Yk is the species mass frac- tion, deﬁned as the mass of the kth species per mixture unit mass, Jk is diffusion ﬂux, Mk is the molecular weight of the species, and ωk is the production rate of the kth species from reactions. δρYk δt + ∇.ρ ⃗VYk = −∇.⃗Jk + Mkωk (3) (3) For ions, the effect of ion drift must also be considered, with ion mass ﬂux ⃗Ji given by Equation 4. 2. SIMULATION MODEL 2. SIMULATION MODEL The CFD-ACE+ package contains different modules that can be selected to analyze different problem types. This rf CCP simula- tion utilizes the ﬂow, turbulence, electric, chemistry and plasma modules and the following is a brief description of the main equa- tions and theory used by the CFD-ACE+ program. A transient simulation is used to allow the rf period behavior of the discharge to be analyzed, as well as the steady state (rf phase averaged) solution. ⃗Ji = −ρDi∇Yi + ρ ⃗UdiYi + ⃗Jc i (4) (4) Here, Di is the diffusion coefﬁcient, ⃗Udi is ion drift velocity rel- ative to ⃗V, and ⃗Jc i is a correction/source term to account for ion creation and loss and ensure species ﬂux conservation holds. The diffusion coefﬁcient (Di) is taken to be the same as the corre- sponding neutral particle, with ion mobility (μi) then solved for Here, Di is the diffusion coefﬁcient, ⃗Udi is ion drift velocity rel- ative to ⃗V, and ⃗Jc i is a correction/source term to account for ion creation and loss and ensure species ﬂux conservation holds. The diffusion coefﬁcient (Di) is taken to be the same as the corre- sponding neutral particle, with ion mobility (μi) then solved for January 2015 \| Volume 2 \| Article 80 \| 2 January 2015 \| Volume 2 \| Article 80 \| 2 Frontiers in Physics \| Plasma Physics Simulation of a plasma micro-thruster Greig et al. where me is the mass of an electron, ke is kinetic energy, νm is electron-ion collision frequency and C is the Maxwellian dis- tribution scaling factor. The electrons are assumed to follow a single temperature Maxwellian distribution due to high collision frequencies within the high pressure discharge, and the electron temperature (Te) is calculated using the electron energy balance equation using Einstein’s relation, μi = Di Ti , where ion temperature (Ti) is approximated as the neutral gas temperature. As the pressures used in Pocket Rocket are around a few Torr, it is assumed the collision frequency of ions is sufﬁciently high to use the drift dif- fusion approximation for ion drift velocity ( ⃗Udi = qiμi⃗E, where qi is the charge of the ion and ⃗E the electric ﬁeld), rather than solving directly for ion momentum. 2. SIMULATION MODEL However, higher operating pressures and self bias that develops on the alumina tube in Pocket Rocket from the large asymmetry of the device means stochastic heating effects are small and the majority of electron heating comes from Joule heating. Heating of the neutrals from ions (through charge exchange or momentum transfer collisions for example) is not included in this simulation, with neutral gas temperature assumed to be a constant 300 K throughout the model. where ϵ is the permittivity of the local medium (taken as 1 for argon and 9.3 for alumina), e is electron charge, qi is the charge on the ith ion and ne and ni are the electron and ion densities, respectively. Particle ﬂuxes are used to calculate space charge (σ) accumulation on the surface of the alumina (dielectric) as shown in Equation 6, where i,n is the ﬂux of ions normal to the surface. δσ δt = e i (qii,n −e,n) (6) (6) To ensure both heavy particles and electron kinetics are cor- rectly captured by the simulation, a dual time-step approach was implemented. The heavy particle parameters were solved using a time step of 7.382 μs, longer than the period of one rf cycle (∼72 ns), as the larger masses do not move sufﬁciently within an rf period to affect the results. Electrons, being much lighter, will exhibit sufﬁcient movement within an rf cycle that must be resolved, therefore electron kinetics were solved using a time scale of 1 20th of an rf period. Electron ﬂux normal to the wall surface (e,n) is given by Equation 7 where ve,th = ( 8kT πme ) 1 2 is the thermal electron velocity and γ is the secondary electron emission coefﬁcient. e,n = 1 4neve,th −γ i (qii,n) (7) (7) 20 As the Pocket Rocket device is cylindrically symmetric, the CFD model used was the top half of an axial 2D cross sec- tion, with an axisymmetry condition applied around the cen- tral axis of the discharge tube. The plenum chamber, discharge tube, alumina tube with electrode surfaces and glass expansion tube were included in the simulation. The mesh was reﬁned to be smaller in regions of interest, such as the discharge tube and near any walls expected to produce sheaths, as shown in Figure 2. 2. SIMULATION MODEL A standard k −ϵ turbulence model is included for all heavy species (refer to Launder and Spaulding [30] for full details of this model), however, the low pressure in the system results in little difference (∼2.4%) between fully laminar and turbulent simu- lations. Heavy particle surface reactions are invoked, such that on collision with a wall an ion or excited neutral will return to the neutral ground state. Four volumetric gas phase reactions are used in the simulation, being elastic collisions between heavy par- ticles and electrons, electron impact ionization, electron impact excitation and stepwise ionization, with rate coefﬁcients and cross sectional data from the JILA database, University of Colorado, as included in the CFD-ACE+ package. 3 2 δ δt (neTe) + ∇(5 2Te ⃗e −χ∇Te) = P −ne r nrcrkr (11) (11) where χ = 5 2neDe is the coefﬁcient of electron conduction. Energy loss from collisions is equated by the sum term with nr, cr and kr being the density, coefﬁcient of collisional energy loss and kinetic energy of the collisional species, respectively. Power density (P) includes stochastic sheath heating and Joule heating. Joule heating is deﬁned as PJoule = ee.(∇φ). Stochastic heating is considered only in the sheath region near the powered electrode and is given by Equation 12 [31], where χ = 5 2neDe is the coefﬁcient of electron conduction. Energy loss from collisions is equated by the sum term with nr, cr and kr being the density, coefﬁcient of collisional energy loss and kinetic energy of the collisional species, respectively. Power density (P) includes stochastic sheath heating and Joule heating. Joule heating is deﬁned as PJoule = ee.(∇φ). Stochastic heating is considered only in the sheath region near the powered electrode and is given by Equation 12 [31], The applied rf ﬁeld and space charge effects from ions and elec- trons, are solved using Poisson’s equation (Equation 5) for the electrostatic potential (φ) Pstoch = 0.61(me e ) 1 2 ϵ0ω2T 1 2e V (12) (12) −∇.ϵ∇φ = e( i qini −ne) (5) (5) where ω is the angular driving frequency at the rf electrode and V is the potential difference between the rf electrode and current grid point. 2. SIMULATION MODEL FIGURE 4 \| Two dimensional, time averaged CFD simulation results for (A) neutral gas density and (B) plasma density within the discharge tube. The full 2D plot has been cropped to show only the region of interest (discharge tube) for ease of viewing. see Figure 2) was set to 0.75 Torr giving a pressure gradient of FIGURE 4 \| Two dimensional, time averaged CFD simulation results for (A) neutral gas density and (B) plasma density within the discharge tube. The full 2D plot has been cropped to show only the region of interest (discharge tube) for ease of viewing. FIGURE 4 \| Two dimensional, time averaged CFD simulation results for (A) neutral gas density and (B) plasma density within the discharge tube. The full 2D plot has been cropped to show only the region of interest (discharge tube) for ease of viewing. FIGURE 3 \| CFD simulation results for a 1D parallel plate CCP over three rf periods showing (A) phase resolved electrode voltage (blue solid line), phase resolved plasma potential at the central point (x = 10 mm) (red dashed line) and phase averaged plasma potential at the central point (x = 10 mm) (black dotted line) and (B) phase averaged ion (solid line) and electron (dashed line) density. see Figure 2) was set to 0.75 Torr giving a pressure gradient of two between the model inlet and outlet, to match experiments and ensure the downstream component of the simulation (within the expansion tube) remained within the ﬂuid regime. maintaining a high degree of accuracy, the 4756 cell mesh was used. Before commencing the more complicated 2D Pocket Rocket simulations, a simple 1D parallel plate CCP was modeled using the same parameters as a previous study performed with the benchmarked 1D PHOENIX PIC code from Laﬂeur et al. [20], to ensure CFD-ACE+ correctly handles the basic CCP features, such as sheaths. An argon CCP discharge at 100 mTorr cre- ated by symmetric electodes placed 20 mm apart, one grounded and one carrying an rf (13.56 MHz) voltage with maximum A 13.56 MHz sinusoidal voltage with maximum amplitude Vrf = 240 V was applied to the powered electrode, which cor- responds to 10 W power input from previous experiments [14]. The alumina tube was speciﬁed as a dielectric with an initial value of 0.1 used for the secondary electron emission coefﬁcient. 2. SIMULATION MODEL To ensure the results of the simulations were inde- pendant of the grid used, a mesh independance study was undertaken using grids with 2054, 4756, and 11075 total cells, using a simple steady state ﬂow simulation, including turbu- lence. The change in values for parameters such as velocity and pressure was less that 1% from the 4756 cell and 11075 cell simulations. Therefore, to save on computing time while Electron behavior is modeled using the electron drift diffusion approximation (Equation 8) for electron density ﬂux (e) and the electron balance equation (Equation 9) ⃗e = μene∇φ −De∇ne (8) δne δt + ∇. ⃗e = Se (9) (8) (9) where Se accounts for electrons produced or consumed in chemi- cal reactions and μe is electron mobility. Electron diffusivity (De) is calculated using De = Teμe = 2e 3mene ∞ 0 Ck 3 2e νm(ke)e( −ke Te )dke (10) (10) January 2015 \| Volume 2 \| Article 80 \| 3 Simulation of a plasma micro-thruster Greig et al. FIGURE 2 \| Pocket Rocket model dimensions and mesh distribution used for CFD simulations. FIGURE 3 \| CFD simulation results for a 1D parallel plate CCP over three rf periods showing (A) phase resolved electrode voltage (blue solid line), phase resolved plasma potential at the central point (x = 10 mm) (red dashed line) and phase averaged plasma potential at the central point (x = 10 mm) (black dotted line) and (B) phase averaged FIGURE 4 \| Two dimensional, time averaged CFD simulation results for (A) neutral gas density and (B) plasma density within the discharge tube. The full 2D plot has been cropped to show only the region of interest (discharge tube) for ease of viewing. FIGURE 2 \| Pocket Rocket model dimensions and mesh distribution used for CFD simulations. FIGURE 2 \| Pocket Rocket model dimensions and mesh distribution used for CFD simulations. FIGURE 2 \| Pocket Rocket model dimensions and mesh distribution used for CFD simulations. FIGURE 3 \| CFD simulation results for a 1D parallel plate CCP over three rf periods showing (A) phase resolved electrode voltage (blue solid line), phase resolved plasma potential at the central point (x = 10 mm) (red dashed line) and phase averaged plasma potential at the central point (x = 10 mm) (black dotted line) and (B) phase averaged ion (solid line) and electron (dashed line) density. Frontiers in Physics \| Plasma Physics 2. SIMULATION MODEL Pure argon was introduced through an inlet on the top of the plenum chamber to give a gas pressure of 1.5 Torr in the plenum. The base pressure at the outlet of the expansion tube (x = 100 mm, January 2015 \| Volume 2 \| Article 80 \| 4 Frontiers in Physics \| Plasma Physics Simulation of a plasma micro-thruster Greig et al. resulting in higher background neutral densities and hence higher plasma densities. However, the global model also did not account for neutral gas heating, so it is unlikely the heating affects are causing the complete disrepancy between the three results. amplitude of 270 V, was modeled using the CFD-ACE+ pro- gram. A Maxwellian electron distribution and secondary elec- tron coefﬁcient of 0.1 were used to match the PHOENIX PIC simulation. While this simulation has a larger physical dimension and lower pressure than Pocket Rocket, applying the scaling law using a factor of 5 gives a 4 mm physical length scale (approximately the same as the Pocket Rocket tube diameter), which when main- taining a constant pD (pressure-distance) value gives a pressure of 0.5 Torr. This is a factor of three higher than the 1.5 Torr pres- sure stated for Pocket Rocket in the upstream plenum, but as the pressure decreases throughout the Pocket Rocket tube to 0.75 Torr downstream, the pressure at the center of the tube is likely to be around 1–1.2 Torr, closer to the scaled pressure value. Although this does not exactly match the Pocket Rocket parameters, the difference is small enough for the benchmarked PIC simula- tion to provide a suitable simpliﬁed test case for the CFD-ACE+ solution. The plasma potential decreases to 33 V at the discharge tube exit, matching experiments done by Dixon et al. [32] on a simi- lar experimental setup, showing a plasma potential around 32 V near the tube exit for an approximately 1.5 Torr argon plasma. Electron temperature within the discharge tube lies between 1.85 and 1.95 eV, slightly lower than but in agreement with global model estimates of 2 eV [14]. Upstream (near x = −10 mm), a sheath forms on the grounded plenum chamber walls, creating hot ion-induced secondary electrons, causing the peak in Te of 2.5 eV. Downstream in the expansion tube the electron temper- ature rapidly decreases to <1 eV as the plasma expands into the larger volume and cools. 2. SIMULATION MODEL FIGURE 5 \| Time averaged CFD simulation results for (A) plasma potential, (B) electron density and (C) electron temperature along the central axis, including the plenum chamber, discharge tube and expansion tube. The resulting phase resolved rf electrode voltage and plasma potential for the 1D simulation are shown in Figure 3A over two rf periods and time averaged electron and ion densities are shown in Figure 3B. The plasma potential in Figure 3A is taken at the midpoint between the two electrodes (at x = 10 mm) and oscil- lates between the maximum rf input voltage of 270 V and just above 0 V. The time averaged plasma potential is represented by the straight dotted line and is found to be 100 V, matching the time averaged plasma potential at the midpoint of the PIC simu- lation [20]. The maximum ion and electron density occurs at the midpoint of the discharge, reaching 9.9 × 1015 m−3 and tapering off by an order of magnitude at the electrodes, again matching the PIC simulation that gave a density in the order of 6 × 1015 m−3. www.frontiersin.org 3. RESULTS AND DISCUSSION A corresponding peak in ion density also occurs during the negative half of the period, whereas the electron density becomes two orders of magnitude lower as the electrons are accelerated away from the sheath region. Although the electron density is much lower during the negative half of the rf period, as the electrons reach temperatures of 33.5 eV, a signiﬁcant number of electrons have energies well above the ﬁrst ionization potential of argon of 15.76 eV, and the corresponding peak in ion density is to be expected. The hot ion induced secondary electrons creating a peak in ion density near the alumina tube surface, also results in a peak in ion density in the center of the tube as the ions diffuse into the plasma bulk, previously noted in experiments by Charles et al. [14] as a peak in ion saturation current. Comparison between ion density calculated by the CFD simulation and ion saturation current previously measured by Charles et al. using a Langmuir probe along the central axis of the discharge tube is shown in Figure 8. Although direct quantitative comparison is not possible, ion current is proportional to ion density [34] and a qualitative comparison is made here. Both the experimental ion current and simulated ion density peak at x = 11 mm corresponding to the center of the rf electrode. Downstream of the peak, the experi- mental ion saturation current values appear to taper off slower than the simulation ion densities due to ﬂow constriction from the Langmuir probe increasing error bars in that region. FIGURE 6 \| Phase resolved voltage at x = 11 mm on the rf electrode (solid blue line), inner alumina tube surface (dashed green line) and plasma potential on the central axis of the tube (dotted red line) over two rf cycles. Phase averaged plasma potential on the central axis is shown as the straight red dotted line and phase averaged inner alumina tube surface voltage is shown as the straight dashed green line. FIGURE 7 \| Electron density (black dotted line), ion density (black dashed line) and electron temperature (blue solid line) with rf phase at a point in the discharge in line with the rf electrode (x = 11 mm) and 0.1 mm from the inner surface of the dielectric tube (y = 2 mm). 3. RESULTS AND DISCUSSION While the focus of this study is the main plasma parameters such as electron density and temperature, plasma potential and bias voltage, the CFD simulation provided full spatial results for a vari- ety of other parameters as well. Although most are not included here, Figure 4 shows 2D phase averaged plots of the neutral gas density (a) and the plasma density (b) within the discharge tube (the full 2D plot has been cropped to show only the region of interest (discharge tube) for ease of viewing). The low ioniza- tion degree in Pocket Rocket (0.44% 15) means the plasma has little affect on the neutral gas density, although direct heating of neutrals from ions is not included here so the effects may be underestimated slightly. Phase averaged results for plasma potential (Vp), electron den- sity (ne) and electron temperature (Te) along the central axis of the thruster are shown in Figure 5. The plasma potential and elec- tron density are both found to peak in the center of the discharge tube, in line with the rf electrode which lies between x = 9.5 mm and 13.5 mm. Peak plasma (electron) density from the simulation measures 4.1 × 1018m−3, approximately a factor of two higher than previous experimental and global model estimates in the same axial location from Charles et al. [14] of 1.8 × 1018 m−3 and 2.4 × 1018 m−3, respectively. The higher density found with the simulation may be caused by the omission of plasma heat- ing effects, giving a lower simulation neutral gas temperature, FIGURE 5 \| Time averaged CFD simulation results for (A) plasma potential, (B) electron density and (C) electron temperature along the central axis, including the plenum chamber, discharge tube and expansion tube. January 2015 \| Volume 2 \| Article 80 \| 5 www.frontiersin.org Simulation of a plasma micro-thruster Greig et al. In the Pocket Rocket device, there is a large asymmetry between the powered rf electrode area and the grounded area (including the grounded electrodes and plenum walls) and a non-uniform axial density proﬁle [14] resulting in a self bias developing on the alumina tube insert. This is clearly visible in Figure 6, showing phase resolved potentials on the powered elec- trode and inner alumina tube surface and the plasma potential on the central axis of the tube, all measured at the lengthwise midpoint of the tube (x = 11 mm). 3. RESULTS AND DISCUSSION dielectric tube with applied rf voltage on the outside [33]. The plasma potential follows the phase of the inner alumina surface voltage and matches the maximum positive voltage as expected for a potential surface in direct contact with a CCP. The time average plasma potential on the central axis is found to be 38 V, somewhat higher than the estimated value of ∼22 V from a global plasma model [14], which only considered the discharge volume and neglected the plenum chamber and downstream expansion area. Figure 7 shows phase resolved results for electron tempera- ture (Te), electron density (ne) and ion density (ni) at a point 0.1 mm from the inner surface of the alumina tube directly in line with the center of the rf electrode (coordinates x = 11 mm, r = 2 mm in Figure 2) over one rf period. During the positive half of the rf period (phase<0.5), the electron temperature sits around 1.4 eV. During the negative half of the period (phase>0.5) the electron temperature increases dramatically up to 33.5 eV. This is due to a large negative sheath potential during the negative half of the rf period from the self bias that develops on the surface of the ceramic tube, acting to accelerate the ion induced secondary electrons through the sheath region and into the plasma bulk. The voltage on the inner alumina tube surface oscillates between 70 V and −180 V, with a DC bias of −33 V. Sheath capacitance model calculations previously performed estimate the inner surface of the alumina tube will develop a bias of −36 V [14], very similar to the −33 V produced by the simulation. A slight phase offset between the inner alumina tube surface and the powered electrode voltage is also visible, which is character- istic of the response voltage on the inner surface of a cylindrical FIGURE 6 \| Phase resolved voltage at x = 11 mm on the rf electrode (solid blue line), inner alumina tube surface (dashed green line) and plasma potential on the central axis of the tube (dotted red line) over two rf cycles. Phase averaged plasma potential on the central axis is shown as the straight red dotted line and phase averaged inner alumina tube surface voltage is shown as the straight dashed green line. 3. RESULTS AND DISCUSSION However, if the secondary electron coefﬁcient is too low the number of ion induced sec- ondary electrons accelerated by the sheath in the vicinity of the rf electrode to energies above the ionization energy of argon is not sufﬁcient to create the main central plasma peak observed in experiments. For secondary electron emission coefﬁcients of 0.02 or above, the resulting axial ion density is consistent with experimental results in terms of a single peak density located at the mid- point of the discharge tube (x = 11 mm), but the peak density varies from 1.3 × 1018m−3 to 4.9 × 1018m−3. It is therefore likely an overestimation of the secondary electron emission coefﬁcient is contributing to the simulation density being higher than the experimental and global model estimates (1.8 × 1018 m−3 and 2.4 × 1018 m−3, respectively 14). For secondary electron coef- ﬁcients of 0.01 or less, the ion density along the central axis develops a two peak distribution, with a drop in density around x = 11 mm, diverging from experimental results. With secondary electrons turned off, the solution converged also with the two Mean free path for electron-argon collisions within the sheath region, using a neutral density in the central region of the tube of 3.5×1022 m−3 (see Figure 4) and cross sectional data from the JILA database, University of Colorado, ranges from 0.57 mm for an electron energy of 2 eV (σ = 3.52 × 10−20 m−2), to 85 μm for an electron energy of 14 eV (σmax = 23.8 × 10−20 m−2) and around 0.91 mm for energetic tail electrons with an electron energy of 180 eV (σ = 6.42 × 10−20 m−2) based on the max- imum sheath potential that develops during the rf cycle (see Figure 6). Sheath width is estimated as ∼0.5 mm, based on the phase-averaged electron and ion density simulation results across the tube radius at x = 11 mm (in line with the center of the rf electrode), taken as the point where ion and electron den- sity distinctly differ as shown in Figure 10. This value is in good agreement with experimental estimates from digital photogra- phy [35] of 0.3 mm to 0.6 mm, but higher than estimates made with the Child sheath law of ∼0.2 mm [14] and is likely slightly overestimated. 3. RESULTS AND DISCUSSION These results show the main plasma bulk appears to be driven by ion induced secondary electrons accelerated through the sheath region by the large negative potential developed on the alumina tube from the self bias formed by asymmetry of the device. Hence, the secondary electron coefﬁcient input into the simulation is one of the most important unknown values in that it can have a dramatic effect on plasma parameters but is not easily measurable from experiments. A recent 2D PIC simulation of a hollow cathode discharge showed the discharge was driven by secondary electrons or so called ‘Gamma’ electrons and sheath heating was not sufﬁcient to sustain the discharge with secondary FIGURE 7 \| Electron density (black dotted line), ion density (black dashed line) and electron temperature (blue solid line) with rf phase at a point in the discharge in line with the rf electrode (x = 11 mm) and 0.1 mm from the inner surface of the dielectric tube (y = 2 mm). January 2015 \| Volume 2 \| Article 80 \| 6 Frontiers in Physics \| Plasma Physics Simulation of a plasma micro-thruster Greig et al. peak distribution, but produced unrealistic densities, suggesting the role of secondary electrons is crucial within the Pocket Rocket discharge, similar to the hollow cathode PIC simulations. electrons turned off [24]. To investigate the role of secondary electrons in the Pocket Rocket device, the above CFD simula- tion was repeated with secondary electron emission coefﬁcients of 0.001, 0.01, 0.02, 0.05, and 0.2, with the resulting axial ion densities shown in Figure 9, including the original results using a secondary electron emission coefﬁcient of 0.1. electrons turned off [24]. To investigate the role of secondary electrons in the Pocket Rocket device, the above CFD simula- tion was repeated with secondary electron emission coefﬁcients of 0.001, 0.01, 0.02, 0.05, and 0.2, with the resulting axial ion densities shown in Figure 9, including the original results using a secondary electron emission coefﬁcient of 0.1. It would seem that with no or low secondary electron effects, plasma breakdown still occurs between the rf and grounded elec- trodes creating low plasma densities in those regions. These ions then bombard the surface of the dielectric, especially in the cen- ter where the large self bias develops. 4. CONCLUSION A computational ﬂuid dynamics simulation of the capacitively coupled plasma micro-thruster Pocket Rocket was performed using CFD-ACE+ for a 10 W, 1.5 Torr argon plasma and has shown good agreement with previous experimental results. Plasma density and plasma potential both reach peak values at the center of the discharge tube, with ion induced secondary elec- trons emitted from the inner surface of the alumina tube near the rf electrode being accelerated by the sheath to temperatures up to 33.5 eV, driving the main plasma density peak in the center of the tube. The peak plasma density found by the simulation of 4.1 × 1018m−3 is approximately a factor of two higher than pre- vious experimental and global model estimates in the same axial location (1.8 × 1018 m−3 and 2.4 × 1018 m−3, respectively 14), most likely caused by an overestimation of the secondary elec- tron emission coefﬁcient. The secondary electron coeffecient has a dramatic effect on ion density, with both the maximum density value and location changing with secondary electron emission coefﬁcient. In addition, the omission of plasma heating effects in the simulation (and global model) resulting in higher background neutral densities and hence higher plasma densities, may also con- tribute to the slightly higher simulation peak plasma density when compared to the experimental value. 16. Greig A, Charles C, Paulin N, Boswell RW. Volume and surface propellant heating in an electrothermal radio-frequency plasma micro-thruster. Appl Phys Lett. (2014) 105:054102. doi: 10.1063/1.4892656 17. Charles C, Boswell RW, Bish A. Low-weight ﬁxed ceramic capacitor impedance matching system for an electrothermal plasma microthruster. J Propul Power (2014) 30:1117–21. doi: 10.2514/1.B35119 18. Gottscho RA, Donnelly VM. Optical emission actinometry and spectral line shapes in rf glow discharges. J Appl Phys. (1984) 56:245–50. doi: 10.1063/1.333954 19. Phillips DM. Determination of gas temperature from unresolved bands in the spectrum from a nitrogen discharge. J Phys D. (1976) 9:507. doi: 10.1088/0022- 3727/9/3/017 20. Laﬂeur T, Boswell RW, Booth JP. Enhanced sheath heating in capacitively coupled discharges due to non-sinusoidal voltage waveforms. Appl Phys Lett. (2012) 100:194101. doi: 10.1063/1.4712128 21. Roberto M, Smith HB, Verboncoeur JP. Inﬂuence of metastable atoms in radio- frequency argon discharges. Plasma Sci IEEE Trans. (2003) 31:1292–8. doi: 10.1109/TPS.2003.820682 22. Vender D, Boswell RW. Electron–sheath interaction in capacitive radio- frequency plasmas. In: 38th National Symposium of the American Vacuum. Vol. 10. (1992). p. 1331–8. Available from: http://link.aip.org/link/?JVA/10/1331/1. 23. Smith HB. 3. RESULTS AND DISCUSSION FIGURE 8 \| Ion saturation currents along the central axis measured using a Langmuir Probe (top) and simulated ion density along the central axis (bottom). Based on the above approximations, low energy and energetic tail electrons will not undergo a sufﬁcient number of collisions within the sheath for the ﬂuid approximation to be valid in that region and will instead enter the main plasma bulk as a mono- energetic beam. While the exact effect of this on the simulation results is indeterminate, the good agreement between the simu- lation and experimental results suggest the effect is small and the FIGURE 8 \| Ion saturation currents along the central axis measured using a Langmuir Probe (top) and simulated ion density along the central axis (bottom). FIGURE 9 \| Ion density along the central axis with changing secondary electron coefﬁcient on the alumina tube and grounded metal walls. Coefﬁcients are 0.001 (solid gray line), 0.01 (solid black line), 0.02 (dot dashed black line), 0.05 (dashed black line), 0.1 (dashed gray line) and 0.2 (dotted black line). FIGURE 10 \| Phase-averaged electron (dashed line) and ion (solid line) density radially across the Pocket Rocket tube, in line with the center of the rf electrode (x = 11 mm). www.frontiersin.org January 2015 \| Volume 2 \| Article 80 \| 7 FIGURE 9 \| Ion density along the central axis with changing secondary electron coefﬁcient on the alumina tube and grounded metal walls. Coefﬁcients are 0.001 (solid gray line), 0.01 (solid black line), 0.02 (dot dashed black line), 0.05 (dashed black line), 0.1 (dashed gray line) and 0.2 (dotted black line). FIGURE 10 \| Phase-averaged electron (dashed line) and ion (solid line) density radially across the Pocket Rocket tube, in line with the center of the rf electrode (x = 11 mm). FIGURE 9 \| Ion density along the central axis with changing secondary electron coefﬁcient on the alumina tube and grounded metal walls. Coefﬁcients are 0.001 (solid gray line), 0.01 (solid black line), 0.02 (dot dashed black line), 0.05 (dashed black line), 0.1 (dashed gray line) and 0.2 (dotted black line). FIGURE 10 \| Phase-averaged electron (dashed line) and ion (solid line) density radially across the Pocket Rocket tube, in line with the center of the rf electrode (x = 11 mm). January 2015 \| Volume 2 \| Article 80 \| 7 www.frontiersin.org Simulation of a plasma micro-thruster Greig et al. REFERENCES 25. O’Connell D, Zorat R, Ellingboe AR, Turner MM. Comparison of measure- ments and particle-in-cell simulations of ion energy distribution functions in a capacitively coupled radio-frequency discharge. Phys Plasmas (2007) 14:103510. doi: 10.1063/1.2795634 1. Goebel DM, Katz I. Fundamentals of electric propulsion: ion and hall thrusters. In: Yuen JH, editor. John Wiley and Sons (2008) Available onlin at: http://descanso.jpl.nasa.gov/SciTechBook/series1/Goebel_cmprsd_opt.pdf. 1. Goebel DM, Katz I. Fundamentals of electric propulsion: ion and hall thrusters. In: Yuen JH, editor. John Wiley and Sons (2008) Available onlin at: http://descanso.jpl.nasa.gov/SciTechBook/series1/Goebel_cmprsd_opt.pdf. 1. Goebel DM, Katz I. Fundamentals of electric propulsion: ion and hall thrusters. In: Yuen JH, editor. John Wiley and Sons (2008) Available onlin at: http://descanso.jpl.nasa.gov/SciTechBook/series1/Goebel_cmprsd_opt.pdf. 2. Charles C. Plasmas for spacecraft propulsion. J Phys D. (2009) 42:163001. doi: 10.1088/0022-3727/42/16/163001 26. Matyash K, Schneider R, Taccogna F, Hatayama A, Longo S, Capitelli M, et al. Particle in cell simulation of low temperature laboratory plas- mas. Contrib Plasma Phys. (2007) 47:595–634. doi: 10.1002/ctpp.2007 10073 3. Frisbee RH. Advanced space propulsion for the 21st century. J Propul Power (2003) 19:1129–54. doi: 10.2514/2.6948 27. Kolobov VI. Fokker-planck modeling of electron kinetics in plasmas and semiconductors. Comput Mater Sci. (2003) 28:302–20. doi: 10.1016/S0927- 0256(03)00115-0 4. Dunaevsky A, Raites Y, Fisch N. Plasma acceleration from radio-frequency discharge in dielectric capillary. Appl Phys Lett. (2006) 88:251502. doi: 10.1063/1.2214127 28. Lu Y, Yan D, Chen Y. 2-D ﬂuid simulation of dual-frequency capacitively coupled plasma. J Hydrodyn Ser B. (2009) 21:814–9. doi: 10.1016/S1001- 6058(08)60217-6 5. Lamprou D, Lappas VJ, Shimizu T, Gibbon D, Perren M. Hollow cathode thruster design and development for small satellites. In: 32nd International Electric Propulsion Conference. Wiesbaden (2011). 6. Martinez-Sanchez M, Pollard JE. Spacecraft electric propulsion - an overview. J Propul Power. (1998) 14:688–99. doi: 10.2514/2.5331 29. Rakhimova TV, Braginsky OV, Ivanov VV, Kim TK, Kong JT, Kovalev AS, et al. Experimental and theoretical study of RF plasma at low and high fre- quency. Plasma Sci IEEE Trans. (2006) 34:867–77. doi: 10.1109/TPS.2006. 875849 7. Blackhall L, Khachan J. A simple electric thruster based on ion charge exchange. J Phys D. (2007) 40:2491. doi: 10.1088/0022-3727/ 40/8/011 7. Blackhall L, Khachan J. A simple electric thruster based on ion charge exchange. J Phys D. (2007) 40:2491. doi: 10.1088/0022-3727/ 40/8/011 30. Launder BE, Spalding DB. The numerical computation of turbulent ﬂows. Comput Methods Appl Mech Eng. (1974) 3:269–89. doi: 10.1016/0045- 7825(74)90029-2 8. Fruchtman A. 4. CONCLUSION Studies of plasma breakdown and decay in a capacitive radiofrequency argon discharge. Phys Plasmas (1998) 5:3469–76. doi: 10.1063/1.873060 24. Laﬂeur T, Boswell RW. Particle-in-cell simulations of hollow cathode enhanced capacitively coupled radio frequency discharges. Phys Plasmas (2012) 19:023508. doi: 10.1063/1.3685709 3. RESULTS AND DISCUSSION simulation results are still reasonable. However, as with all model- ing applications, the simulation results are an approximation and should be taken as a guideline only. 14. Charles C, Boswell RW. Measurement and modeling of a radiofre- quency micro-thruster. Plasma Sources Sci Technol. (2012) 21:022002. doi: 10.1088/0963-0252/21/2/022002 15. Greig A, Charles C, Hawkins R, Boswell RW. Direct measurement of neutral gas heating in a radio-frequency electrothermal plasma micro-thruster. Appl Phys Lett. (2013) 103:074101. doi: 10.1063/1.48 18657 Received: 23 September 2014; accepted: 05 December 2014; published online: 06 January 2015. Citation: Greig A, Charles C and Boswell RW (2015) Simulation of main plasma parameters of a cylindrical asymmetric capacitively coupled plasma micro-thruster using computational ﬂuid dynamics. Front. Phys. 2:80. doi: 10.3389/fphy.2014.00080 January 2015 \| Volume 2 \| Article 80 \| 9 This article was submitted to Plasma Physics, a section of the journal Frontiers in Physics. Copyright © 2015 Greig, Charles and Boswell. This is an open-access article dis- tributed 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 publica- tion 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. This article was submitted to Plasma Physics, a section of the journal Frontiers in Physics. Copyright © 2015 Greig, Charles and Boswell. This is an open-access article dis- tributed 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 publica- tion in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Conﬂict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Received: 23 September 2014; accepted: 05 December 2014; published online: 06 January 2015. REFERENCES Energizing and depletion of neutrals by a collisional plasma. Plasma Sources Sci Technol. (2008) 17:024016. doi: 10.1088/0963- 0252/17/2/024016 31. Lee I, Graves DB, Lieberman MA. Modeling electromagnetic effects in capacitive discharges. Plasma Sources Sci Technol. (2008) 17:015018. doi: 10.1088/0963-0252/17/1/015018 9. Fruchtman A. The thrust of a collisional-plasma source. IEEE Trans Plasma Sci. (2011) 39:530–9. doi: 10.1109/TPS.2010.2089067 32. Dixon S, Charles C, Boswell R. Spatial evolution of EEPFs in a millimetre scale radio frequency argon plume. J Phys D. (2013) 46:365202. doi: 10.1088/0022- 3727/46/36/365202 10. Fruchtman A. Neutral depletion in a collisionless plasma. IEEE Trans Plasma Sci. (2008) 36:403–13. doi: 10.1109/TPS.2008.918777 11. Charles C. A review of recent laboratory double layer experiments. Plasma Sources Sci Technol. (2007) 16:R1. doi: 10.1088/0963-0252/16/4/R01 33. Butler HS, Kino GS. Plasma sheath formation by radio-frequency ﬁelds. Phys Fluids (1963) 6:1346–1355. doi: 10.1063/1.1706905 12. Charles C, Boswell R. Current-free double-layer formation in a high- density helicon discharge. Appl Phys Lett. (2003) 82:1356–8. doi: 10.1063/1. 1557319 34. Sheridan TE. How big is a small Langmuir probe? Phys Plasmas (2000) 7:3084– 8. doi: 10.1063/1.874162 13. Boswell R, Charles C, Alexander P, Dedrick J, Takahashi K. Plasma expan- sion from a radio frequency microdischarge. Plasma Sci IEEE Trans. (2011) 39:2512–3. doi: 10.1109/TPS.2011.2143434 35. Greig A, Charles C, Boswell R. Plume characteristics of an electrother- mal Plasma microthruster. Plasma Sci IEEE Trans. (2014) 42:2728–9. doi: 10.1109/TPS.2014.2321176 January 2015 \| Volume 2 \| Article 80 \| 8 Frontiers in Physics \| Plasma Physics Simulation of a plasma micro-thruster Greig et al. January 2015 \| Volume 2 \| Article 80 \| 9 www.frontiersin.org www.frontiersin.org
https://openalex.org/W2061881258	https://genomebiology.biomedcentral.com/counter/pdf/10.1186/gb-2007-8-11-r251	English	null	Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish	Genome biology	2,007	cc-by	52,643	Correspondence: Maria Leptin. Email: mleptin@uni-koeln.de Correspondence: Maria Leptin. Email: mleptin@uni-koeln.de Received: 20 April 2007 Revised: 30 October 2007 Accepted: 27 November 2007 Published: 27 November 2007 Genome Biology 2007, 8:R251 (doi:10.1186/gb-2007-8-11-r251) Published: 27 November 2007 Genome Biology 2007, 8:R251 (doi:10.1186/gb-2007-8-11-r251) The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/11/R251 © 2007 Stein et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. i i b fi h l i f l fi h l f h i i d id ifi h l l i hi b f ili f fi h d l / 2007 Stein et al. Volume 8, Issue Research Addresses: *Institute for Genetics, University of Cologne, Zuelpicher Str. 47, 50674 Cologne, Germany. †The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK. Open Access Open Access Abstract Background: The zebrafish has become a widely used model to study disease resistance and immunity. Although the genes encoding many components of immune signaling pathways have been found in teleost fish, it is not clear whether all components are present or whether the complexity of the signaling mechanisms employed by mammals is similar in fish. Results: We searched the genomes of the zebrafish Danio rerio and two pufferfish for genes encoding components of the Toll-like receptor and interferon signaling pathways, the NLR (NACHT-domain and leucine rich repeat containing) protein family, and related proteins. We find that most of the components known in mammals are also present in fish, with clearly recognizable orthologous relationships. The class II cytokines and their receptors have diverged extensively, obscuring orthologies, but the number of receptors is similar in all species analyzed. In the family of the NLR proteins, the canonical members are conserved. We also found a conserved NACHT- domain protein with WD40 repeats that had previously not been described in mammals. Additionally, we have identified in each of the three fish a large species-specific subgroup of NLR proteins that contain a novel amino-terminal domain that is not found in mammalian genomes. Conclusion: The main innate immune signaling pathways are conserved in mammals and teleost fish. Whereas the components that act downstream of the receptors are highly conserved, with orthologous sets of genes in mammals and teleosts, components that are known or assumed to interact with pathogens are more divergent and have undergone lineage-specific expansions. for the genetic analysis of human immunity, knowledge of components of immune defense systems in the zebrafish would also aid our understanding of the evolution of immunity. Background g With the sequence of the zebrafish genome as well as the sequences of two pufferfish genomes nearly completed, and in view of the widespread use of the zebrafish as a model to study immunity [1], it is both pertinent and feasible to deter- mine which of the genes that encode components of the mam- malian immune system are also found in fish. In addition to being a prerequisite for using the zebrafish as a model system Zebrafish are a member of the large group of teleost fish that, together with a small nonteleost sister group, constitute the ray-finned fishes. The ray-finned fishes diverged from the Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.2 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.2 http://genomebiology.com/2007/8/11/R251 skin defense peptides), and future genetic research may well reveal additional fish-specific molecules and mechanisms. common ancestor of other bony vertebrates, which include tetrapods as well as lungfishes and coelacanths, 450 million years ago. They appear to have undergone a massive radiation about 235 million years ago, resulting in as many teleost spe- cies as there are species represented by all other vertebrates together (approximately 24,000 species in each case). One genetic event that has been regarded to be associated with the radiation of the teleosts in particular is a whole genome dupli- cation event early in the teleost lineage. Although some genes or regions of the genome, most notably the Hox gene clusters, have been maintained in multiple copies, others have under- gone re-diploidization. The availability of additional gene copies has been proposed to have facilitated the evolution of the high level of diversity in morphology and behavior in the teleost fish [2,3]. To be able to judge orthologous relationships properly, we also included protein family members that have not been shown to have immune signaling functions, in particular because it cannot be excluded that these may have as yet uni- dentified roles in immune signaling, as has recently been dis- covered for TNF-receptor associated factor (Traf)3 [22]. We find that the families of intracellular signaling adaptors and enzymes are largely conserved. By contrast, the class II cytokines and their receptors have diverged significantly, and the NLR (NACHT-domain and leucine rich repeat contain- ing) proteins exhibit extensive, species-specific gene amplifi- cation and diversification. Background Components of the adaptive immune system have been stud- ied intensively in many fish species and have been analyzed molecularly and genetically (for review [4]). Unlike the adap- tive immune system, some of the systems that contribute to innate immunity are conserved throughout the animal king- dom. The presence of genes encoding components of these systems in the zebrafish and other fish was therefore not unexpected. In addition to the well studied adaptive immune genes, protein and gene families involved in innate immune mechanisms that have been analyzed in detail include the complement gene family (for review [5]), the Toll-like recep- tors (TLRs) [6,7], and two sets of receptor genes that encode proteins structurally similar to the immunoglobulin-type and C-type lectin domain-type of mammalian NK (natural killer cell) receptors [8-11]. Similarly, genes encoding tumor necro- sis factors (TNF), ILs, IFNs, and their respective receptors have been identified in various fish species [12-18]. Together with studies on subsets of intracellular signaling molecules [19-23], these findings indicate that many components of innate immune signaling pathways known from mammals are conserved in the teleost fish. However, it is not clear whether all components are present or whether, in general, the complexity of the signaling mechanisms employed by mammals is similar in fish. For example, whereas some mem- bers of the TLR family exhibit orthologous relationships between zebrafish and mammals, there are also expansions within the TLR gene family that are specific for the zebrafish or the mammals [6,7]. Similarly, the novel immune-type receptors, which share several common features with mam- malian immunoglobulin-type natural killer cell receptors, exhibit species-specific expansions and diversifications [8,10]. Compone Figure 1 Components of the TLR and IFN signaling pathways and intracellular pattern recognition receptors Figure 1 Components of the TLR and IFN signaling pathways and intracellular pattern recognition receptors. The molecules analyzed in this study are shown in color. For simplicity, not all members of each protein family are shown. IFN, interferon; TLR, Toll-like receptor. p g g p y p g p g Components of the TLR and IFN signaling pathways and intracellular pattern recognition receptors. The molecules analy color. For simplicity, not all members of each protein family are shown. IFN, interferon; TLR, Toll-like receptor. Each of these groups of proteins is discussed individually below. pufferfish are closely related to each other, and the zebrafish is more closely related to the pufferfish than to mammals and therefore shares a branch with the pufferfish on the phyloge- netic tree. In several cases, for example Tab1 and Tab2, the Tetraodon sequences do not group with their counterparts from Takifugu. In most of these cases this is due to internal deletions or insertions, or terminal deletions or extensions in the Tetraodon genes, which are most easily explained by unreliable predictions for these genes based on faulty assem- bly of the genome (see below for specific cases). We have not investigated these cases further. Results and discussion As the basis for our search, we first assembled a set of sequences of mammalian genes that encode components of the TNF, IFN, and TLR pathways, and the NLR proteins in mice and humans (Figure 1). We then identified homologs of these genes in the zebrafish genome. We first checked whether Ensembl [24] or ZFIN [25] listed potential homologs and added these to our list. In cases in which putative homologs were not found in Ensembl or ZFIN, we used TBLASTN [26] to screen unfinished clones from the genome sequencing project and trace sequences from the whole genome shotgun project. If matching sequences were found, they were analyzed in detail in their genomic context and were manually annotated to generate a gene prediction, using the available mammalian sequences and any existing expressed sequence tags (ESTs) as evidence. Where gene pre- dictions were available from the Tetraodon nigroviridis or Takifugu rubripes genomes, we also included these in our analyses, but we did not make any assemblies or annotations ourselves. A complete list of all sequences used in this study is provided in Additional data files 1-9. We used MEGA software [27] to compare the encoded fish proteins with their mammalian counterparts. For some pro- teins, the annotated sequences were not complete and could not be completed because the available DNA sequence was not sufficiently reliable or had gaps. We therefore point out that the phylogenetic trees we present show relationships, but are not intended to show precise evolutionary distances. For most of the core signal transduction components of each pathway we found clear orthologous relationships between the mammalian and the zebrafish genes (see Gene families with largely orthologous relationships between teleosts and mammals, below), as illustrated for example by the branches for Tollip (Toll-interacting protein) or Tab (Tak1-binding protein)3 in Figure 2. These branches reflect the known evo- lutionary relationships between the five species. Mouse and human exhibit the highest level of similarity, the two This report concentrates on identifying those molecules known from mammalian innate immune signaling systems that are conserved between teleost fish and mammals. The study is restricted to the pathways that have not been exten- sively studied by others previously. Gene families with largely orthologous relationships between teleosts and mammals In the protein families of the immune kinases, the adaptors in the TLR signaling pathway, the interferon response factors (IRFs), the signal transducers and activators of transcription (Stats), and the Trafs we found orthologous genes in fish for almost all of the mammalian genes. This is summarized in Figures 2 to 6. However, there were also occasional duplica- tions or losses either in the fish or in the mammalian lineage. The findings are briefly summarized below and in the figure legends. For the class II cytokine receptor family the orthology was less clear (see Class II cytokines and their receptors, below) or nonexistent, as has previously been noted [17]. For one group of proteins, those containing NLRs, our comparison reveals extensive, species-specific expansion of subfamilies (see Intracellular pathogen sensors: the NACHT-domain family, below). Results and discussion It is likely that there are also nonconserved defense systems associated with the char- acteristic physiologies of fish and mammals (for example, Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.3 http://genomebiology.com/2007/8/11/R251 Components of the TLR and IFN signaling pathways and intracellular pattern recognition receptors Figure 1 Components of the TLR and IFN signaling pathways and intracellular pattern recognition receptors. The molecules analyzed in this study are shown in color. For simplicity, not all members of each protein family are shown. IFN, interferon; TLR, Toll-like receptor. Kinases The kinases were the family that exhibited the most apparent orthologies between fish and mammals. For all of the essen- tial kinases involved in signal transduction mediated by TLR, Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.4 http://genomebiology.com/2007/8/11/R251 Phylogenetic trees of the innate immune signaling adaptors and diagrams of their protein structures Figure 2 Phylogenetic trees of the innate immune signaling adaptors and diagrams of their protein structures. The fish protein names are highlighted in blue (Dr [Danio rerio]) or green (Fr [Takifugu rubripes] and Tn [Tetraodon nigroviridis]). The numbers in the tree indicate the bootstrap values. Scale: interval of 0.1 amino acid substitutions. Hs, Homo sapiens, Mm, Mus musculus. Protein domains are shown as boxes based on identification by Pfam [55] or Smart [56]. Some domains were not recognized by these programs, although manual inspection indicated clear conservation of the domain within the protein family. These domains are also shown as boxes in the diagrams. The identities of the domains are listed at the bottom. Scale bar = 100 amino acids. The Tetraodon version of the Ikap (IKK [Inhibitor of nuclear factor-κB kinase] complex associated protein) gene contains two full repeats of the IKI3 domain. It is not clear whether this prediction is due to an error in the genome assembly or whether the gene does indeed contain an internal duplication covering the whole length of the gene found in other species. The two halves of the predicted gene were treated as separate peptides in the phylogenetic tree and the diagram. Kinases HsMyd88 MmMyd88 DrMyd88 FrMyd88 TnMyd88 HsSarm1 MmSarm1 DrSarm1 FrSarm1 TnSarm1 HsNEMO MmNEMO DrNEMO FrNEMO HsTab1 MmTab1 DrTab1 FrTab1 TnTab1 HsIKAP MmIKAP DrIKAP FrIKAP TnIKAPa TnIKAPb HsTab2 MmTab2 DrTab2 FrTab2 TnTab2 100 100 100 100 HsTollip MmTollip DrTollip FrTollip TnTollip 100 55 85 100 100 100 100 100 100 100 100 99 100 100 100 92 99 100 100 51 100 100 0.1 CUE zinc finger RanBP2 coiled-coil TIR SAM PP2C IKI3 Death C2 100 aa HsTab3 MmTab3 DrTab3 FrTab3 TnTab3 100 100 100 100 HsTirap MmTirap DrTirap FrTirap HsTicam1 MmTicam1 HsTicam2 MmTicam2 DrTicam FrTicam TnTicam 98 100 79 100 100 100 55 36 61 100 100 100 TnNEMO and diagrams of their protein structures d di f h i i Th fi h i hi hli h d i bl (D CUE zinc finger RanBP2 coiled-coil TIR SAM PP2C IKI3 Death C2 100 aa HsMyd88 MmMyd88 DrMyd88 FrMyd88 TnMyd88 HsSarm1 MmSarm1 DrSarm1 FrSarm1 TnSarm1 HsNEMO MmNEMO DrNEMO FrNEMO HsTab1 MmTab1 DrTab1 FrTab1 TnTab1 HsIKAP MmIKAP DrIKAP FrIKAP TnIKAPa TnIKAPb HsTab2 MmTab2 DrTab2 FrTab2 TnTab2 100 100 100 100 HsTollip MmTollip DrTollip FrTollip TnTollip 100 55 85 100 100 100 100 100 100 100 100 99 100 100 100 92 99 100 100 51 100 100 0.1 HsTab3 MmTab3 DrTab3 FrTab3 TnTab3 100 100 100 100 HsTirap MmTirap DrTirap FrTirap HsTicam1 MmTicam1 HsTicam2 MmTicam2 DrTicam FrTicam TnTicam 98 100 79 100 100 100 55 36 61 100 100 100 TnNEMO Phylogenet Figure 2 Phylogenetic trees of the innate immune signaling adaptors and diagrams of their protein structures Figure 2 Phylogenetic trees of the innate immune signaling adaptors and diagrams of their protein structures. The fish protein names are highlighted in blue (Dr [Danio rerio]) or green (Fr [Takifugu rubripes] and Tn [Tetraodon nigroviridis]). The numbers in the tree indicate the bootstrap values. Scale: interval of 0.1 amino acid substitutions. Hs, Homo sapiens, Mm, Mus musculus. Protein domains are shown as boxes based on identification by Pfam [55] or Smart [56]. Some domains were not recognized by these programs, although manual inspection indicated clear conservation of the domain within the protein family. These domains are also shown as boxes in the diagrams. The identities of the domains are listed at the bottom. Scale bar = 100 amino acids. The Tetraodon version of the Ikap (IKK [Inhibitor of nuclear factor-κB kinase] complex associated protein) gene contains two full repeats of the IKI3 domain. It is not clear whether this prediction is due to an error in the genome assembly or whether the gene does indeed contain an internal duplication covering the whole length of the gene found in other species. The two halves of the predicted gene were treated as separate peptides in the phylogenetic tree and the diagram. tion with IRAK1, was not found in any of the three fish. This suggests that it has arisen from a duplication event that occurred only within the mammalian lineage (Figure 3). The alternative, loss of IRAK2, for example in the teleost lineage TNF, and nucleotide oligomerization domain containing pro- tein (Nod), we find orthologs in zebrafish and in most cases also in pufferfish. IL-1 receptor associated kinase (IRAK)2, which is thought to serve as an accessory protein in combina- Genome Biology 2007, 8:R251 Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.5 http://genomebiology.com/2007/8/11/R251 Phylogenetic tree of the interferon response factors Figure 4 Phylogenetic tree of the interferon response factors. Details are as in Figure 2. The chicken (Gg [Gallus gallus]) IRF10 was included to show its relationship to fish IRF10, because no ortholog for this gene is found in mammals. IRF, interferon response factor. Phylogenet Figure 2 HsIRF6 MmIRF6 DrIRF6 FrIRF6 TnIRF6 100 100 100 100 DrIRF11 FrIRF11 TnIRF11 100 100 100 HsIRF5 MmIRF5 DrIRF5 FrIRF5 TnIRF5 100 100 91 100 HsIRF1 MmIRF1 DrIRF1 FrIRF1 TnIRF1 100 99 56 78 HsIRF2 MmIRF2 DrIRF2a FrIRF2 DrIRF2b 100 65 94 99 43 100 HsIRF4 MmIRF4 DrIRF4a DrIRF4b FrIRF4 TnIRF4 DrIRF4c 100 100 98 98 100 99 GgIRF10 DrIRF10 FrIRF10 TnIRF10 100 100 95 99 HsIRF8 MmIRF8 DrIRF8 FrIRF8 TnIRF8 100 100 100 100 100 HsIRF9 MmIRF9 DrIRF9 FrIRF9 TnIRF9 100 100 100 92 95 HsIRF3 MmIRF3 DrIRF3 FrIRF3 TnIRF3 100 100 100 96 97 HsIRF7 MmIRF7 DrIRF7 FrIRF7 TnIRF7 100 100 100 98 99 0.1 Phylogenetic tree of the kinases Figure 3 HsIKKa M m IKKa DrIKKa FrIKKa1 TnIKKa1 FrIKKa2 TnIKKa2 100 100 99 100 100 100 100 HsIKKb M m IKKb DrIKKb FrIKKb TnIKKb 100 100 96 100 HsTBK1 M m TBK1 DrTBK1 TnTBK1b FrTBK1 TnTBK1a 100 100 79 100 100 100 HsIKKe M m IKKe DrIKKe FrIKKe TnIKKe 100 50 100 84 100 HsTak1 M m Tak1 DrTak1 FrTak1 TnTak1 100 100 100 100 42 HsRipk1 M m Ripk1 DrRipk1 FrRipk1 TnRipk1 100 100 100 100 100 HsRipk5 M m Ripk5 DrRipk5 FrRipk5a FrRipk5b TnRipk5b TnRipk5a 100 47 100 75 45 100 HsRipk3 M m Ripk3 DrRipk3 100 100 100 99 HsRipk2 M m Ripk2 DrRipk2 FrRipk2 TnRipk2 100 100 100 100 99 100 HsRipk4 M m Ripk4 DrRipk4 FrRipk4 TnRipk4 100 100 100 100 HsNLK M m NLK DrNLKa FrNLKa TnNLKa DrNLKb FrNLKb TnNLKb 100 76 75 100 54 100 100 HsIRAK1 M m IRAK1 DrIRAK1 FrIRAK1 TnIRAK1 100 100 100 100 HsIRAK3 M m IRAK3 DrIRAK3 100 100 57 32 HsIRAK4 M m IRAK4 FrIRAK4 TnIRAK4 DrIRAK4 100 100 99 100 HsIRAK2 M m IRAK2 100 100 100 100 HsJAK3 M m JAK3 DrJAK3 FrJAK3 TnJAK3 100 100 100 79 100 HsJAK2 M m JAK2 FrJAK2a TnJAK2a DrJAK2b DrJAK2a FrJAK2b TnJAK2b 100 100 61 98 100 47 100 0.1 HsTyk2 M m Tyk2 DrTyk2 FrTyk2 TnTyk2 100 100 100 100 HsJAK1 M m JAK1 DrJAK1 FrJAK1 TnJAK1 100 100 100 100 100 Phylogenet Figure 4 y g p g Phylogenetic tree of the interferon response factors. Details are as in Figure 2. The chicken (Gg [Gallus gallus]) IRF10 was included to show its relationship to fish IRF10, because no ortholog for this gene is found in mammals. IRF, interferon response factor. Adaptors Phylogenetic tree of the STAT proteins Figure 5 Phylogenetic tree of the STAT proteins. Details are as in Figure 2. STAT, signal transducer and activator of transcription. HsSTAT1 MmSTAT1 FrSTAT1 TnSTAT1 DrSTAT1a DrSTAT1b HsSTAT3 MmSTAT3 DrSTAT3 FrSTAT3 TnSTAT3 HsSTAT4 MmSTAT4 DrSTAT4 FrSTAT4 TnSTAT4 HsSTAT2 MmSTAT2 DrSTAT2 FrSTAT2 TnSTAT2 HsSTAT5a MmSTAT5a HsSTAT5b MmSTAT5b DrSTAT5.1 FrSTAT5.1 TnSTAT5.1 DrSTAT5.2 HsSTAT6 MmSTAT6 DrSTAT6 FrSTAT6 TnSTAT6 100 100 100 100 100 100 100 99 100 100 100 100 100 100 100 100 100 100 100 100 100 87 100 100 76 99 100 100 66 100 47 99 0.1 p The adaptors that are involved in innate immune signaling cascades are well conserved in fish, as was previously observed for those interacting with the TLRs [6,7,23]. We find orthologous genes in each of the three fish species for Myd88 (myeloid differentiation factor 88), Sarm1 (sterile α and HEAT/armadillo motif containing protein 1), Tollip, IKAP (IKK complex associated protein), NEMO (NF-κB essential modulator), Tab1, Tab2, and Tab3, and in the zebrafish and Takifugu for Tirap (Toll/IL-1 receptor associated protein). For the mammalian Ticam (Toll-like receptor adaptor mole- cule)1 and Ticam2 (also named TRIF and TRAM) genes, there is only one homologous gene in each of the three fish, which is equally distant to Ticam1 and Ticam2, indicating a duplica- tion of an ancestral gene in the mammalian lineage and sub- sequent divergence of the two copies (Figure 2). The alternative interpretation, that Ticam2 was lost specifically in the teleost lineage, does not fit with the fact that it is also not present in the genomes of Xenopus and chicken [28]. An apparent contradiction to our observation is a report of both Ticam1 and Ticam2 in Hydra [29]. However, cnidarians too have only one Ticam, because the gene cited as Tram is in fact not the TRAM (TRIF-related adaptor molecule) that is synon- ymous with Ticam2, but encodes an unrelated protein, the translocation-associated membrane protein, which has the same acronym. Phylogenet Figure 2 Phylogenetic tree of the kinases Figure 3 Phylogenetic tree of the kinases. Details of the tree are as in Figure 2. Phylogenetic tree of the kinases Figure 3 Phylogenetic tree of the kinases. Details of the tree are as in Figure 2. Phylogenetic tree of the kinases Figure 3 Phylogenetic tree of the kinases. Details of the tree are as in Figure 2. Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.6 http://genomebiology.com/2007/8/11/R251 IFN response factors p For IRF1, IRF3, and IRF5 to IRF9, clear orthologous relation- ships are found between mammals and fish. In each fish spe- cies we also find an additional gene, which we call IRF11 and which is equally distant to both IRF1 and IRF2. DrIRF4b, which is most closely related to the IRF4s found in the puffer- fish, maps to a region of the genome that is syntenic with the region containing IRF4 in mammals and in the two puffer- fish, indicating that these are orthologous genes. In addition to the homologs of the IRFs in mammals, we find an addi- tional IRF in each of the fish, which we named IRF10, because it groups with a similar gene from chicken. It appears that this gene has been lost in mammals (Figure 4). Signal transducers and activators of transcription Mammalian Stat2, Stat3, Stat4, and Stat6 have clear orthologs in all three fish species (Figure 5). Stat5 has been independently duplicated in mammals and in zebrafish [21]. The group of Stat1 genes contains one gene from each puffer- fish with a good match to mammalian Stat1, but two genes from zebrafish that are surprisingly divergent but still resem- ble Stat1 more than the other Stats. The duplication event that led to this situation is recognizable in the genome, because the whole region containing the gene is duplicated and syn- tenic with the same region in human (Figure 7). The positions of flanking genes in the human and zebrafish genome are indications of a number of rearrangements. On chromosome 9 in the zebrafish these have been associated with a further Phylogenet Figure 5 y g p g Phylogenetic tree of the STAT proteins. Details are as in Figure 2. STAT, signal transducer and activator of transcription. (it is also absent in Medaka and stickleback), is less likely because a search of the ray and shark genomes did not iden- tify any sequences for IRAK2. Conversely, we find duplica- tions in the fish lineage for Jak2 (Janus kinase 2) and NLK (nuclear factor-κB [NF-κB] essential modulator-like kinase), and duplications in both pufferfish for IKKa (inhibitor of NF- κB kinase) and Ripk5 (receptor-interacting protein kinase 5). (it is also absent in Medaka and stickleback), is less likely because a search of the ray and shark genomes did not iden- tify any sequences for IRAK2. IFN response factors Conversely, we find duplica- tions in the fish lineage for Jak2 (Janus kinase 2) and NLK (nuclear factor-κB [NF-κB] essential modulator-like kinase), and duplications in both pufferfish for IKKa (inhibitor of NF- κB kinase) and Ripk5 (receptor-interacting protein kinase 5). Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.7 http://genomebiology.com/2007/8/11/R251 Phylogenetic tree of the TRAFs and diagrams of their protein domain structure Figure 6 Phylogenetic tree of the TRAFs and diagrams of their protein domain structure. Details are as in Figure 2, except that the scale shows 0.2 amino acid substitutions. TRAF, tumor necrosis factor receptor-associated factor. 98 HsTraf7 MmTraf7 DrTraf7 FrTraf7 TnTraf7 95 50 100 HsTraf4 MmTraf4 DrTraf4a FrTraf4 TnTraf4 DrTraf4b 100 100 100 80 100 HsTraf6 MmTraf6 DrTraf6 FrTraf6 TnTraf6 100 100 98 100 DrTraf5 HsTraf5 MmTraf5 100 87 DrTraf1 HsTraf1 MmTraf1 100 99 99 HsTraf2 MmTraf2 DrTraf2a FrTraf2a1 TnTraf2a1 FrTraf2a2 TnTraf2a2 DrTraf2b FrTraf2b TnTraf2b 100 100 100 100 70 100 100 83 99 78 HsTraf3 MmTraf3 DrTraf3 FrTraf3 TnTraf3 100 100 100 100 93 38 0.2 100aa RING zinc finger coiled-coil MATH WD40 tails are as in Figure 2 except that the scale shows 0 2 amino acid 100aa RING zinc finger coiled-coil MATH WD40 etails are as in Figure 2, except that the scale shows 0.2 amino acid 100aa RING zinc finger coiled-coil MATH WD40 Phylogenetic tree of the TRAFs and diagrams of their protein domain structure Figure 6 Phylogenetic tree of the TRAFs and diagrams of their protein domain structure. De substitutions. TRAF, tumor necrosis factor receptor-associated factor. 98 HsTraf7 MmTraf7 DrTraf7 FrTraf7 TnTraf7 95 50 100 HsTraf4 MmTraf4 DrTraf4a FrTraf4 TnTraf4 DrTraf4b 100 100 100 80 100 HsTraf6 MmTraf6 DrTraf6 FrTraf6 TnTraf6 100 100 98 100 DrTraf5 HsTraf5 MmTraf5 100 87 DrTraf1 HsTraf1 MmTraf1 100 99 99 HsTraf2 MmTraf2 DrTraf2a FrTraf2a1 TnTraf2a1 FrTraf2a2 TnTraf2a2 DrTraf2b FrTraf2b TnTraf2b 100 100 100 100 70 100 100 83 99 78 HsTraf3 MmTraf3 DrTraf3 FrTraf3 TnTraf3 100 100 100 100 93 38 0.2 HsTraf2 MmTraf2 Phylogenet Figure 6 y g g p g Phylogenetic tree of the TRAFs and diagrams of their protein domain structure. Details are as in Figure 2, except that the scale shows 0.2 amino acid substitutions. TRAF, tumor necrosis factor receptor-associated factor. duplication of part of the Stat1 gene, resulting in a pseudog- ene (ENSDARG00000040710; STAT1Ψ in Figure 7). same protein structure and a high degree of similarity. Traf1 and Traf5 are present in zebrafish, but no predictions exist for these genes in the pufferfish genomes. It is interesting that zebrafish Traf1 differs from mammalian Traf1 in that, like the other family members, it contains a Ring finger and a zinc fin- ger (Figure 6), indicating that the absence of these domains in mammalian Traf1 is due to a loss that occurred specifically in TNF-receptor associated factors All of the Traf protein family members Traf1 to Traf7 are rep- resented in fish (Figure 6). For Traf3, Traf6, and Traf7 we find one gene in each of the three fish species, in all cases with the TNF-receptor associated factors All of the Traf protein family members Traf1 to Traf7 are rep- resented in fish (Figure 6). For Traf3, Traf6, and Traf7 we find one gene in each of the three fish species, in all cases with the Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.8 http://genomebiology.com/2007/8/11/R251 Synteny between regions containing STAT4 and STAT1 genes on human chromosome 2 and Danio rerio chromosomes 22 and 9 Figure 7 Synteny between regions containing STAT4 and STAT1 genes on human chromosome 2 and Danio rerio chromosomes 22 and 9. Genes transcribed on the top or bottom strands are shown above and below the lines representing the chromosomes. Homologous regions are shown by colored arrows. A further duplication of the region containing HIBCH and GDF8 is found on Danio rerio chromosome 11. Numbers represent nucleotide positions in the genome in megabases based on the Zv6 assembly. Gene names are Swissprot, Zebrafish Information Network, or Ensembl identifiers. 191 192 HIBCH GDF8 STAT4 STAT1 Nab1 INPP1 NP060164 GLS Myo1B SPEG RQCD1 PLCD4 219 HIBCH GDF8 STAT4 STAT1b Nab1 zgc92925 GLS 39 STAT1Ψ 40 T000059586 HIBCH GDF8 RQCD1 PLCD4 13 14 GDF8 STAT1 Nab1 Q1LUQ4 Q1LWI2 addit ional copy of t his on Dr chrom. Phylogenet Figure 6 11 Dr chromosome 22 Hs chromosome 2 Dr chromosome 9 13 14 Synteny be Figure 7 y y g g g g Synteny between regions containing STAT4 and STAT1 genes on human chromosome 2 and Danio rerio chromosomes 22 and 9. Genes transcribed on the top or bottom strands are shown above and below the lines representing the chromosomes. Homologous regions are shown by colored arrows. A further duplication of the region containing HIBCH and GDF8 is found on Danio rerio chromosome 11. Numbers represent nucleotide positions in the genome in megabases based on the Zv6 assembly. Gene names are Swissprot, Zebrafish Information Network, or Ensembl identifiers. the mammalian lineage. Traf4 is duplicated only in zebrafish [30], whereas there have been several duplication events in the fish lineage for Traf2. authors subdivided the genes into groups encoding ligand- binding and non-ligand-binding chains before conducting their phylogenetic analysis. However, the justification for the assignment of particular fish genes that have no clear orthologs in mammals to one or other group is not obvious, especially because no sequence data were given in this study that unambiguously identify the genes analyzed. We therefore revisited the phylogeny of class II cytokine recep- tors in teleosts and mammals. In summary, for the families described thus far, clear orthol- ogies exist between the teleost and mammalian lineages, with a few duplications for some of the gene family members. Class II cytokines and their receptors Class II cytokine receptors The family is defined by the presence of the D200 domain, which consists of two immunoglobulin domain-like sub- domains of the fibronectin type III class, SD100A and SD100B. As has previously been pointed out [17], the bioin- formatic identification of class II cytokine receptor genes is not trivial, and it is therefore unsurprising that Ensembl [24] contained predictions for only ten such genes in zebrafish. Three of these do not encode class II cytokine receptors but for thrombopoietin and titins, which have similar domains. To identify further receptor genes we searched the zebrafish genome and all available zebrafish ESTs for the subdomains SD100A and SD100B (see Materials and methods, below). Mammals have two distinct, heterodimeric receptors for type I and type II IFNs, as well as a set of closely related receptors for other class II helical cytokines. Although a large group of this type is found in fish, there are no simple orthologies between the receptors of this class in mammals and teleost fish [16,17,31]. A previous analysis identified 11 genes in Tetraodon, named cytokine receptor family B (CRFB)1 to CRFB11 [17]. The authors found that the genomic region con- taining IFN-α receptor (IFNAR)chain 2, IL-10 receptor (IL10R)chain 2, IFNAR1, and IFN-γ receptor (IFNGR)chain 2 in mammals is syntenic with a region containing six class II cytokine receptor genes in Tetraodon [17] (see Figure 8). However, sequence comparison allowed no clear assignment of the fish genes to their mammalian counterparts, with the exception of the genes encoding tissue factor (TF), which is duplicated in Tetraodon (TF1 and TF2). A subsequent study [31], which included all available sequences throughout the animal kingdom, came to a slightly different conclusion regarding the phylogenetic relationships. In this study the We identified 22 candidates, of which seven had incomplete D200 domains or exhibited only spurious resemblance to D200 domains. These and the three genes encoding the D200-containing proteins thrombopoietin and titin were eliminated from further analysis. Gene predictions were available for eight of the remaining 12 genes. Of the four genes Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.9 http://genomebiology.com/2007/8/11/R251 Syntenic organization of classII cytokine receptor genes Figure 8 Syntenic organization of classII cytokine receptor genes. Class II cytokines and their receptors Class II cytokine receptors Round brackets denote groups of genes in cases where there are no clear orthologous relationships of individual members with genes in the other species. that had not been predicted by automated annotation tools, two (CRFB15 and CRFB16) were found only in the as yet unplaced whole genome shotgun sequences. We re-annotated all 12 genes using the known gene structure of class II cytokine receptor genes and homology to known class II receptor genes as support. We used these sequences for a phy- logenetic analysis, which, in addition to the mouse and human sequences, also included Takifugu rubripes and Tetraodon nigroviridis CRFB1 to CRFB11 and IL20R2, as well as an additional gene, the product of which we shall call CRFB13 (Ensembl: NEWSINFRUG00000164405 and GSTENG0003154300). A set of recently described zebrafish class II cytokine receptor genes included two genes not iden- tified by us (DrCRFB2 and DrCRFB6), which we have added to our analysis [18]. Finally, DrCRFB14 was found by Georges Lutfalla, who generously contributed its sequence for inclu- sion in this analysis. four exceptions in which high bootstrap values justify the interpretation of the genes sharing direct common ancestors. The genes encoding TF in mammals cluster with two genes from each fish. The phylogeny indicates independent duplica- tion events in the pufferfish and zebrafish lineage. The other set of genes that reliably group together are those encoding IL20R1, IL20R2, and IL-22 binding protein (IL22BP), with one representative in each of the mammals and fish. For the other relationships between mammalian and fish genes the bootstrap values are so low that the relationships discussed below must be considered with caution. Several mammalian genes have no plausible orthologs in the three fish genomes analyzed here, and others have more than one. We therefore sought further evidence for evolutionary rela- tionships by analyzing the genomic context of the genes. A summary is shown in Figure 8. Two sets of genes are linked both in mammals and in the two pufferfish. The first is the IFNAR2, IL10R2, IFNAR1, and IFNGR2 complex and its syntenic complex described by Lutfalla and colleagues [17] for Tetraodon. This synteny is also maintained in Takifugu and in all three cases continues outside the class II cytokine recep- The phylogram of the class II cytokine receptors (Figure 9) corroborates previous conclusions that this gene family has undergone independent gene duplications and divergence in teleost fish and mammals. Class II cytokines and their receptors Class II cytokine receptors (a) Diagram of the structures of the mammalian receptor chains, with the blue and green rectangles representing the S100A and S100B domains, the red rectangle the intracellular domain of the ligand binding chains, and the gray rectangle the intracellular domain of the non-ligand-binding chains and TF (after Renauld [57]). (b) Synteny between regions containing class II cytokine receptor genes in mammals and fish. Fat horizontal lines indicate chromosomes in the four species. The brackets above the human genes show evolutionary relationships between the paralogs. Vertical broken lines indicate suggested evolutionary relationships between the genes in the different species, based on the tree in Figure 9. Color coding of names: red = long intracellular domain; black = short intracellular domain; blue = no intracellular domain; and pink = intermediate length intracellular domain. Circled names indicate ligand binding chains. Round brackets denote groups of genes in cases where there are no clear orthologous relationships of individual members with genes in the other species. IL20R2 IFNGR1 IL20R1 IL22BP IL10R1 IL22R1 IL28R1 TF IFNGR2 IFNAR1 IFNAR2 IL10R2 IL20R2 TF2 TF1 CRFB8 CRFB9 CRFB7 CRFB4 CRFB13 CRFB3 CRFB2 CRFB1 CRFB5 CRFB6 Tn IL20R2 TF2 TF1 CRFB8 CRFB9 CRFB7 CRFB4 CRFB13 CRFB3 CRFB1 CRFB5 CRFB6 Fr CRFB16 TFa TFb CRFB15 CRFB14 Hs Dr CRFB2 CRFB6 CRFB9 CRFB7 CRFB1 CRFB4 CRFB5 CRFB8 (a) Structure of the mammalian receptor chains (b) Chromosomal organization CRFB12 CRFB13 (a) (b) Chromosomal organization b) Chromosomal organization Syntenic o Figure 8 Syntenic organization of classII cytokine receptor genes Figure 8 Syntenic organization of classII cytokine receptor genes. (a) Diagram of the structures of the mammalian receptor chains, with the blue and green rectangles representing the S100A and S100B domains, the red rectangle the intracellular domain of the ligand binding chains, and the gray rectangle the intracellular domain of the non-ligand-binding chains and TF (after Renauld [57]). (b) Synteny between regions containing class II cytokine receptor genes in mammals and fish. Fat horizontal lines indicate chromosomes in the four species. The brackets above the human genes show evolutionary relationships between the paralogs. Vertical broken lines indicate suggested evolutionary relationships between the genes in the different species, based on the tree in Figure 9. Color coding of names: red = long intracellular domain; black = short intracellular domain; blue = no intracellular domain; and pink = intermediate length intracellular domain. Circled names indicate ligand binding chains. Class II cytokines and their receptors Class II cytokine receptors Some of the fish genes cannot be matched to likely orthologs in mammals, and vice versa, with Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.10 http://genomebiology.com/2007/8/11/R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.10 tor complex, in that the gene neighboring IFNGR2 is Tm50b in all cases, followed by Nnp1. However, the corresponding genes in the zebrafish are no longer linked (although they all lie on the same chromosome). Phylogenetic tree of the class II cytokine receptors Figure 9 HsIL20R2 MmIL20R2 DrCRFB16 FrIL20R2 TnIL20R2 HsIL22BP MmIL22BP DrCRFB9 FrCRFB9 TnCRFB9 HsIFNAR2 MmIFNAR2 DrCRFB1 FrCRFB1 TnCRFB1 DrCRFB2 TnCRFB2 HsIL20R1 MmIL20R1 DrCRFB8 FrCRFB8 TnCRFB8 HsTF MmTF FrTF2 TnTF2 DrTFb DrTFa FrTF1 TnTF1 HsIL28R1 MmIL28R1 DrCRFB14 HsIL10R1 MmIL10R1 HsIL22R1 MmIL22R1 DrCRFB12 HsIFNGR1 MmIFNGR1 DrCRFB13 FrCRFB13 TnCRFB13 DrCRFB7 FrCRFB7 TnCRFB7 DrCRFB6 FrCRFB6 TnCRFB6 FrCRFB3 TnCRFB3 HsIFNGR2 MmIFNGR2 HsIFNAR1 MmIFNAR1 HsIL10R2 MmIL10R2 FrCRFB4 TnCRFB4 DrCRFB4 DrCRFB15 DrCRFB5 FrCRFB5 TnCRFB5 100 100 98 86 93 68 100 100 76 100 75 100 100 99 100 62 100 100 82 100 34 100 100 100 91 100 100 100 12 100 48 100 100 80 91 83 100 100 79 94 100 100 49 100 44 100 100 100 100 100 79 48 39 100 100 46 84 66 53 100 32 0.1 The synteny is roughly reflected in the sequence similarities, in that IFNAR2 is most similar to CRFB1 and CRFB2 and that the IL10R2/IFNAR1/IFNGR2 group clusters with the CRFB3/4/5/6/15 group. In particular, the IL10R2/IFNAR1/ IFNGR2 and CRFB3/4/5/6/15 genes encode receptors with short cytoplasmic domains, whereas IFNAR2 and CRFB1 and CRFB2 have long cytoplasmic tails. However, within the group orthologies are not clear. It is therefore not possible to conclude whether the ancestral complex that existed before the split of the teleosts and tetrapods contained two genes (a precursor for IFNAR2 and a precursor of the IL10R2/ IFNAR1/IFNGR2 group) with subsequent independent duplications in teleosts and mammals, or four genes, with fast divergence in the IL10R2/IFNAR1/IFNGR2 and the CRFB3/ 4/5/6/15 groups obscuring their common origin. The second region in which a syntenic arrangement of genes is retained is the one containing IFNGR1, IL20R1 and IL22BP in mammals, and CRFB9 and the previously undetected CRFB13 in Tetraodon and Takifugu. Again, the closest rela- tives of these genes (CRFB9 and CRFB13, respectively) are not syntenic in zebrafish. Class II cytokines and their receptors Class II cytokine receptors Notably, fish CRFB9 proteins share the absence of a transmembrane domain with the mamma- lian IL22BPs. In view of this and the syntenic arrangement, the most reasonable interpretation is a homology of IFNGR1/ CRFB13 and IL22BP/CRFB9. In summary, teleost fish have approximately the same number of class II cytokine receptors as mammals, but the genes have evolved rapidly and independently since the sepa- ration of the species. We shall leave the discussion at this point, because the current set of data does not support further speculation. A statement about which of these receptors are functionally equivalent will have to await experimental anal- ysis, as has been conducted for two of the zebrafish CRFBs [18]. It will be interesting to determine whether fish distin- guish between viral and bacterial induced IFN signaling path- ways in the same way as mammals. Class II cytokines y IFNs have been reported in several fish species with an ambiguous nomenclature [32-40]. We find ten class II cytokine genes in zebrafish, and five in each pufferfish (Figure 10). The large group of mammalian type I IFNs cluster together on one branch of the phylogenetic tree that does not include any fish cytokines. This fits with the view that the gen- erally intronless type I IFN genes are the product of a retro- transposition event [17], which occurred after the split of teleosts and tetrapods. Apart from the clear fish orthologs of the mammalian type II IFNs, the remaining fish class II cytokines are more similar to the mammalian ILs and type III Phylogenetic tree of the class II cytokine receptors Figure 9 Phylogenetic tree of the class II cytokine receptors. Details are as in figure 2. y g y p g Phylogenetic tree of the class II cytokine receptors. Details are as in figure 2. Genome Biology 2007, 8:R251 http://genomebiology.com/2007/8/11/R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.11 IFNs. Like these, they are mostly encoded by genes with four phase 0 introns, supporting the view that this constitutes the gene structure of the ancestral class II cytokine gene. Phylogenetic tree for the classII cytokines Figure 10 Phylogenetic tree for the classII cytokines Details are as in Figure 2 See HsIFNα8 HsIFNα17 HsIFNα4 HsIFNα10 HsIFNα21 HsIFNα7 HsIFNα14 HsIFNα1 HsIFNα2 HsIFNα5 HsIFNα6 MmIFNα2 MmIFNα11 MmIFNα4 MmIFNα9 MmIFNα1 MmIFNα5 MmIFNα6 MmIFNα7 MmIFNα10 MmIFNα12 HsIFNω HsIFNκ MmIFNκ HsIFNε1 MmIFNε1 HsIFNβ MmIFNβ DrIFNφ6 HsIFNλ1 HsIFNλ2 HsIFNλ3 MmIFNλ1 MmIFNλ3 MmIFNλ2 HsIL10 MmIL10 DrIL10 FrIL10 TnIL10 HsIL19 MmIL19 HsIL20 MmIL20 DrIL34 FrIL34 TnIL34 HsIL26 HsIL24 MmIL24 HsIL22 MmIL22 FrIL35 TnIL35 HsIFNγ MmIFNγ DrIFNγ1 DrIFNγ2 FrIFNγ TnIFNγ DrIFNφ2 DrIFNφ3 DrIFNφ4 DrIFNφ1 FrIFNφ TnIFNφ DrIFNφ5 100 100 100 100 99 100 100 100 100 100 100 100 100 100 100 99 100 99 87 84 100 98 15 18 38 99 53 86 17 14 15 34 40 100 26 19 28 39 41 100 95 100 99 99 100 100 62 69 41 50 100 68 97 100 100 42 34 97 36 29 20 17 26 13 0.1 Among these class II cytokines, IL-10 exhibits an apparent orthology between fish and mammals [41,42]. Class II cytokines This is also supported by the genomic locations of the IL-10 genes, which are situated adjacent to and on the opposite strand of the Mapkap2 genes in all five species (Figure 11). The genes that had been annotated as IL-20 in the zebrafish (Refseq: NP_001076424.1) and Tetraodon (Uniprot: Q7SX60), and initially as IL-19 and then changed to IL-24 in Takifugu (Ensembl: SINFRUG00000154816) are equally related to mammalian IL-19 and IL-20. The previous automated nam- ing of the fish genes should therefore be amended. In con- cordance with the nomenclature rules for vertebrate gene families, this gene has therefore been given the next available number in the IL series (IL-34). The fish IL-34 genes and the mammalian IL-19, IL-20, and IL-24 genes are located in the vicinity of the IL-10 genes (in the zebrafish this gene has not yet been placed on a chromosome), but duplications and inversions have broken up the syntenic relationships down- stream of IL-10. The phylogenetic tree argues for a common precursor for these genes that has duplicated in mammals, yielding IL-19 and IL-20. Whether IL-24 is the product of a second local duplication or of an older duplication of a larger segment of the genome is not clear, but it shows a higher degree of similarity to the class II cytokine genes found in a complex on a different chromosome in all five species (Figure 11). A second group of class II cytokines exhibiting high sequence similarity are the mammalian IL-22, IL-24 and IL-26, and two pufferfish interleukins annotated as 'IL-24' in Tetraodon (Uniprot: Q7SX82) and 'homologous to IL-24' in Takifugu (Ensembl: SINFRUG00000156387). Again, the phylogram shows that this name is problematic, because if anything these proteins are more similar to IL-22, and their genes exhibit the same syntenic relation to the flanking MDM1 gene as the IL-22 genes do in mammals (Figure 11). However, the zebrafish gene in the same position (RefSeq: NP_001018628), annotated as IL-22 [33], is highly divergent in sequence. Because frequent duplications and loss of genes as well as rapid sequence divergence appear to operate within this family, originally orthologous genes may no longer be recognizable. This is further illustrated by the flanking IL-26 gene in the human genome. Class II cytokines The mouse genome has lost this gene; in the zebrafish a class II cytokine gene described as IL- 26 [33] is present in this position, but it does not cluster with the IL-22/24/26 group. Although the IL genes between MDM1 and IFN-γ are in apparently orthologous positions in all five species, there is no indication that the mammalian arrangement MDM1/IL-22/IL-26/IFN-γ represents the ancestral cluster, rather than the IL genes having arisen by independent duplications in mammals and teleosts. Because the names given to the fish cytokines of this group are Phylogenetic tree for the classII cytokines Figure 10 Phylogenetic tree for the classII cytokines. Details are as in Figure 2. See text for gene names. Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.12 Genome Biology 2007, Volume 8, Issue 11, Article R25 http://genomebiology.com/2007/8/11/R251 Genomic organization of two class II cytokine gene clusters Figure 11 Genomic organization of two class II cytokine gene clusters. Chromosomes are shown as lines with the positions of the region marked in megabase pairs underneath. Genes transcribed on the top strand are shown above the line, and those transcribed in the opposite direction are shown below. Class II cytokine encoding genes are shaded in gray. In the left diagram the syntenic regions and duplications, and inversions surrounding the IL-10 locus are shaded in red and blue. The human IL-10 gene is located on chromosome 1 and the region shows the same arrangement as in the mouse. The current zebrafish genome assembly Zv7 does not yet contain the recently sequenced clone CU459075, which places IL-34 into the interval between IL-10 and prolargin (IL- 34 is included in Zv7 on the unplaced contig Zv7_NA1656). There are therefore no coordinates for the right end of the interval. The two pufferfish show the same arrangement both for the region around IL-10 and for the MDM1/cytokine/IFN-γ region. The names for the fish genes are explained in the text. IFN, interferon; IL, interleukin. Genomic or Figure 11 Genomic organization of two class II cytokine gene clusters Figure 11 Genomic organization of two class II cytokine gene clusters. Chromosomes are shown as lines with the positions of the region marked in megabase pairs underneath. Genes transcribed on the top strand are shown above the line, and those transcribed in the opposite direction are shown below. Class II cytokine encoding genes are shaded in gray. In the left diagram the syntenic regions and duplications, and inversions surrounding the IL-10 locus are shaded in red and blue. The human IL-10 gene is located on chromosome 1 and the region shows the same arrangement as in the mouse. The current zebrafish genome assembly Zv7 does not yet contain the recently sequenced clone CU459075, which places IL-34 into the interval between IL-10 and prolargin (IL- 34 is included in Zv7 on the unplaced contig Zv7_NA1656). There are therefore no coordinates for the right end of the interval. The two pufferfish show the same arrangement both for the region around IL-10 and for the MDM1/cytokine/IFN-γ region. The names for the fish genes are explained in the text. IFN, interferon; IL, interleukin. In summary, like the receptors, the class II cytokine genes have duplicated and diverged independently in fish and mammals. It remains to be tested experimentally which class II cytokines are responsible for which immune function. extremely confusing and suggest relationships for which there is no evidence, we again propose a new nomenclature, as shown in Figures 10 and 11 (IFN-ϕ6 for zebrafish IL-22, IFN-ϕ5 for zebrafish IL-26, and IL-35 for the pufferfish IL- 24). Class II cytokines 20.3 mapkap2 Ikbke prolargin fibromodulin IL10 Dyrk3 Rassf5 IL34 20.5 8.96 Mb 8.86 mapkap2 Ikbke IL10 Dyrk3 prolargin fibromodulin IL34 133.1 132.7 Mb mapkap2 Ikbke IL10 Dyrk3 IL19 IL20 IL24 Rassf5 135.7 135.8 prolargin fibromodulin Dr chromosome 11 Mm chromosome 1 Tn chromosome 11 (and Fr scaff_79) 117.9 Mb IL22 IFN-g MDM1 117.5 19.246 IL35 IFNg MDM1 19.260 Mb 14.06 13.99 Mb IFN-phi6 IFNg2 MDM1 IFN-phi5 IFNg1 IL22 IFN-g MDM1 67.00 IL26 66.80 Mb Dr chromosome 4 Mm chromosome 10 Tn Un_random (and Fr scaff_167) Hs chromosome 12 117.9 Mb IL22 IFN-g MDM1 117.5 19.246 IL35 IFNg MDM1 19.260 Mb 14.06 13.99 Mb IFN-phi6 IFNg2 MDM1 IFN-phi5 IFNg1 IL22 IFN-g MDM1 67.00 IL26 66.80 Mb Dr chromosome 4 Mm chromosome 10 Tn Un_random (and Fr scaff_167) Hs chromosome 12 20.3 mapkap2 Ikbke prolargin fibromodulin IL10 Dyrk3 Rassf5 IL34 20.5 8.96 Mb 8.86 mapkap2 Ikbke IL10 Dyrk3 prolargin fibromodulin IL34 133.1 132.7 Mb mapkap2 Ikbke IL10 Dyrk3 IL19 IL20 IL24 Rassf5 135.7 135.8 prolargin fibromodulin Dr chromosome 11 Mm chromosome 1 Tn chromosome 11 (and Fr scaff_79) IL10 Overview o Figure 12 Overview of a phylogenetic tree of 277 NLR proteins Figure 12 Overview of a phylogenetic tree of 277 NLR proteins. Each sequence is assigned a background color to illustrate species relationships: pink = human, yellow = mouse, blue = zebrafish, green = Takifugu, and turquoise = Tetraodon. The 'canonical' proteins Nod1, Nod2, Nod3, Nod9, CIITA, and Apaf, which show clear homologous relationships between the five species, cluster at the top (rainbow colors). The mammalian Nalp proteins cluster together (pink/yellow region). Each fish has a large group of species-specific proteins (blue, green, and turquoise regions). In addition, Takifugu and Tetraodon share several apparently orthologous gene pairs (green and turquoise region). Apaf, apoptotic protease activating factor; CIITA, major histocompatibility complex class II, transactivator; Nalp, NACHT, leucine rich repeat and PYD containing protein; NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family; Nod, nucleotide oligomerization domain containing protein. lian Nalps on a phylogenetic tree. We found no homologs for any of the mammalian Nalps in fish. We therefore screened the whole zebrafish genome for sequences encoding NACHT- domains. This revealed a large number of additional sequences encoding NACHT domains. Most of these were not within genes found by the automated gene prediction algo- rithms, because the number of and similarity between the genes was so high that they had been masked as repeats. We therefore annotated these genes manually using ESTs as guides and identified a large set of novel NACHT-domain containing genes. After we had completed our initial annota- tions, automated predictions for 205 NACHT-domain encod- ing genes were deposited at the National Center for Biotechnology Information (NCBI). These showed only a par- tial overlap with our sequences. Many were incomplete or contained two NACHT domains, indicating incorrect annota- tions. We therefore re-screened and re-annotated the zebrafish genome and have found more than 200 genes of this class (the complete list is given in Additional data file 9). These are numbered sequentially by chromosome number and by their order on the chromosome. We have not been able to produce perfect gene models for all of them. As discussed below, they have novel amino-terminal sequences, and in the absence of sufficient EST evidence we were unable in all cases to draw reliable conclusions regarding the 5' end of the gene. Similarly, the LRRs in the carboxyl-terminal region are diffi- cult to predict reliably. Intracellular pathogen sensors: the NACHT-domain family Four of the remaining fish class II cytokine genes cluster with the mammalian INF-γ genes and the rest do not group with any of the mammalian genes. The pufferfish each have one IFN-γ gene, whereas the zebrafish has two, namely IFN-γ1 and IFN-γ2 [33,34], which lie in tandem in a position in the genome that has retained its synteny between mammals and teleosts (Figure 11). A large family of cytoplasmic proteins, characterized by the presence of a nucleotide-binding domain, the NACHT domain [44,45] or the closely related NB-ARC domain [46], has been implicated in inflammation and innate immune sig- naling in animals and plants. Some of them have been shown to recognize intracellular pathogen-associated molecular pat- terns through their carboxyl-terminal leucine-rich repeats (LRRs). They differ in their amino-terminal effector domains (for example, CARD or pyrin domains), which mediate signal transduction to downstream targets, leading to the activation of NF-κB or the apoptotic pathway. Finally, a group of teleost class II cytokines, some of which had previously been called IFN-λ, cluster on a branch without mammalian cytokines. Because they are not more related to mammalian IFN-λ than to other cytokines, we call them IFN- ϕ1 to IFN-ϕ4. IFN-ϕ1 has previously been described as 'zebrafish interferon', 'IFNab', and 'IFN-λ' [17,18,32], and IFN-ϕ2 and IFN-ϕ3 as 'type I IFN 2' and 'type I IFN 3' [43]. Only one gene of this type, most closely related to the zebrafish IFN-ϕ1 gene, is found in the two pufferfish. This may be due to the difficulty in identifying these genes, and it would not be surprising if further class II cytokine genes were found in the pufferfish genomes. An initial search in the fish genomes for homologs of the known mammalian NLR proteins of the Nod subfamily found homologs for Nod3 and Nod9 in all three fish species: Nod2 in zebrafish and Takifugu, and Nod1 in Takifugu. Three genes in zebrafish, two in Takifugu, and one in Tetraodon were annotated as 'Nalps' (NACHT, leucine rich repeat and PYD containing proteins) but did not group with the mamma- Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein http://genomebiology.com/2007/8/11/R251 Figure 12 Hs Tn Fr Dr Mm Nod1 Nod2 CIITA Nod9 mammalian Nalp Apaf1 NACHT-P1 Ipaf Nod3 Fr/ Tn expansion Dr expansion Fr expansion Tn expansion Fr/ Tn expansion Phylogenetic relationships of NLR protein families in mammals and fish Fish-specific properties of novel fish NLR proteins The large groups of novel, fish-specific NLR proteins are highly conserved in each species, indicating recent species- specific expansions (Figure 12 and additional file 10). Like other NLR proteins, they contain LRRs at the carboxyl-termi- nus, but the majority does not contain any of the amino-ter- minal effector domains that have been found in conjunction with NACHT-domains in mammals or plants (such as CARD, pyrin or TIR domains). However, the region immediately upstream of the NACHT domain is highly conserved in all of the fish proteins (Figure 14). A phylogenetic tree of all NLR containing predicted peptides from human, mouse, and the three fish species reveals the fol- lowing relationships (Figure 13). The canonical Nod proteins Nod1, Nod2, Nod3 (recently renamed as Nlrc3) and Nod9 (recently renamed as NlrIX), as well as Apaf1 (apoptotic pro- tease activating factor 1) and CIITA (major histocompatibility complex class II, transactivator), are present in all five species and exhibit clear orthologous relationships (Figure 14). The Nalp proteins (which have recently been renamed Nlrp) form a separate branch, representing a mammalian expansion of NLR proteins. For most of the genes on this branch, there are closely related pairs of mouse and human genes, but several cases of mouse-specific or human-specific duplications can also be found, notably the mouse Nalp4 genes. Two zebrafish sequences that cluster with this group, 2.03 and 2.05, encode only a NACHT domain with a divergent P-loop and should therefore not be considered Nalp-like proteins. To find out whether this region corresponded to other known peptide motifs, we used a hidden Markov model built from the zebrafish sequences for a BLAST search of the mamma- lian genomes. No good matches were found. We then searched the three fish genomes. In the zebrafish and in Tak- ifugu we found only those genes we had already identified via their NACHT domains. In the Tetraodon genome many but not all of the matches we found were upstream of NACHT domains or were part of our previous gene predictions. As the remaining ones were again located mainly in the Un-random set, we did not attempt to link them to the predictions for the NACHT domains, for the reasons discussed above. As in the other two fish genomes, none of the matches were within gene predictions for other (non-NACHT-domain) genes. Overview o Figure 12 Extensive experimental work will be needed to characterize these genes. For our analysis here we have selected a set of 70 representative sequences. Fr/ Tn expansion Dr expansion Fr/ Tn expansion We also searched the two pufferfish genomes for members of this gene family to find out whether the group we found in zebrafish was specific to this species, or whether the massive gene duplication had occurred early in the fish lineage. We found 70 members of this family among the annotated genes in the genome of Takifugu rubripes. A large number of matches found in the Tetraodon genome were not parts of predicted or annotated genes, as had been the case in the zebrafish. Again, these sequences had been masked as repeats. We manually assembled a set of sequences using Figure 12 Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.14 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.14 http://genomebiology.com/2007/8/11/R251 homology to the zebrafish and Takifugu sequences as guides. It is striking that the majority of the members of this gene family (40/49) are located within incompletely assembled contigs/scaffolds that have not been assigned to chromosomes (the 'Un_random' set). Initially, our searches for NACHT-domain encoding genes resulted in a number of predictions that spanned separate contigs, but which had additional fragments of genes of this family interspersed within their predicted introns. This suggests that these predictions were not correct, but were due to accidental occurrence of apparently spliceable gene fragments in neigh- boring contigs of this assembly that are in fact not located next to each other in the genome. This view is supported by the finding that three sequences, which are very closely related to consecutive parts of the other fish Nod2 genes, were positioned on widely separated contigs in the Un_random assembly. We have combined these three fragments into one sequence, which we call TnNod2. The high proportion of genes from this family in the nonassembled part of the genome might be an indication that the proper assembly of these contigs is made difficult or impossible precisely because of the repetitive nature of this family. before the split of the two species and suggesting conserva- tion of their function. We note again that the Tetraodon gene predictions are less reliable and are often incomplete, leading to spurious homology assignments. Overview o Figure 12 The relationship of these sequences to the other fish sequences therefore represents an approximate picture that must be interpreted with caution. Whereas most of the novel fish NLR proteins are more related to each other than to mammalian NLR proteins, there are exceptions (apart from the canonical proteins mentioned above). One group of new fish proteins, which we named NACHT-P1, clustered with Apaf1. We wished to know whether this was a fish-specific NACHT protein and searched the mouse and human genomes for similar sequences. We found one ortholog in each case, neither of which had been characterized previously. Their amino-terminal parts contain no motifs known from other proteins. Like the Apaf proteins, these sequences contain WD40 repeats instead of LRRs. FrNACHT-P2 and TnNACHT-P2 have an unusual amino-ter- minal addition, a filament domain. We found no other sequence in any organism that encodes a protein composed of a filament domain and a NACHT domain. Phylogenetic relationships of NLR protein families in mammals and fish R251.16 Genome Biology 2007, Volume 8, Issue 11, Article R25 http://genomebiology.com/2007/8/11/R251 Phylogenetic tree of NACHT proteins shared by mammals and fish and diagram of their protein structures Figure 13 (see previous page) Phylogenetic tree of NACHT proteins shared by mammals and fish and diagram of their protein structures. In addition to the known proteins Nod1, Nod2, Nod3, Nod9, CIITA, and Apaf, this tree shows that a new protein is shared by all five species, which we have named NACHT-P1. The protein domain structure diagram shown next to NACHT-P3 is representative of the majority of the novel fish proteins. Apaf, apoptotic protease activating factor; CIITA, major histocompatibility complex class II, transactivator; Nod, nucleotide oligomerization domain containing protein. fish and Medaka, also associated with NACHT-domain encoding genes, which we did not follow up further. can be subdivided into four groups (Figure 14). Each of these groups has further shared motifs upstream of the Fisna- domain (Figure 15). The amino-terminal sequences in group 1 are highly conserved and not found in any of the other fam- ilies (darker green shading in Figure 15). A comparison with mammalian proteins showed that it has significant similarity with the pyrin-domain found in mammalian Nalp and MEFV (mediterranean fever)proteins. Group 2 has a 101 amino acid stretch upstream of the Fisna domain that is shared by all members of this group (lighter green shading in Figure 15). It shows a distant resemblance to the pyrin domain of group 1. The most amino-terminal sequences in this group contain motifs shared with members from groups 3 and 4. A motif shared by members from these three groups is a repeat (dif- ferent hues of blue shading in Figure 15 indicate different ver- sions of the repeat), which occurs in one, two, or three copies per protein, or in one case, in ten copies. Group 2 has a ver- sion of this repeat with a four-amino-acid insertion, which is also found in some members of group 3. These repeats are usually combined with a specific amino-terminal peptide of 14 amino acids (pink shading). Other conserved amino-ter- minal peptides (yellow or orange shading) are associated with a particular type of repeat. Group 4 is the least homogeneous, showing divergence both within the group and in comparison with the other groups, in the repeats as well as in the Fisna and NACHT domains. The fish-specific domain upstream of the NACHT domain Figure 14 (see following page) The fish-specific domain upstream of the NACHT domain. (a) Alignment of a representative subset of the Fisna domain (the region upstream of the NACHT domain that is shared by all of the novel fish NLR proteins. The group names on the right refer to the subdivision of the Danio rerio groups according to similarities in the NACHT-domain and the Fisna domain (also see Figure 15) or indicate which species form the group. Peptide motifs with similarity to Nod2 and Nod3 are underlined. (b) Hidden Markov model (HMM) logo representing the consensus sequence of the Fisna domain in all three fish species. The logo has been generated using the software HMMER [58,59] and visualized using the HMM-Logo web server [60,61]. Peptide sequences from human Nod2 and Nod3 with similarity to short stretches of the Fisna consensus, color coded to highlight conserved residues, are listed underneath, as are stretches from the regions upstream of the NACHT domain present in 140 sea urchin NLR proteins. NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family; Nod, nucleotide oligomerization domain containing protein. Genome Biology 2007, Volume 8, Issue 11, Article R251 Phylogenetic relationships of NLR protein families in mammals and fish This indi- cates that this domain, which we will call the Fisna (fish-spe- cific NACHT associated) domain, has been recruited specifically by a common ancestor of the novel NLR proteins in the fish lineage. Confirming this view, a cursory search of other fish genomes showed highly similar sequences in cat- Most strikingly, the large groups of newly identified fish sequences lie on mostly species-specific branches. The major- ity of the zebrafish genes form a branch of their own, which includes no genes from either of the two pufferfish. Consist- ent with the closer relationship between the two pufferfish, the genes from these two species are less clearly separated. Whereas one branch contains exclusively a subset of genes from Takifugu, the branch that contains the majority of Tetraodon genes also includes several Takifugu genes. There are two branches with several cases of apparent orthologies between Takifugu and Tetraodon (genes from the two species that are more similar to each other than to any other gene in their own species), indicating the existence of these genes Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.15 http://genomebiology.com/2007/8/11/R251 HsNod1 MmNod1 DrNod1 FrNod1 TnNod1 HsNod2 MmNod2 DrNod2 FrNod2 TnNod2 HsCIITA MmCIITA FrCIITA TnCIITA DrCIITA HsIpaf MmIpaf HsNaip MmNaip1 MmNaip6 HsApaf1 MmApaf1 DrApaf1 FrApaf1 TnApaf1 HsNACHT-P1 MmNACHT-P1 DrNACHT-P1 FrNACHT-P1 TnNACHT-P1 HsNod9 MmNod9 DrNod9 FrNod9 TnNod9 HsNod3 MmNod3 DrNod3 FrNod3 TnNod3 FrNACHT-P2 TnNACHT-P2 HsNalp10 MmNalp10 FrNACHT-P3a FrNACHT-P3b HsNalp6 MmNalp6 HsNalp1 MmNalp1 MmNalp1 like MmNalp1c HsNalp3 MmNalp3 HsNalp12 MmNalp12 HsNalp14 MmNalp14 HsNalp5 MmNalp5 HsNalp13 HsNalp8 HsNalp2 HsNalp7 MmNalp2 HsNalp11 HsNalp9 MmNalp9a MmNalp9c MmNalp9b HsNalp4 MmNalp4b MmNalp4d MmNalp4f MmNalp4a MmNalp4c MmNalp4e MmNalp4e like 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 97 100 100 100 100 100 100 100 77 100 92 100 100 100 100 100 100 100 100 100 100 100 99 100 100 100 100 100 100 99 82 94 87 52 35 84 85 39 99 75 34 23 62 98 60 33 95 0.1 100aa CARD NB-ARC NACHT LRR WD 40 PYD BIR Filament fish-specific HsNalp10 MmNalp10 Figure 13 (see legend on next page) Figure 13 (see legend on next page) Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. Phylogenetic relationships of NLR protein families in mammals and fish No significant homologies to the repeat sequence are found in mammals. Although, as mentioned above, there is no evidence for the presence of this domain other than in fish, we noticed that a short peptide motif within this domain (LK/E/NQ/K/ RYITE/D) is also found in mammalian Nod2 (LEDYITE), and another (LYIIEGESEGVNEEHEVLQ) just downstream of the first, in Nod3 (LLLVD/EGLSDLQQK/REHDLM/V/TQ). The region containing these sequences in Nod2 and Nod3 is neither part of the NACHT nor of the CARD domain and has not been assigned a cell biologic function. Their conservation in the new NLR gene families might indicate a shared origin and possibly shared functions. A similar expansion of NLR-encoding genes was recently described in the sea urchin [47,48]. We compared the pre- dicted sea urchin protein sequences with our sequences. In addition to sharing high similarity with the fish proteins in the NACHT domain and the LRRs, the sea urchin proteins also have a region upstream of the NACHT domain that is highly conserved among the sea urchin set of proteins, and includes sequence motifs similar to those in the fish proteins and in mammalian Nod2. Conclusion Our findings show that the components of the TLR and class II cytokine signaling systems known from mammals are also found in teleosts. Although all of the main constituents are present, there are differences in the degree to which the vari- ous functional groups are conserved. This is the case both for the divergence in sequence as well as for the creation of new genes by duplications. The greatest divergence is found in the NLR protein family, with lineage-specific expansions in each organism, as has also been found for this type of protein in echinoderms [47,48]. Similar, if less extreme, situations are found for the TLRs [6,7] and the novel immune-type receptors [8-10], gene fam- ilies that also have sets of orthologous receptors in fish and mammals as well as fish-specific expansions. Thus, the ele- ments of the systems that are directly involved in interactions with pathogen components are those that are most likely to diversify by undergoing lineage-specific expansions. Indeed, a study that specifically tested the role of lineage-specific gene families in five eukaryotic species found that the genes that were particularly prone to such expansions included those involved in responses to pathogens [50]. Furthermore, our results are in concordance with recent findings from a com- parison of three insect genomes that showed the following [51]: first, the genes associated with immune functions are on average more divergent than the rest of the genome; and sec- ond, that the divergence occurs primarily in those genes whose products interact with the pathogen. This study found that in addition to pathogen recognition proteins, this was also the case for the effectors, a set of proteins we have not analyzed in the zebrafish. The most highly conserved group of proteins are those involved in intracellular signal transduction downstream of the transmembrane receptors: the kinases, adaptors, Stats, Trafs and transcriptional regulators. They exhibit high sequence conservation and largely orthologous relationships, such that for each gene there is one copy in each species, and these genes are more closely related to each other than to other genes of the family. We see only a few cases of duplica- tions. In some cases (Ticam-1, Ticam-2, and IRAK2) there appear to have been gene duplications only in mammals, but more often we find additional genes in the fish genomes. Conclusion Additional copies of genes in the teleosts need not necessarily be generated by lineage-specific individual gene duplications, but may instead be remnants of the third whole genome duplication postulated for the teleost lineage [3]. We do not see as a general rule that for each mammalian gene there is more than one copy in the zebrafish genome. However, in the highly conserved gene groups we do in fact see more duplica- tions of fish genes than of mammalian genes (additional cop- ies for 12 genes in the case of teleosts, although not always in all three species, and only three duplicates in the two mammals). This suggests that at least some of these may indeed be remnants of the third whole genome duplication in teleosts, as is supported by the syntenic organization of the duplicated genes and the flanking genes in the case of the Stat genes. The expansion of gene families involved in pathogen recogni- tion is likely to reflect adaptations of the species to new pathogen environments. We have not yet tested whether there is a particularly high level of sequence variability asso- ciated with particular parts of the NLR proteins. The number of LRRs varies greatly, but it will be necessary to validate the gene models for each gene before any reliable conclusions can be drawn. It will also be interesting to see whether the genes are more polymorphic than other genes in the genome. The fact that the few ESTs that are available, which are derived from a different strain of zebrafish, do not correspond 100% to any of the gene models is a hint that this might be the case. The function of the NLR genes and the significance of their species-specific expansion will be an exciting topic for exper- imental analysis. The family of the class II cytokine receptors is neither highly conserved, nor does it exhibit species-specific expansions. The five species we compared have approximately the same number of receptor chain genes, but the divergence is so great that no reliable orthologies can be established. A similar lack of orthology is seen for the ligands. Apart from the lineage specific expansions of the type I IFNs, there are similar num- bers of class II cytokine genes in the five species, but they can- not be assigned into orthologous groups (with the exception of IL-10 and IFN-γ). Distribution in the genome for INF-ϕ1 and are involved in defense against viruses [18]. Similar studies will be necessary to determine the functions of the remaining ligands and receptors. The rapid evolution of the gene families for the class II cytokines and their receptors probably reflects the fact that the IFN system is frequently subverted by pathogens, resulting in the need for compensa- tory mutations to escape inactivation. Significantly, the receptor family member that is not primarily associated with pathogen defense, TF, does not exhibit this high level of divergence. for INF-ϕ1 and are involved in defense against viruses [18]. Similar studies will be necessary to determine the functions of the remaining ligands and receptors. The rapid evolution of the gene families for the class II cytokines and their receptors probably reflects the fact that the IFN system is frequently subverted by pathogens, resulting in the need for compensa- tory mutations to escape inactivation. Significantly, the receptor family member that is not primarily associated with pathogen defense, TF, does not exhibit this high level of divergence. The genes encoding the novel proteins are distributed throughout the genome. Some chromosomes contain single genes, or a few, widely spaced genes, but many of the genes occur in large tandem clusters (Figure 16). Peptide motifs in the amino-terminal part of the zebrafish NLR proteins Peptide motifs in the amino-terminal part of the zebrafish NLR proteins Further study of the amino-terminal regions of the new zebrafish NLR proteins showed that many of them contained considerable stretches of predicted peptide sequences upstream of the conserved fish domain, in some cases with multiple, related sequence repeats. Manual editing of the automated alignment created by ClustalW [49] revealed the following structure of the amino-terminal regions of this pro- tein family (Figure 15). In summary, the amino-terminal parts of the novel NLR pro- teins contain up to three different motifs, two of which are found only in fish. The Fisna domain is found in all of the pro- teins and is located immediately upstream of the NACHT domain. It is specific for this protein family in fish. Groups 1 and 2 contain a pyrin-related domain upstream of the NACHT domain. Members of groups 2 to 4 can in addition contain one or more copies of a motif that is also specific for the novel fish NLR proteins. Members of groups 3 and 4 contain multiple variants of this motif but no pyrin-domain-like sequences. Based on sequence similarity in the NACHT-domain, which is equally recognizable in the Fisna domain, the protein family Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.17 http://genomebiology.com/2007/8/11/R251 (a) (a) Figure 14 (see legend on previous page) (b) Genome Biology 2007, 8:R251 nd on previous page) (b) G Bi l 2007 8 R251 Figure 14 (see legend on previous page) (b) Genome Biology 2007, 8:R251 Figure 14 (see legend on previous page) (b) (b) Figure 14 (see legend on previous page) Genome Biology 2007, 8:R251 Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.18 http://genomebiology.com/2007/8/11/R251 Conclusion The strong divergence also prohibits speculations on which ligand might bind to which receptor in the zebrafish. For one pair this has recently been established experimentally; CRFB1 and CRFB5 are the receptor chains Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.19 http://genomebiology.com/2007/8/11/R251 Structure of the amino-termini of the new zebrafish NLR proteins Figure 15 Structure of the amino-termini of the new zebrafish NLR proteins. ClustalW alignment of the set of 70 predicted NLR proteins was truncated four amino acids downstream of the start of the Fisna domain, and the alignment of the remaining amino-terminal sequences was edited manually using Jalview [62]. Sequences that did not extend significantly beyond the Fisna domain were deleted, as were some sequences in groups with many similar or identical sequences. The remaining sequences represent a set of characteristic compositions of motifs found in the amino-terminal part of this family of proteins. (a) Overview of the alignment with characteristic sequence motifs shaded in color: green = pyrin-like domain in groups 1 and 2; blue = repeated motif (different shades of blue mark different versions of the repeat); yellow/orange tones = conserved amino-terminal amino acids; and pink = specific amino- terminal peptide of 14 amino acids. (b) Details of the alignment in panel a in which amino acid similarities and identities are highlighted in ClustalW colors. A set of mammalian PYD domains are aligned above the zebrafish group 1 and group 2 pyrin-like domains to illustrate the similarity. NLR, nucleotide- binding domain/NACHT domain and leucine rich repeat containing family. Structure o Figure 15 p g Structure of the amino-termini of the new zebrafish NLR proteins. ClustalW alignment of the set of 70 predicted NLR proteins was truncated four amino acids downstream of the start of the Fisna domain, and the alignment of the remaining amino-terminal sequences was edited manually using Jalview [62]. Sequences that did not extend significantly beyond the Fisna domain were deleted, as were some sequences in groups with many similar or identical sequences. The remaining sequences represent a set of characteristic compositions of motifs found in the amino-terminal part of this family of proteins. (a) Overview of the alignment with characteristic sequence motifs shaded in color: green = pyrin-like domain in groups 1 and 2; blue = repeated motif (different shades of blue mark different versions of the repeat); yellow/orange tones = conserved amino-terminal amino acids; and pink = specific amino- terminal peptide of 14 amino acids. (b) Details of the alignment in panel a in which amino acid similarities and identities are highlighted in ClustalW colors. A set of mammalian PYD domains are aligned above the zebrafish group 1 and group 2 pyrin-like domains to illustrate the similarity. NLR, nucleotide- binding domain/NACHT domain and leucine rich repeat containing family. Manual annotations of genes were carried out by the Havana group at the Sanger Institute, in accordance with human annotation workshop guidelines [53]. Materials and methods Software Standard web-based programs were used for sequence com- parisons, alignments, and phylogenies. The phylogenetic trees in the figures were generated using the MEGA software package [27]. Conclusion 4.08 4.44 4.41 14.14 23.05 4.42 14.40 4.30 4.14 4.31 4.21 4.46 4.50 4.19 4.12 4.49 4.38 4.34 4.02 4.01 18.03 4.28 18.11 4.17 4.15 4.09 3.06 3.08 1.25 1.27 1.26 1.20 1.28 1.31 1.13 1.21 1.18 1.22 1.24 1.17 1.01 up06 15.07a 25.01 15.03 15.04 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MA E E RV K GS LS E K H - - - - - - - - - - - S V RS GS F V S S S V S LK S DWS K GGP P P D LRGK T P S S V K S - - - - - MA E E RV K DS LS E K H - - - - - - - - - - - S V RS GS RV S S S V S LK S DHS K DGRP P NF RE K T P S S V K S - - - - - MA E E RV K DS LS E K H - - - - - - - - - - - S V RS GS CV S S S V S LK S DRS K DP - P P GF S GE RA S S A QS - - - - - MA E E RV K DS LS E K H - - - - - - - - - - - S V RS GS CV S S S V S LK S DRS K DR - P P E F S GE T P S S A QS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ME DT HT - - D LD LS T GC - - S S V HQK RA E A E P S CV S - - - - MK S DA S MT - P P V K F K S GNT GA A V S S V H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MA E E RV K DS LS E K H - - - - - - - - - - - S V RS GS C - - - - - - MK S DWS K E D - P P E F S GE T P S P A Q I - - - - - MA E E RV K DS LS E K H - - - - - - - - - - - S V RS GS CV S S A V S LK S DGS MGP P P E LS E K S P S S A - - - - - - - - MA E E RV K DS LS E K H - - - - - - - - - - - S V RS GS RV CS S V S LK S NRS K DNP P I F RE K T QS F A K S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V RS GS CV S S S V S MK S DGS MGHP P D LRE K T LS S A K S - - - - - - - - - - - - - - - - - - - - - - - - - S V HQK RA E A E P S CV S - - - - MK S DA S ME RP - I A F K S GNT GP A V S - - - - - - - - - - - - - - - - - - - - A A V RRRA E A E P S CV S - - - - LK S DA S MGHP E NNF RS E HT P P A LS - S QK - ME DT HS S GDQD LS T GC - - S P RK RK RE E A E P S CV S - - - - MRS DQS MGE P - LT F K T E NT QP V V S S CK - MGNT HT A GDQD LS T GC - - S S V HQK RA E A E P S CV S - - - - MK S DK S MA LP - I NF K S GNT RP A V S S V H - MGNT HT A GDQD LS T GC - - S S V HQK RA E A E P S CV S - - - - MK S DA S MH - P P I HF K S GNT GP A V S LV H - - - - - - - - - - - - - - - - - - - - - - MQK RA E A E P S CV S - - - - MK S DA S MGV P - I T F K S E NT GP A V S S V H - ME DK HT P GD LD LS T GC - - S S V HQNRA E A E P S CV S - - - - MK S DV S MD - P P T NF K S GNT GP A V S S RK - - - - - - - - - - - - - - - - - - - - - V RNGHRS P LS S CV S - - - - LK S NQS I GV RP D LS DGA V NS DS V K S S K K - - - - - - - - - - - - - - - - - - - - LK RK RE E S S LS S S MF - - - - I K S DHP F G LP LY LS DRA V NS DS V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MS LK K RE DE DS DS E M - - - - - - - - S S A S P GS I CGS - - - - V E S DGS I E K T P P A LNNA S V T S D LR - - - - - - - QNK RE DV DP T F E M - - - - - - - - S S A S P GS GCV S - - - - LK S DRS ME K A P LA LNDE S V T S E LRK - - - MS LQNE S E DE DS A CK M - - - - - - - - S S A S P GS S CV S - - - - MK S DQS I LMP P - N LS DV S V T S D LS R - - - - MS K RK RE DE DA A S E I - - - - - - - - S S A S P GS S CV S - - - - V K S DQS MK K N LP DF S DA S V T S D LS I S K T - MS QK K S E DV DS DS E M - - - - - - - - NS V S P GS A CE S - - - - LK S DWS ME K T P P N LGDR - - - - - - - - - - - - MK RP - - - - - - - - - - - - - - - - - - - - - E S P E S S S V S - - - - MK S GRS ME QP M - RF S DDP M I S DP R I N - - - MK RP - - - - - - - - - - - - - - - - - - - - - E S P E S S S V S - - - - MK S GRS ME QP M - RF S DDP V T S DP R I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - NV S - - - - V K S DQS MGY HP - N LS DE T LT S DF R - RK - - - - - - - - - - - - - - - - - - - - - - - - - - - E S S GP S GV S - - - - V K S DWS ME RP P - A LS NQP V T S DP R LRK - LQK K NE DE DE E S A S E M - - - - - - - - S Y P P P E S S CV S - - - - MK S DWS MV Y P P - D LS DA S V T S DP NNS K R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P S GV S - - - - I RS NT S LF LP P - N I S DGA V T S DP - - S RH - MQK S K - - - - - - - - - - - - - - - - - - - - - S P E S S V A S - - - - V T S DQS T D - P P QNY RG - - - - - - - - - - - - - MHRP D - - - - - - - - - - - - - - - - - - - - - P P GP S CV S - - - - I RS DWS MN - P P HN LS GDS DA RS - - - - - - - MDQP I HF K S GDT K S D L - - S S V HQK S T E S E S S CV S - - - - MRS NDK S V HQP L - - - - - - - - - - - - - - - - - MDDT Q I S RDE NV S P GC - - S S V HQK S T E S E P S CV S - - - - MRS DDE S V DQP L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HV P DQRHHS P DP S A V S - - - - LK S DK S MK T P I E LQNGHNP GDQS F - - - - - - - - - - - - - - - - - - - - - - - - V QQK RS DS P V HS V LP - - - - MK RHK S MK T P DQ LQNE DP L LNQS S - - - - MA E E RV K GS LS E K H MA E E RV K DS LS E K H MA E E RV K DS LS E K H MA E E RV K DS LS E K H MA E E RV K DS LS E K H MA E E RV K DS LS E K H MA E E RV K DS LS E K H ME DT HS S GDQD LS T GC MGNT HT A GDQD LS T GC MGNT HT A GDQD LS T GC ME DK HT P GD LD LS T GC ME DT HT - - D LD LS T GC S V RS GS F V S S S V S LK S DWS K W GGP P P D LRGK T S S V K S P S V RS GS RV S S S V S LK S DHS K DGRP P NF RE K T S S V K S P S V RS GS CV S S S V S LK S DRS K DP - P P GF S E G RA S S A QS S V RS GS CV S S S V S LK S DRS K DR - P P E F S GE T S S A Q P S S V RS GS C - - - - - - MK S DWS K E D W - P P E F S GE T A Q P S P I S V RS GS CV S S A V S LK S DGS MGP P P E L S S A S E K S P - - - S V RS GS RV CS S V S LK S NRS K DNP I P F RE K T QS F A K S - - V RS GS CV S S S V S MK S DGS MGH D P P LRE K T LS S A K S - S S V HQK RA E A E P S CV S - - - - MK S DA S MT - P P V K F K S GNT GA A V S S V H - S V H - - S V HQK RA E A E P S CV S - - - - MK S DA S ME RP - I A F K S GNT G A V S P - A A V RRRA E A E P S CV S - - - - LK S DA S MGHP E NNF RS E HT A P P LS S QK S P RK RK RE E A E P S CV S - - - - MRS DQS MGE P - LT F K T E NT Q V V S P S CK S S V HQK RA E A E P S CV S - - - - MK S DK S MA LP - I NF K S GNT R A V S P S V H S S V HQK RA E A E P S CV S - - - - MK S DA S MH - P P I HF K S GNT G A V S P LV H - - - MQK RA E A E P S CV S - - - - MK S DA S MGV P - I T F K S E NT G A V S P S V H S S V HQNRA E A E P S CV S - - - - MK S DV S MD - P P T NF K S GNT G A V S P S RK S V H - S S V HQK S T E S E S S CV S - - - - MRS NDK S V HQP L S S V HQK S T E S E P S CV S - - - - MRS DDE S V DQP L - - - - V P DQRHHS P DP S A V S - - - - LK S DK S MK T P I E LQNGHNP D G QS F V QQK RS DS P V HS V LP - - - - MK RHK S MK T P DQ LQNE D L P LNQS S - V RNGHRS P LS S CV S - - - - LK S NQS I GV RP D LS DGA V NS DS V K S S K K LK RK RE E S S LS S S MF - - - - I K S DHP F G LP Y L LS DRA V NS DS V - - - - - S P E S S V A S - - - - V T S DQS T D - P P QNY RG - - - - - - P P GP S CV S - - - - I RS DWS M W N - P P HN LS GDS DA RS S S A S P GS I CGS - - - - V E S DGS I E K T A P P LNNA S V T S D LR - - - - S S A S P GS GCV S - - - - LK S DRS ME K A P LA LNDE S V T S E LRK - - - S S A S P GS S CV S - - - - MK S DQS I LMP - P N LS DV S V T S D LS R - - - S S A S P GS S CV S - - - - V K S DQS MK K N LP DF S DA S V T S D LS I S K T NS V S P GS A CE S - - - - LK S DWS ME K T P P N LGDR - - - - - - - - - - - - - E S P E S S S V S - - - - MK S GRS ME QP M - RF S DDP M I S D R P I N - - - - E S P E S S S V S - - - - MK S GRS ME QP M - RF S DDP V T S D R P I - - - - - - - - - - - NV S - - - - V K S DQS MGY H - P N LS DE T LT S DF R - RK - - - E S S GP S GV S - - - - V K S DWS ME RP P - A LS NQP V T S D R P LRK - S Y P P P E S S CV S - - - - MK S DWS MV Y P P - D LS DA S V T S DP NNS K R - - - - - - P S GV S - - - - I RS NT S LF LP P - N I S DGA V T S DP - - S RH - MS LK K RE DE DS DS E M - - - QNK RE DV DP T F E M MS LQNE S E DE DS A CK M - MS K RK RE DE DA A S E I - MS QK K S E DV DS DS E M LQK K NE DE DE E S A S E M - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Group 1 2 3 4 NALP4_MOUS E NALP5_HUMAN NALP7_HUMAN MEFV_MOUS E MEFV_RAT MEFV_HUMAN S DF G LMWY LK E LNK K E F I K F K E F L I QE I LK LK LK Q I S WT E V K K A S RE D LA N L L LK CY E E NQA WDMT F N I LQK I NRK D LT E RA T E S S Y G LQWC LY E LDK E E F QT F K E L LK K K S S E S T T CS I P QF E I E NA NV E C LA L L LHE Y Y GA S LA WA T S I S I F E NMN LRT LS E K A RD E WT LQT L LE Q LNE DE LK S F K S L LWA F P LE DV LQK T P WS E V E E A DGK K LA E I LV NT S S E NWI RNA T V N I LE E MN LT E LCK MA K A GDH L LNT LE E L LP Y DF E K F K F K LQNT S LE K GHS K I P RGHMQMA RP V K LA S L L I T Y Y GE E Y A V R LT LQ I LRA T NQRQ LA E E LRK RV DH L LNT LE E L LP Y E LE K F K F K LHT T S LE K GHS R I P LS LV K MA RP I K LT R L L LT Y Y GE E Y A V R LT LQ I LRA T NQRQ LA E E LHK P S DH L LS T LE E LV P Y DF E K F K F K LQNT S V QK E HS R I P RS Q I QRA RP V K MA T L LV T Y Y GE E Y A V Q LT LQV LRA I NQR L LA E E LHR L L K E K L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 4.08 4.44 4.41 14.14 23.05 4.42 14.40 4.30 4.14 4.31 4.21 4.46 4.50 4.12 4.49 4.19 4.38 4.34 4.02 4.01 18.03 4.28 18.11 4.17 4.15 4.09 3.06 3.08 1.25 1.27 1.26 1.20 1.28 1.31 1.13 1.21 1.18 1.22 1.24 1.17 1.01 up06 15.07a 25.01 15.03 15.04 - 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- - - M (a) (b) - - - - - - - - - - - - - - - - - - - - - - MA NA K Q L LK NS LDE LV DA E LK E F QWY L - - - - - I NDHRD I S K A E ME NA DR LK T V DK MV S CF GP K GA V K T T V D I LRK I NQNE LA E E LE NK HK QGA V LE T CK S P P F DY T NT S RE LK - - - - - - - - - - - - - - - - - - - - - - MA NV K Q L LK K S LDE LV E DT LK DF QWH L - - - - - MNDHRE I S K A E ME NA DRRNT V DK LV S CF GS E RA V K I T V DT LRK LNQNQ LA E D LE NT QK QGA A S E T CK S P P V DY T HT S HE LK - - - - - - - - - - - - - - - - - - - - - - MA NV K QR LK DS LDE LK E DT LK DF QWH L - - - - - M I DHRE I S T GE LE NA DRRK T V DK LV S CF GS E RA V K I T V DT LRK I K QNQ LA E E LE K K QQQGA A S E T CK S P P V DY T NT S HE LK - - - - - - - - - - - - - - - - - - - - - - MA NV K Q L LDNS LDE L LE A E LK K F QRC L - - - - - V NDHRD I S K A E ME NA DR LDT V DK MV S CF GS E RA V K I T V NT LRK I K QNQ LA E E LE NT QK QGA A S E S CK S P P V DY T NT S RE LK - - - - - - - - - - - - - - - - - - - - - - MA NV K Q L LDNS LDE L LE A E LK K F QWC L - - - - - V NDHNE I S K A E LE NA DR LDT V DK MV S CF GS E RA V K V T V DT LRK I K QND LA DQ LE NT QNQGT A LE NCK T LP LDY T N I S HE LK - - - - - - - - - - - - - - - - - - - - - - MA NV K Q L LK NS LDE LRE A E LK E F QWC L - - - - - V NDHRE I S T A E LE NA DR LK T V DK LV S CF K P E RA LK T T V DT LRK I K QNE LA E E LE NT QK QGA A S E T CNS P P V DY T NT S RE LK - - - - - - - - - - - - - - - - - - - - - - ME NV K Q L LK NS LK E LV E V E LK E F QWC L - - - - - RNDY RC I S K S E ME NA DR LE T V DK ME S CF GP E GA V K I T V D I LRK I NQND LA E K LE NT QK Q - - A S E NS K T P - LDY T N I S HE LK - - - - - - - - - - - - - - - - - - - - - - MA S V E E L L LK S LE D LE NP E LK K F QWH L - - - - - K K Y P K R I Y K CE ME K A DR LDT V DK MV E CF GA E DA V NNT V S I LRK I NQNN LA E Q LE NE HK NQGS A S A DS K QV LQE N - - S K R LK - - - - - - - - - RRH LV A D LV S Y S - - - - - - - - - DN LQWI F QN LE S K M I RF L - - - - - K NQ LE NF RK I LQHK NRQE F I K E F I E NRS I LT E A A LD LT LF F LRE MK QDQA A DT LQG - - - - - - - - - - - - - - - - - E LF F I NQ LK - - - - - - - - - RRH LV A D LV S Y S - - - - - - - - - DN LQWI F QN LE S K MF RF L - - - - - K NQ LE NF RK I LQHK NRQE F I K E F NE NRS G I T E A A LD LT LF F LRE MK QDK A A DT LE G - - - - - - - - - - - - - - - - - E LF F I NHF K - - - - - - - - - V E Y E S - DS GDE T HRRHK S F T - DN LQS I F QN LE S K M I RF L - - - - - K NE LE K F K K I LK E E NRQE F V K E F NE NRS I I T E A A LD LT L LF LRE MK QDQA A DT LQG - - - - - - - - - - - - - - - - - E L LF NNQ LK - - - - - - - - - V E Y E S A DS GDE T HRRHK S F T - DN LQS I F QN LE S K M I RF L - - - - - K NQ LE NF K K I LQE E NRQE F V K E F NE NRS I I T E A A LD LT LF F LRE MT QDQA A DT LQG - - - - - - - - - - - - - - - - - E L LY I NQ LK - - - - - - - - - V E Y E A A D LGDE T HRRHK S F T - DN LQS I F QN LE S K M I RF L - - - - - K NE LE K F K K I LQE E NRE E F V K E F NE NRS I I T E A A LD LT LF F LRE MK QDQA A DT LQG - - - - - - - - - - - - - - - - - E LF F I NQ LK - - - - - - - - - V E Y E S A DS GDE T HRRHK S F T - DN LQS I F QN LE S K M I RF L - - - - - K NE LGNF K K I LQE E NS QE F V K E F T E NRS I I RE A A LD LT LF F LRE MK QDQA A D I LE D - - - - - - - - - - - - - - - - - E LF F I NQ LK - - - - - - - - - V E Y E LP Y S GDK T HRS HK GF T - K D LP R I F QN LE S K I I RY L - - - - - K NE LE K F K K I LQE E NRQDF V K HF NE NRS I I T E A A LD LT LF F LRE MK QDE A A DT LE G - - - - - - - - - - - - - - - - - E LF F I NQ LK - - - - - - - - - V E Y E LP E P RHRT HRRHK S F T - DN LV WI F QN LE NK I DRF L - - - - - K NE LK K F K K I LQE E NRQDF V K NF NDNRCR I T E A A LD LT LF F LRDMK QDE V A DT LQG - - - - - - - - - - - - - - - - - E LF F I NQ LK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QS V DGDDQT GD LQQDS LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K S V DGDDQT GD LQQDS LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E S V DGDDQT GD LQQDS LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V S V DGDDQT GD LQQDS LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - F S V DGDDQT GD LQQDS LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V - - - RDDQT GD LQQD L LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V S V DGDDQT GD LQQDS LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V S V DGDDQT GD LQQDS LQP E HDE LQRV K E QHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V S V DRDGQT GD LQQDS LQP E HDE LQRV K E QHK V S V DRDDQS GD L LQDS LQP E HDE LQRV K E QHK K K A E S L LS S CV S LK S DQS I GV RP D LS DGP V NS E S V K S S K K RK K A E S L LS S CV S LK S NK S I G I RP D LS DGP V NS DS V S S S Y QK HT S HY K T E A Y I Q I E S QQP V N - - - - - - - - - - - - - - - - - - - - DD LQRV K E QHK - RK RE E S S LS S S MS MK S DHY I D LP P - - - - - - - - - - - CK L I S NRK RE S S P LS S CV S MK S NHS MS LP P Y LS DGA V NS DS V S P Y Y QK H I S HF K T E A C LM I DS QQ LV H - - - - - - - - - - - - - - - - - - - - DD LQRV K DQHR - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - RRT K I LK L I T P V P NS T P NY QT HN I QDNT DA - - - - HM LE T GD LQRV K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QRK K I RK L I T P V P NS T P NY QT P I I QDNT DA - - - - HM LE T GD LQRV K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QRK K T HK P I T - - - - - - S NY QT H I I QDNT E A - - - - HT LE T GD LQRV K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S S QK K P K V V A F V QS S K S K NE T C I I QE NT E T P LQRQA LE T GD LQRV K DHHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S DA LDK P DY E K LH - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R I K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I K LQK I K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - CQNQRHM - - - - - - - - - - - - I QE NT E T V LQRQT LE T GD LQRV K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - RRE R LE S P E F S NV S V K S S RS ME QP MRF S GE P V T S DP HMMGF T S S F HY K NQGH I - - - - - - - - - - - - S QDNT A T I LQMQT LE T V N LQRV K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HF DQE I I E P V F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QT QA LE T GD LQR I K DQY K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T P R LE S P E LS GV S V K S DWS I E RP P A F S NE P V T S DP RRCD I HE RCV N I T QS N I GS - - - - - - - - - - - - - - - - - - - - - QT LK T RD LQRV K DQ LK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - NM LE K I K NK HK - - - - - - - - - - - - - - - - - - - - - - - - - - - GRK K E N LS QS QS RCGV S E S RP T E D I DCP P CRK RCRWGS S F S GS A HG I T RA DQQE NP G I LE K S V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HDE V QRV K DQHK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HT E LT F S HDH - - - - - - - - - LHA E V HK T F R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - RT E LRF S HGP - - - - - - - - - QHA E A T K T Y R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HV RS GDT E T D - - - - - - - - - LS HE A LNT F R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - HV RS GDT E T D - - - - - - - - - LS HK A LNT F R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T K Y Y QD I LT A DS LP GRF I HQQDS E T HRQK I QR - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - F K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - QQT K V S E I Q - - QA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - LK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - RK K A E S L LS S CV S LK S DQS I GV RP D LS DGP V NS E S V K S S K K RK RE E S S LS S S MS MK S DHY I D LP P - - - - - - - - - - - CK L I S N R LK NS LDE LV DA E LK E F QWY L W - - - - - I NDHRD I S K A E ME NA DR LK T V DK MV S CF GP K GA V K T T V D I LRK I NQNE LA E E LE LK K S LDE LV E DT LK DF QWH L - - - - - MNDHRE I S K A E ME NA DRRNT V DK LV S CF GS E RA V K I T V DT LRK LNQNQ LA E D LE LK DS LDE LK E DT LK DF QWH L - - - - - M I DHRE I S T GE LE NA DRRK T V DK LV S CF GS E RA V K I T V DT LRK I K QNQ LA E E LE LDNS LDE L LE A E LK K F QRC L - - - - - V NDHRD I S K A E ME NA DR LDT V DK MV S CF GS E RA V K I T V NT LRK I K QNQ LA E E LE LDNS LDE L LE A E LK K F QWC L W - - - - - V NDHNE I S K A E LE NA DR LDT V DK MV S CF GS E RA V K V T V DT LRK I K QND LA DQ LE LK NS LDE LRE A E LK E F QWC L W - - - - - V NDHRE I S T A E LE NA DR LK T V DK LV S CF K P E RA LK T T V DT LRK I K QNE LA E E LE LK NS LK E LV E V E LK E F QWC L W - - - - - RNDY RC I S K S E ME NA DR LE T V DK ME S CF GP E GA V K I T V D I LRK I NQND LA E K LE L LK S LE D LE NP E LK K F QWH L W - - - - - K K Y P K R I Y K CE ME K A DR LDT V DK MV E CF GA E DA V NNT V S I LRK I NQNN LA E Q LE DN LQWI F QN LE S K M I RF L - - - - - K NQ LE NF RK I LQHK NRQE F I K E F I E NRS I LT E A A LD LT LF F LRE MK QDQA A DT LQ DN LQWI F QN LE S K MF RF L - - - - - K NQ LE NF RK I LQHK NRQE F I K E F NE NRS G I T E A A LD LT LF F LRE MK QDK A A DT LE DN LQS I F QN LE S K M I RF L - - - - - K NE LE K F K K I LK E E NRQE F V K E F NE NRS I I T E A A LD LT L LF LRE MK QDQA A DT LQ DN LQS I F QN LE S K M I RF L - - - - - K NQ LE NF K K I LQE E NRQE F V K E F NE NRS I I T E A A LD LT LF F LRE MT QDQA A DT LQ DN LQS I F QN LE S K M I RF L - - - - - K NE LE K F K K I LQE E NRE E F V K E F NE NRS I I T E A A LD LT LF F LRE MK QDQA A DT LQ DN LQS I F QN LE S K M I RF L - - - - - K NE LGNF K K I LQE E NS QE F V K E F T E NRS I I RE A A LD LT LF F LRE MK QDQA A D I LE K D LP R I F QN LE S K I I RY L - - - - - K NE LE K F K K I LQE E NRQDF V K HF NE NRS I I T E A A LD LT LF F LRE MK QDE A A DT LE DN LV WI F QN LE NK I DRF L - - - - - K NE LK K F K K I LQE E NRQDF V K NF NDNRCR I T E A A LD LT LF F LRDMK QDE V A DT LQ RK K A E S L LS S CV S LK S NK S I G I RP D LS DGP V NS DS V S S RK RE S S P LS S CV S MK S NHS MS LP P Y LS DGA V NS DS V S P Y 4.08 - - - - - - - - - - - - - - - - - - - (a) 4.08 4.44 4.41 14.14 23.05 4.42 14.40 4.30 4.14 4.31 4.21 4.46 4.50 4.19 4.12 4.49 4.38 4.34 4.02 4.01 18.03 4.28 18.11 4.17 4.15 4.09 3.06 3.08 1.25 1.27 1.26 1.20 1.28 1.31 1.13 1.21 1.18 1.22 1.24 1.17 1.01 up06 15.07a 25.01 15.03 15.04 NALP4_MO NALP5_HU NALP7_HU MEFV_MO MEFV_RAT MEFV_HUM (b NALP4 MOUSE (b) Structure of Figure 15 Search for class II cytokine receptor genes To identify class II cytokine receptor genes we searched the zebrafish genome and all available zebrafish ESTs for the sub- domains SD100A and SD100B running the Prosite protein annotation [54] with the hidden Markov model matrices with accession numbers PS50299 (SD100A) and PS50300 (SD100B). In all phylogenetic trees presented in this study complete sequences were used rather than only the conserved domains. The alignments for generating the phylogenetic trees were performed with ClustalW using the Blosum matrix with standard parameters. For the phylogenetic reconstruction the neighbor-joining method [52] was used with a bootstrap test of 1,000 replicates. Gaps and missing data were treated as pair-wise deletions. The screen of genomic sequences encoding SD100A or SD100B domains identified 12 genes, of which two encoded titins, one encoded thrombopoeitin, eight encoded cytokine class II receptor genes that previously were found to belong to the Interpro IPR000282 family, and one Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.20 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.20 http://genomebiology.com/2007/8/11/R251 Chromosomal locations of zebrafish NLR proteins Figure 16 Chromosomal locations of zebrafish NLR proteins. The 11 chromosomes containing the main clusters of NLR genes are shown. The number of NLR genes on each chromosome is listed below the chromosome number. A further 42 genes) are distributed on 11 other chromosomes, and 20 genes are on as yet unplaced contigs. This list includes a compilation of all predictions (automated as well as manually annotated) and locations of hits from a TBLASTN search for NACHT domains. Future improvements of the genome assembly and further manual annotations will most likely result in minor changes of this map. Genes are denoted by lines on the right of the chromosome irrespective of orientation. NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family. Search for class II cytokine receptor genes 1.01- 1.03 1.04 1.05- 1.32 1 (32) 3.09 3.01 3.03 3.06- 3.08 3.04, 3.05 3 (9) 4.08- 4.10 4.02 - 4.05 4.01 4.06 4.07 4.28- 4.31 4.14 - 4.18 4.11 4.13 4.27 4.12 4.19 - 4.23 4.24 - 4.26 4.32 4.33- 4.36 4.37- 4.39 4.40 4.41- 4.43 4.46- 4.52 4.44 4.45 4.53- 4.56 4 (50) 14.0X 14.01 14.09 14.03 - 14.08 14.10 14.11 14.14 14.13 14.12 14.15 14.23 14.16 -14.22 14.24 14.25 - 14.28 14.29 - 14.31 14.32 - 14.36 14.37 - 14.43 14.44 14.45 14.46 - 14.47 14 (47) 20.18 20.19 20.20 20.01 20.02 20.03 20.04 - 20.17 20 (20) 22.01- 22.08 22.09- 22.11 22.12 22.13 22.14 22.15 22.16 - 22.19 22.20 - 22.23 22.24, 22.25 22 (25) 15.01 15.02 15.03 - 15.08 15.09 - 15.11 15 (11) 17.01 - 17.04 17.05 - 17.10 17.11 - 17.12 17 (12) 18.01 18.02 - 18.11 18 (11) 21.03- 21.05 21.01- 21.02 21.06- 21.09 21.10 21 (10) 5.01 5.02 5.03- 5.07 5.08- 5.10 5 (10) 0 20 30 40 50 60 70 80 10 90 Mbp chromosome: (number of NACHT domain genes) Chromosom Figure 16 Chromosom Figure 16 Chromosomal locations of zebrafish NLR proteins Figure 16 Chromosomal locations of zebrafish NLR proteins. The 11 chromosomes containing the main clusters of NLR genes are shown. The number of NLR genes on each chromosome is listed below the chromosome number. A further 42 genes) are distributed on 11 other chromosomes, and 20 genes are on as yet unplaced contigs. This list includes a compilation of all predictions (automated as well as manually annotated) and locations of hits from a TBLASTN search for NACHT domains. Future improvements of the genome assembly and further manual annotations will most likely result in minor changes of this map. Genes are denoted by lines on the right of the chromosome irrespective of orientation. NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family. Chromosomal locations of zebrafish NLR proteins Figure 16 Chromosomal locations of zebrafish NLR proteins. The 11 chromosomes containing the main clusters of NLR genes are shown. The number of NLR genes on each chromosome is listed below the chromosome number. A further 42 genes) are distributed on 11 other chromosomes, and 20 genes are on as yet unplaced contigs. This list includes a compilation of all predictions (automated as well as manually annotated) and locations of hits from a TBLASTN search for NACHT domains. Future improvements of the genome assembly and further manual annotations will most likely result in minor changes of this map. Genes are denoted by lines on the right of the chromosome irrespective of orientation. NLR, nucleotide-binding domain/NACHT domain and leucine rich repeat containing family. (GENSCAN0000036149) encoded a previously unidentified gene of this class. Search for new NLR proteins For the manual annotation of NLR genes in the zebrafish genome, we initially used the ESTs with the accession num- bers CF347458.1, CD284951.1, CO915312.1, CF266152.1, BM534859.1, and DT055906.1 as guides. The ESTs were not 100% identical to any of the genomic sequences we identified, which may be due to polymorphisms between the strains from which the genome sequence and the ESTs were derived. The NLR proteins were identified as follows. A TBLASTN search of the Ensembl zebrafish genome assembly Zv4 with the mammalian Nalp3 gene identified more than 200 sites in the genome encoding complete or partial NACHT domains. A collection of 170 NACHT-domain encoding zebrafish genes from the NCBI database, which only partly overlapped the set identified by TBLASTN, were also mapped onto the genome. The merged list of the two nonoverlapping sets of sites in the genome were sorted by chromosomal location, each site was given a number (chromosome number plus numerical order- ing). The regions containing the potential genes were then further refined using available ESTs and gene predictions as guides. The resulting sequences were blasted against the fin- ished and unfinished clone sequences and the hits on finished clones were finally manually annotated. For further refine- ment of annotations we also used the motifs identified in Figure 15 in particular to improve the predictions for the full amino-terminal extensions of the genes. The following additional data are available with the online version of this paper. Additional file 1 lists the kinase protein sequences in FASTA format. Additional file 2 lists the adaptor protein sequences in FASTA format. Additional file 3 lists the IRF protein sequences in FASTA format. Additional file 4 lists the Stat protein sequences in FASTA format. Additional file 5 lists the Traf protein sequences in FASTA format. Additional file 6 lists the class II cytokine receptor protein sequences in FASTA format. Additional file 7 lists the class II cytokine pro- tein sequences in FASTA format. Additional data file 8 lists the NLR protein sequences in FASTA format, except for the zebrafish-specific NLRs. Additional data file 9 lists the zebrafish-specific NLR protein sequences in FASTA format. Additional data file 10 is a high resolution of the large phylo- gram of 277 NLRs presented in Figure 12. Search for new NLR proteins Addi i l d fil Ki i Li d h ki i i FASTA f Cli k h f fil Addi i l d fil Ad i Li d h d i i FASTA f Cli k h f fil Addi i l d fil IRF i Li d h IRF i i FASTA f Cli k h f fil Addi i l d fil S i Li d h S i i FASTA f Cli k h f fil Addi i l d fil T f i Li d h T f i i FASTA f Cli k h f fil Addi i l d fil 6 Cl II ki i Li d h l II ki i i FASTA f Cli k h f fil Addi i l d fil Cl II ki i Li d h l II ki i i FASTA f Cli k h f fil Addi i l d fil 8 NLR i Li d h NLR i i FASTA f f h b fi h ifi NLR Cli k h f fil Addi i l d fil Z b fi h ifi NLR i Li d h b fi h ifi NLR i i FASTA fCli k h f fil Addi i l d fil A hi h l i f h l h l f NLR d i Fi A hi h l i f h l h l f NLR d i Fi Cli k h f fil Authors' contributions named these candidates zf17 and zf18. We then assessed the annotations of zf1 to zf18, and annotated or re-annotated the sequences manually, if no annotations existed (zf1, zf2, zf6, and zf14) or the previous annotations appeared incomplete or incorrect. This analysis showed that twelve of the genes encoded proteins with the characteristics of class II cytokine receptors. CS and ML conducted BLAST searches, made alignments and phylogenetic trees, made the figures and wrote the text. MC identified the cytokine and cytokine receptor genes and analyzed their genomic contexts, GL made the manual anno- tations of the novel NLR genes and cytokine receptor genes. (GENSCAN0000036149) encoded a previously unidentified gene of this class. for which we analyzed the known or predicted full-length sequences in more detail. One of the ESTs (accession CK692344) was not represented in the zebrafish genome (neither assembly Zv6 nor trace sequences) and turned out to correspond to a mouse gene. Three sequences had only spuri- ous resemblances to SD100A or SD100B encoding sequences, often over very short stretches, and encoded known proteins with other functions. This left 16 potential candidates for cytokine class II receptor encoding genes, which we named zf1 to zf16. Six of these had also been identified by the genomic screen. Two candidates from the genomic screen were not in this group, because no ESTs exist for them. We To screen the ESTs, we first translated every EST sequence in the six possible frames and then searched for the sub- domains. We followed a similar procedure with all the ab initio predictions (Genscan and Fgenesh) obtained in the analysis of the zebrafish Zv6 assembly [24]. From the EST analysis we obtained 69 different sequences, of which 14 encoded both subdomains. Comparison of the 69 sequences showed that they represented 20 different genes, Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.21 http://genomebiology.com/2007/8/11/R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251. Stein et al. R251.21 Acknowledgements This work was supported by the Wellcome Trust and the European Molec- ular Biology Organization. ML thanks Richard Durbin, Kerstin Jekosch, and staff at the Sanger Center for providing space and a stimulating sabbatical environment. We thank our colleagues, in particular Jonathan Howard, for discussions and suggestions, Dale Richardson for assembling the set of NCBI NACHT-domain predictions, and Jane Parker and Jeff Dangl for com- ments on the manuscript. Jonathan Rast very kindly provided a file with the sequences of the sea urchin NACHT domain proteins. We are especially thankful to Georges Lutfalla and Dina Aggad for sharing ideas and informa- tion, and for generously providing the sequences of DrCRFB14 and IFN-ϕ4. References Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 http://genomebiology.com/2007/8/11/R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.22 Danio rerio. J Virol 2003, 77:1992-2002. 10. Yoder JA, Litman RT, Mueller MG, Desai S, Dobrinski KP, Mont- gomery JS, Buzzeo MP, Ota T, Amemiya CT, Trede NS, et al.: Reso- lution of the novel immune-type receptor gene cluster in zebrafish. Proc Natl Acad Sci USA 2004, 101:15706-15711. 33. 33. Igawa D, Sakai M, Savan R: An unexpected discovery of two interferon gamma-like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals. Mol Immunol 2006, 43:999-1009. 11. Panagos PG, Dobrinski KP, Chen X, Grant AW, Traver D, Djeu JY, Wei S, Yoder JA: Immune-related, lectin-like receptors are dif- ferentially expressed in the myeloid and lymphoid lineages of zebrafish. Immunogenetics 2006, 58:31-40. 34. Chen JY, You YK, Chen JC, Huang TC, Kuo CM: Organization and promoter analysis of the zebrafish (Danio rerio) interferon gene. DNA Cell Biol 2005, 24:641-650. g 12. Bobe J, Goetz FW: Molecular cloning and expression of a TNF receptor and two TNF ligands in the fish ovary. Comp Biochem Physiol B Biochem Mol Biol 2001, 129:475-481. g 35. Milev-Milovanovic I, Long S, Wilson M, Bengten E, Miller NW, Chin- char VG: Identification and expression analysis of interferon gamma genes in channel catfish. Immunogenetics 2006, 58:70-80. 13. Engelsma MY, Huising MO, van Muiswinkel WB, Flik G, Kwang J, Savelkoul HF, Verburg-van Kemenade BM: Neuroendocrine- immune interactions in fish: a role for interleukin-1. Vet Immunol Immunopathol 2002, 87:467-479. 36. Long S, Milev-Milovanovic I, Wilson M, Bengten E, Clem LW, Miller NW, Chinchar VG: Identification and expression analysis of cDNAs encoding channel catfish type I interferons. Fish Shell- fish Immunol 2006, 21:42-59. p 14. Zou J, Secombes CJ, Long S, Miller N, Clem LW, Chinchar VG: Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus). Dev Comp Immunol 2003, 27:845-858. f 37. Zou J, Carrington A, Collet B, Dijkstra JM, Yoshiura Y, Bols N, Secombes C: Identification and bioactivities of IFN-gamma in rainbow trout Oncorhynchus mykiss: the first Th1-type cytokine characterized functionally in fish. J Immunol 2005, 175:2484-2494. p 15. Huising MO, Stet RJ, Savelkoul HF, Verburg-van Kemenade BM: The molecular evolution of the interleukin-1 family of cytokines; IL-18 in teleost fish. References Lewis RS, Ward AC: Conservation, duplication and divergence of the zebrafish stat5 genes. Gene 2004, 338:65-74. p J 45. Koonin EV, Aravind L: The NACHT family: a new group of pre- dicted NTPases implicated in apoptosis and MHC transcription activation. Trends Biochem Sci 2000, 25:223-224. 46 C i i 22. Oganesyan G, Saha SK, Guo B, He JQ, Shahangian A, Zarnegar B, Perry A, Cheng G: Critical role of TRAF3 in the Toll-like recep- tor-dependent and -independent antiviral response. Nature 2006, 439:208-211. 46. van der Biezen EA, Jones JD: The NB-ARC domain: a novel sig- nalling motif shared by plant resistance gene products and regulators of cell death in animals. Curr Biol 1998, 8:R226-227. 23. van der Sar AM, Stockhammer OW, van der Laan C, Spaink HP, Bitter W, Meijer AH: MyD88 innate immune function in a zebrafish embryo infection model. Infect Immun 2006, 74:2436-2441. g 47. Hibino T, Loza-Coll M, Messier C, Majeske AJ, Cohen AH, Terwilliger DP, Buckley KM, Brockton V, Nair SV, Berney K, et al.: The immune gene repertoire encoded in the purple sea urchin genome. Dev Biol 2006, 300:349-365. 24. Birney E, Andrews D, Caccamo M, Chen Y, Clarke L, Coates G, Cox T, Cunningham F, Curwen V, Cutts T, et al.: Ensembl 2006. Nucleic Acids Res 2006, 34:D556-561. 48. Rast JP, Smith LC, Loza-Coll M, Hibino T, Litman GW: Genomic insights into the immune system of the sea urchin. Science 2006, 314:952-956. 25. Sprague J, Doerry E, Douglas S, Westerfield M: The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental research. Nucleic Acids Res 2001, 29:87-90. 49. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. 50. Lespinet O, Wolf YI, Koonin EV, Aravind L: The role of lineage- specific gene family expansion in the evolution of eukaryotes. Genome Res 2002 12:1048-1059 49. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. 26. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410. g 50. Lespinet O, Wolf YI, Koonin EV, Aravind L: The role of lineage- specific gene family expansion in the evolution of eukaryotes. References Dev Comp Immunol 2004, 28:395-413. 38. Zou J, Yoshiura Y, Dijkstra JM, Sakai M, Ototake M, Secombes C: Identification of an interferon gamma homologue in Fugu, Takifugu rubripes. Fish Shellfish Immunol 2004, 17:403-409. p 16. Reboul J, Gardiner K, Monneron D, Uze G, Lutfalla G: Comparative genomic analysis of the interferon/interleukin-10 receptor gene cluster. Genome Res 1999, 9:242-250. 39. Long S, Wilson M, Bengten E, Bryan L, Clem LW, Miller NW, Chin- char VG: Identification of a cDNA encoding channel catfish interferon. Dev Comp Immunol 2004, 28:97-111. 17. Lutfalla G, Roest Crollius H, Stange-Thomann N, Jaillon O, Mogensen K, Monneron D: Comparative genomic analysis reveals inde- pendent expansion of a lineage-specific gene family in verte- brates: the class II cytokine receptors and their ligands in mammals and fish. BMC Genomics 2003, 4:29. p 40. Robertsen B, Bergan V, Rokenes T, Larsen R, Albuquerque A: Atlan- tic salmon interferon genes: cloning, sequence analysis, expression, and biological activity. J Interferon Cytokine Res 2003, 23:601-612. 18. Levraud JP, Boudinot P, Colin I, Benmansour A, Peyrieras N, Her- bomel P, Lutfalla G: Identification of the zebrafish IFN recep- tor: implications for the origin of the vertebrate IFN system. J Immunol 2007, 178:4385-4394. 41. Zhang DC, Shao YQ, Huang YQ, Jiang SG: Cloning, characteriza- tion and expression analysis of interleukin-10 from the zebrafish (Danio rerio). J Biochem Mol Biol 2005, 38:571-576. J 19. Baoprasertkul P, Peatman E, Somridhivej B, Liu Z: Toll-like recep- tor 3 and TICAM genes in catfish: species-specific expression profiles following infection with Edwardsiella ictaluri. Immuno- genetics 2006, 58:817-830. ( ) J 42. Zou J, Clark MS, Secombes CJ: Characterisation, expression and promoter analysis of an interleukin 10 homologue in the puffer fish, Fugu rubripes. Immunogenetics 2003, 55:325-335. p g p g 43. Zou J, Tafalla C, Truckle J, Secombes CJ: Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates. J Immunol 2007, 179:3859-3871. 20. Ben J, Jabs EW, Chong SS: Genomic, cDNA and embryonic expression analysis of zebrafish IRF6, the gene mutated in the human oral clefting disorders Van der Woude and pop- liteal pterygium syndromes. Gene Expr Patterns 2005, 5:629-638. J 44. Damiano JS, Oliveira V, Welsh K, Reed JC: Heterotypic interac- tions among NACHT domains: implications for regulation of innate immune responses. Biochem J 2004, 381:213-219. p yg y p , 21. References Apaf1, apoptotic protease activating factor 1; CRFB, cytokine receptor family B; EST, expressed sequence tag; Fisna, fish- specific NACHT associated; IL, interleukin; IFN, interferon; IFNAR, interferon-α receptor; IFNGR, interferon-γ receptor; IL10R, interleukin-10 receptor; IL22BP, interleukin-22 bind- ing protein; IRAK, interleukin-1 receptor associated kinase; IRF, interferon response factor; LRR, leucine-rich repeat; NF-κB, nuclear factor-κB; NLR, NACHT-domain and leucine rich repeat containing; Nalp, NACHT, leucine rich repeat and PYD containing protein; Nod, nucleotide oligomerization domain containing protein; Stat, signal transducer and acti- vator of transcription; Tab, Tak1-binding protein; TF, tissue factor; Ticam, Toll-interleukin 1 receptor domain (TIR) con- taining adaptor molecule; TLR, Toll-like receptor; TNF, tumor necrosis factor; Traf, TNF-receptor associated factor. 1. Trede NS, Langenau DM, Traver D, Look AT, Zon LI: The use of zebrafish to understand immunity. Immunity 2004, 20:367-379. 2. Venkatesh B: Evolution and diversity of fish genomes. Curr Opin Genet Dev 2003, 13:588-592. 3. Volff JN: Genome evolution and biodiversity in teleost fish. Heredity 2005, 94:280-294. 4. Traver D, Herbomel P, Patton EE, Murphey RD, Yoder JA, Litman GW, Catic A, Amemiya CT, Zon LI, Trede NS: The zebrafish as a model organism to study development of the immune system. Adv Immunol 2003, 81:253-330. 5. Nonaka M, Kimura A: Genomic view of the evolution of the complement system. Immunogenetics 2006, 58:701-713. 5. Nonaka M, Kimura A: Genomic view of the evolution of the complement system. Immunogenetics 2006, 58:701-713. 6. Meijer AH, Gabby Krens SF, Medina Rodriguez IA, He S, Bitter W, Ewa Snaar-Jagalska B, Spaink HP: Expression analysis of the Toll- like receptor and TIR domain adaptor families of zebrafish. Mol Immunol 2004, 40:773-783. 6. Meijer AH, Gabby Krens SF, Medina Rodriguez IA, He S, Bitter W, Ewa Snaar-Jagalska B, Spaink HP: Expression analysis of the Toll- like receptor and TIR domain adaptor families of zebrafish. Mol Immunol 2004, 40:773-783. 7. Jault C, Pichon L, Chluba J: Toll-like receptor gene family and TIR-domain adapters in Danio rerio. Mol Immunol 2004, 40:759-771. 8. Litman GW, Hawke NA, Yoder JA: Novel immune-type receptor genes. Immunol Rev 2001, 181:250-259. 9. Yoder JA, Mueller MG, Wei S, Corliss BC, Prather DM, Willis T, Lit- man RT, Djeu JY, Litman GW: Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster. Proc Natl Acad Sci USA 2001, 98:6771-6776. 58. Eddy SR: Profile hidden Markov models. Bioinformatics 1998, 14:755-763. 59. HMMER [http://bioweb.pasteur.fr/seqanal/interfaces/ hmmbuild.html] 60. Schuster-Bockler B, Schultz J, Rahmann S: HMM Logos for visuali- zation of protein families. BMC Bioinformatics 2004, 5:7. 61. HMM logo web server [http://www.sanger.ac.uk/cgi-bin/software/ analysis/logomat-m.cgi] 62. Clamp M, Cuff J, Searle SM, Barton GJ: The Jalview Java alignment editor. Bioinformatics 2004, 20:426-427. Genome Biology 2007, 8:R251 References Genome Res 2002, 12:1048-1059. 27. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 2004, 5:150-163. g 28. Sullivan C, Postlethwait JH, Lage CR, Millard PJ, Kim CH: Evidence for evolving Toll-IL-1 receptor-containing adaptor molecule function in vertebrates. J Immunol 2007, 178:4517-4527. 51. Waterhouse RM, Kriventseva EV, Meister S, Xi Z, Alvarez KS, Bar- tholomay LC, Barillas-Mury C, Bian G, Blandin S, Christensen BM, et al.: Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Science 2007, 316:1738-1743. 29. Miller DJ, Hemmrich G, Ball EE, Hayward DC, Khalturin K, Funayama N, Agata K, Bosch TC: The innate immune repertoire in cni- daria - ancestral complexity and stochastic gene loss. Genome Biol 2007, 8:R59. 52. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425. 30. Kedinger V, Alpy F, Tomasetto C, Thisse C, Thisse B, Rio MC: Spa- tial and temporal distribution of the traf4 genes during zebrafish development. Gene Expr Patterns 2005, 5:545-552. 53. Havana [http://www.sanger.ac.uk/HGP/havana/hawk.shtml] 54 P i [h // / /] 54. Prosite [http://www.expasy.org/prosite/] 31. Krause CD, Pestka S: Evolution of the Class 2 cytokines and receptors, and discovery of new friends and relatives. Pharma- col Ther 2005, 106:299-346. 55. Pfam [http://www.sanger.ac.uk/Software/Pfam] 56. Smart [http://smart.embl-heidelberg.de] [ p g ] 57. Renauld JC: Class II cytokine receptors and their ligands: key antiviral and inflammatory modulators. Nat Rev Immunol 2003, 3:667-676. 57. Renauld JC: Class II cytokine receptors and their ligands: key antiviral and inflammatory modulators. Nat Rev Immunol 2003, 3:667-676. 32. Altmann SM, Mellon MT, Distel DL, Kim CH: Molecular and func- tional analysis of an interferon gene from the zebrafish, Genome Biology 2007, 8:R251 Genome Biology 2007, Volume 8, Issue 11, Article R251 Stein et al. R251.23 http://genomebiology.com/2007/8/11/R251 58. Eddy SR: Profile hidden Markov models. Bioinformatics 1998, 14:755-763. 59. HMMER [http://bioweb.pasteur.fr/seqanal/interfaces/ hmmbuild.html] 60. Schuster-Bockler B, Schultz J, Rahmann S: HMM Logos for visuali- zation of protein families. BMC Bioinformatics 2004, 5:7. 61. HMM logo web server [http://www.sanger.ac.uk/cgi-bin/software/ analysis/logomat-m.cgi] 62. Clamp M, Cuff J, Searle SM, Barton GJ: The Jalview Java alignment editor. Bioinformatics 2004, 20:426-427. Genome Biology 2007, 8:R251
https://openalex.org/W4384922700	https://www.ajnr.org/content/ajnr/early/2023/07/20/ajnr.A7929.full.pdf	English	null	Epileptogenic Tubers Are Associated with Increased Kurtosis of Susceptibility Values: A Combined Quantitative Susceptibility Mapping and Stereoelectroencephalography Pilot Study	American journal of neuroradiology	2,023	cc-by	6,886	Received December 11, 2022; accepted after revision June 7, 2023. From Developmental Neurosciences (A.C., K. Seunarine, R.J.P., M.M.T., J.H.C.), Great Ormond Street Institute of Child Health and Department of Medical Physics and Bioengineering (K. Shmueli), University College London, London, UK; Departments of Neurosurgery (A.C., K. Seunarine, R.J.P. M.M.T.), Neuroradiology (J.S., P.H., K.M., U.L.), Neurology (C.E., R.C.S., J.H.C.), and Neurophysiology (F.M.), Great Ormond Street Hospital, London, UK; Department of Pediatric Neurology (R.C.S.), Nemours Children’s Hospital, Wilmington, Delaware; and Engineering and Physical Sciences Research Council/Wellcome Centre for Medical Engineering and Department of Biomedical Engineering (D.W.C.), School of Biomedical Engineering and Imaging Sciences, King’s College London, London, UK. Epileptogenic Tubers Are Associated with Increased Kurtosis of Susceptibility Values: A Combined Quantitative Susceptibility Mapping and Stereoelectroencephalography Pilot Study A. Chari, J. Sedlacik, K. Seunarine, R.J. Piper, P. Hales, K. Shmueli, K. Mankad, U. Löbel, C. Eltze, F. Moeller, R.C. Scott, M.M. Tisdall, J.H. Cross, and D.W. Carmichael A. Chari, J. Sedlacik, K. Seunarine, R.J. Piper, P. Hales, K. Shmueli, K. Mankad, U. Löbel, C. Eltze, F. Moeller, R.C. Scott, M.M. Tisdall, J.H. Cross, and D.W. Carmichael ABSTRACT From Developmental Neurosciences (A.C., K. Seunarine, R.J.P., M.M.T., J.H.C.), Great Ormond Street Institute of Child Health and Department of Medical Physics and Bioengineering (K. Shmueli), University College London, London, UK; Departments of Neurosurgery (A.C., K. Seunarine, R.J.P. M.M.T.), Neuroradiology (J.S., P.H., K.M., U.L.), Neurology (C.E., R.C.S., J.H.C.), and Neurophysiology (F.M.), Great Ormond Street Hospital, London, UK; Department of Pediatric Neurology (R.C.S.), Nemours Children’s Hospital, Wilmington, Delaware; and Engineering and Physical Sciences Research Council/Wellcome Centre for Medical Engineering and Department of Biomedical Engineering (D.W.C.), School of Biomedical Engineering and Imaging Sciences, King’s College London, London, UK. Please address correspondence to Aswin Chari, MD, Developmental Neurosciences, Great Ormond Street Institute of Child Health, University College London, 30 Guilford St, London, WC1N 1EH UK; e-mail: Aswin.chari.18@ucl.ac.uk; @aswinchari Indicates open access to non-subscribers at www.ajnr.org Indicates article with online supplemental data. http://dx.doi.org/10.3174/ajnr.A7929 AJNR Am J Neuroradiol : 2023 www.ajnr.org 1 A. Chari is supported by a Great Ormond Street Hospital Children’s Charity Surgeon Scientist Fellowship. This work has been supported by the Great Ormond Street Hospital-National Institute of Health Research Biomedical Research Center and, in part, by the Henry Smith Charity and Action Medical Research (GN2214). D.W.C. was supported by the King’s College London Wellcome/Engineering and Physical Sciences Research Council Center for Medical Engineering (WT 203148/Z/16/Z). Indicates article with online supplemental data. http://dx.doi.org/10.3174/ajnr.A7929 AJNR Am J Neuroradiol : 2023 www.ajnr.org 1 A. Chari is supported by a Great Ormond Street Hospital Children’s Charity Surgeon Scientist Fellowship. This work has been supported by the Great Ormond Street Hospital-National Institute of Health Research Biomedical Research Center and, in part, by the Henry Smith Charity and Action Medical Research (GN2214). D.W.C. was supported by the King’s College London Wellcome/Engineering and Physical Sciences Research Council Center for Medical Engineering (WT 203148/Z/16/Z). Pilot Study Mapping and Stereoelectroencephalography Combined Quantitative Susceptibility A Increased Kurtosis of Susceptibility Values: Epileptogenic Tubers Are Associated with Scott, M.M. Tisdall, J.H. Cross and D.W. Carmichael Shmueli, K. Mankad, U. Löbel, C. Eltze, F. Moeller, R.C. A. Chari, J. Sedlacik, K. Seunarine, R.J. Piper, P. Hales, K. Scott, M.M. Tisdall, J.H. Cross and D.W. Carmichael Shmueli, K. Mankad, U. Löbel, C. Eltze, F. Moeller, R.C. A. Chari, J. Sedlacik, K. Seunarine, R.J. Piper, P. Hales, K. of October 23, 2024. This information is current as of October 23, 2024. This information is current as http://www.ajnr.org/content/early/2023/07/20/ajnr.A7929 published online 20 July 2023 AJNR Am J Neuroradiol http://www.ajnr.org/content/early/2023/07/20/ajnr.A7929 published online 20 July 2023 AJNR Am J Neuroradiol http://www.ajnr.org/content/early/2023/07/20/ajnr.A7929 published online 20 July 2023 AJNR Am J Neuroradiol Published July 20, 2023 as 10.3174/ajnr.A7929 ORIGINAL RESEARCH PEDIATRICS ORIGINAL RESEARCH PEDIATRICS ABSTRACT BACKGROUND AND PURPOSE: Prior studies have found an association between calciﬁcation and the epileptogenicity of tubers in tuberous sclerosis complex. Quantitative susceptibility mapping is a novel tool sensitive to magnetic susceptibility alterations due to tissue calciﬁcation. We assessed the utility of quantitative susceptibility mapping in identifying putative epileptogenic tubers in tuberous sclerosis complex using stereoelectroencephalography data as ground truth. MATERIALS AND METHODS: We studied patients with tuberous sclerosis complex undergoing stereoelectroencephalography at a single center who had multiecho gradient-echo sequences available. Quantitative susceptibility mapping and R2* values were extracted for all tubers on the basis of manually drawn 3D ROIs using T1- and T2-FLAIR sequences. Characteristics of quantitative susceptibility mapping and R2* distributions from implanted tubers were compared using binary logistic generalized estimating equation models designed to identify ictal (involved in seizure onset) and interictal (persistent interictal epileptiform activity) tubers. These models were then applied to the unimplanted tubers to identify potential ictal and interictal tubers that were not sampled by stereoelectroencephalography. RESULTS: A total of 146 tubers were identiﬁed in 10 patients, 76 of which were sampled using stereoelectroencephalography. Increased kurtosis of the tuber quantitative susceptibility mapping values was associated with epileptogenicity (P ¼ .04 for the ictal group and P ¼ .005 for the interictal group) by the generalized estimating equation model. Both groups had poor sensitivity (35.0% and 44.1%, respectively) but high speciﬁcity (94.6% and 78.6%, respectively). CONCLUSIONS: Our ﬁnding of increased kurtosis of quantitative susceptibility mapping values (heavy-tailed distribution) was highly speciﬁc, suggesting that it may be a useful biomarker to identify putative epileptogenic tubers in tuberous sclerosis complex. This ﬁnding motivates the investigation of underlying tuber mineralization and other properties driving kurtosis changes in quantitative susceptibility mapping values. T uberous sclerosis complex (TSC) is a genetic disorder often associated with difficult-to-treat drug-resistant epilepsy T uberous sclerosis complex (TSC) is a genetic disorder often associated with difficult-to-treat drug-resistant epilepsy (DRE).1 While some studies report favorable outcomes following resective epilepsy surgery to treat TSC-associated DRE, others report that only about 50% become seizure-free following resec- tive surgery, especially in complex cases with no clear dominant tuber.2-6 There is an increasing practice of using stereoelectroen- cephalography (SEEG) to guide surgery in these patients, with a recognition that the best outcomes are achieved in patients with a clear “dominant” tuber and a focal putative seizure onset zone.7 T Received December 11, 2022; accepted after revision June 7, 2023. ABBREVIATIONS: DRE ¼ drug-resistant epilepsy; GEE ¼ generalized estimating equation; QSM ¼ quantitative susceptibility mapping; SEEG ¼ stereoelec- troencephalography; TSC ¼ tuberous sclerosis complex Image Acquisition Images were acquired using a 3T MR imaging scanner (Magnetom Prisma; Siemens) with a 20-channel head and neck receive coil. The image-acquisition parameters for the anatomic T1-weighted 3D-MPRAGE scan were the following: TI ¼ 900 ms, TR ¼ 2300 ms, TE ¼ 2.74 ms, flip angle ¼ 8°, readout bandwidth¼ 200Hz/ pixel, 1-mm3 isotropic voxel size, acquisition matrix ¼ 256 256 240, parallel acquisition acceleration factor¼ 2, coronal orienta- tion, total scan time¼ 5 minutes 19 seconds. Studies have identified calcification as an indicator of DRE and epileptic foci in TSC, though these studies have predominantly used CT scans to assess whether there was calcification as a binary variable.10,11 In the past few years, quantitative susceptibility map- ping (QSM) has become more prevalent as a MR imaging tech- nique, with the ability to detect intracranial calcification with high levels of sensitivity and specificity.12 QSM is an advancement of SWI in which postprocessing techniques are applied to quantify the magnetic susceptibility of tissue. Its advantage is that it removes the blooming artifacts in SWI that are a factor of tissue geometry and orientation, leading to precise local quantification of magnetic susceptibility.13 Magnetic susceptibility is increased by paramagnetic materials (hemorrhage, iron, gadolinium con- trast) and decreased by diamagnetic materials (eg, calcification), and levels of these minerals have previously been shown to be altered in focal cortical dysplasia.14 As part of the QSM processing, an R2* map is also generated, which is a measure of the amount of dephasing caused by B0 field inhomogeneities either from mac- roscopic field perturbations or those due to local susceptibility effects.13 In contrast to QSM, local field perturbations due to para- magnetic or diamagnetic materials have a similar effect. The image acquisition parameters for the 3D T2-FLAIR were the following: TI ¼ 1800 ms, TR ¼ 5000 ms, TE ¼ 395 ms, echo-train length ¼ 233 with variable flip angle optimized for T2-weighting, readout bandwidth ¼ 650 Hz/pixel, acquisition matrix ¼ 384 291 240 reconstructed to 0.65 0.65 1 mm3 voxel size, parallel acquisition acceleration factor ¼ 2, cor- onal orientation, total scan time ¼ 6 minutes 10 seconds. QSM Processing In this exploratory pilot study, we sought to assess whether the QSM signal in tubers was able to identify putative epilepto- genic tubers in a cohort of children with TSC undergoing SEEG as part of their presurgical evaluation for DRE. To explore the characteristics of the QSM distribution within each tuber, we assessed summary statistics of the histograms in each tuber (me- dian, upper quartile, lower quartile, skewness, and kurtosis). We hypothesized that QSM and the associated R2* values would dif- fer between epileptogenic and nonepileptogenic tubers, with epi- leptogenic tubers having lower QSM and higher R2* values as a result of increased calcium content and that this difference may affect both the median values and skewness of the distributions. Single-channel image data were combined for each TE by the sum of squares of all single-channel magnitude images. The com- bined phase image w was calculated for each TE by the sum of conjugate complex multiplication between the previous and cur- rent TEs. The conjugate complex multiplication eliminates the incongruous spatial phase sensitivities of each coil element, allow- ing the phase correct summation of the complex image data. The combined phase image of the first TE was set to zero. f TE1 ¼ 0 f TEn ¼ arg X zTEn1 zTEn 8n.1 : Image Acquisition The image acquisition parameters for the R2/QSM 3D multiecho gradient-echo sequence were the following: TEs ¼ 3, 7, 11, 15, 19, 23, and 27 ms, TR ¼ 38 ms, flip angle ¼15°, readout bandwidth ¼ 360Hz/pixel, acquisition matrix ¼ 192 156 144 reconstructed to 0.6-mm3 isotropic voxel size, 6/8 partial Fourier factor in phase- and section-encoding directions, parallel acquisition acceleration factor ¼ 2 in a phase-encoding direction, transverse orientation, total scan time ¼ 5 minutes 41 seconds. Copyright 2023 by American Society of Neuroradiology. However, targeting tubers as part of an SEEG implantation plan in these patients can be difficult because there is often a high tuber burden, semiology can be difficult to interpret, and video- electroencephalography lateralization and localization are often poor. The interpretation of neuroimaging is further complicated by heterogeneity in the appearance of tubers, with some imaging fea- tures more associated with epilepsy than others. One study identi- fied 3 radiologically different tuber types based on T1-, T2-, and T2-FLAIR characteristics; the dominant type of tuber in a patient was associated with the likelihood of autism spectrum disorder, in- fantile spasms, and seizures, though no insights were drawn about the epileptogenicity of specific tubers.8 Another study used this classification to quantitatively assess the epileptogenicity of tubers and the surrounding cortex during SEEG. It concluded that resec- tion of the dominant tuber associated with a T2-FLAIR hypoin- tense center, a higher epileptogenicity index compared with other tubers and the perituberal cortex, continuous interictal epileptiform discharges, and stimulation-induced seizures were associated with 80% seizure freedom. Outcomes were less favorable when there was a more complex organization of the epileptogenic zone.9 studies in Epidemiology (STROBE) guidelines. Because it used routinely collected clinical data, ethics approval and the need for individual patient informed consent was waived by the research and development department at Great Ormond Street Hospital, and this study was registered as a clinical audit with the clinical audit department at Great Ormond Street Hospital. We studied a series of consecutive patients with TSC under- going SEEG evaluation between January 2016 and December 2020 and who also had multiecho gradient-echo imaging suitable for QSM as part of their routine clinical MR imaging before SEEG was available for study inclusion. The decision to perform SEEG, the interpretation of the SEEG results, and offers of resec- tive surgery were collected by the epilepsy surgery multidiscipli- nary team without reference to the QSM data. 2 Chari 2023 www.ajnr.org Software, Data, and Code Availability Statement Software, Data, and Code Availability Statement All image processing was performed using the tools described above and in-house scripts in Matlab, Release 2020b (MathWorks) for QSM processing and data extraction and SPSS (IBM) for GEE modeling. Code is available at www.github.com/aswinchari/QSM. The GitHub repository contains the tuber information used to construct the GEE models. Coregistered imaging data are available from the corresponding authors on reasonable request. QSM and R2 Results Of the 14 patients, 11 had undergone QSM as part of their preoperative scans, but 1 patient had to be excluded due to artifacts from dental braces. Therefore, the scans of 10 patients were included in the subsequent analyses (Online Supplemental Data). From these patients, a total of 146 tubers were masked (range, 6–23 tubers per patient), of which 76 were sampled by SEEG electrodes (range, 4–13 tubers per patient) and 70 were not. Of the sampled tubers, 20 were la- beled as ictal, and 34, as interictal. By means of the 76 tubers sampled by SEEG electrodes, 2 bi- nary logistic GEE models were constructed to predict whether tubers were involved in ictal onset (ictal model) or interictal activity (interictal model). For both models, the only factor inde- pendently associated with ictal or interictal status was the kurtosis of the QSM histogram (P ¼ .04 for the ictal model and P ¼ .005 for the interictal model) (Online Supplemental Data). The predic- tions of the models were used to assess whether they correctly categorized the implanted tubers; both models had poor sensitiv- ity (35.0% and 44.1%, respectively) but high specificity (94.6% and 78.6%, respectively) (Fig 2A). Image Processing Manual segmentation of all visible tubers was performed using coregistered volumetric T1- and T2-FLAIR MR imaging using ITK-SNAP (www.itksnap.org).19 This was performed by the first author (A.C.), who is a neurosurgical resident with experi- ence in pediatric epilepsy, under the supervision of a neuroradi- ologist with a special interest in pediatric epilepsy (K.M.). To reduce QSM artifacts from the pial surface vessels, we limited the manual segmentations to 1–2 mm away from the pial sur- face (Fig 1A). Manual visual checks were performed to ensure that all implanted tubers were segmented. AJNR Am J Neuroradiol : 2023 www.ajnr.org Clinical Results During the 5-year period, 14 children with TSC underwent SEEG. One child had previously undergone tuber resection, while another had undergone resective surgery for a subependy- mal giant cell astrocytoma. In 12 children (85.7%), a seizure- onset zone was identified following SEEG, and resective surgery was offered. All except 1 child had undergone resective surgery, including resection of single tuber and multiple tubers 6 mesial temporal structures. Imaging from the SEEG (including the electrode locations), the QSM and R2* maps, and postoperative scans were coregis- tered to the original volumetric T1 and validated visually (Fig 1A). Using these coregistered maps and the report from the SEEG procedure, we classified each segmented tuber as being implanted or not and, if implanted, whether it was labeled as having ictal epileptiform activity (ie, part of the seizure onset zone, “ictal”) or interictal epileptiform activity (“interictal”). This classification was based on the SEEG report, which was completed by the clinical team including a consultant neuro- physiologist, consultant neurologist, and consultant neurosur- geon with experience in SEEG. The definition of “ictal” was that there was electrophysiologic change in the tuber at seizure onset, and the tuber was identified as one that should be resected as part of any surgery were it to be offered. The definition of “interictal” was persistent interictal epileptiform discharges in the tuber. In addition, note was made of whether the tuber was resected from the postoperative imaging. At a median follow-up of 2.0 years (range, 1.0–4.3 years), 2 patients (18.2%) had an Engel Class I outcome, 3 (27.3%) had an Engel Class II outcome, 5 (45.4%) had an Engel Class III out- come, and 1 (9.1%) had an Engel Class IV outcome. MATERIALS AND METHODS A brain mask was computed on the first echo magnitude image using the FSL Brain Extraction Tool (http://fsl.fmrib.ox.ac.uk/fsl/ fslwiki/BET).15 The R2* map was calculated on the logarithmic This was a single-center retrospective cohort study reported according to the STrengthening the Reporting of OBservational 2 Chari 2023 www.ajnr.org magnitude images using the Moore-Penrose pseudoinverse implementation. The frequency shift was calculated from the combined phase images of all TEs using the Fit_ppm_complex.m function in the MEDI toolbox (https://github.com/huawu02/ MEDI_toolbox/blob/main/UPDATES.m).16 The local frequency shift was calculated using the projection onto dipole fields method with an eroded brain mask (85% of the original size of the FSL BET brain mask) to minimize nonlocal phase contribu- tions.17 The QSM map was then calculated from the local fre- quency shift using the iterative Tikhonov dipole inversion method.18 then applied to the unimplanted tubers to predict ictal and inter- ictal tubers from the unimplanted tubers and assess whether the predicted unresected ictal and interictal tuber burden correlated with the outcome following SEEG-guided resective epilepsy surgery. Statistical Analysis QSM and R2* values from each tuber were extracted on the basis of the coregistered QSM and R2* maps. To characterize the dis- tribution of quantitative data from each tuber, we extracted the size of the tuber (number of voxels) and the median, upper quar- tile, lower quartile, skewness, and kurtosis of the QSM and R2* maps for each tuber (Fig 1B). Using the values above (11 variables in total), 2 generalized estimating equation (GEE) binary logistic regression models were constructed using only the implanted tubers to assess whether there were factors that were predictive of whether the tuber was ictal or interictal. GEE models accommodate for repeated meas- ures, which, in this case, are the potential intrasubject correlation between many tubers in the same patient. Following evaluation of model specificity and sensitivity, these model parameters were The developed model parameters were subsequently applied to the test data set of unimplanted tubers. They identified 10 tubers as ictal and 27 as interictal, of which 7 were overlapping. These were spread across most of the subjects with a range of 0–4 addi- tional ictal tubers and 0–6 additional interictal tubers identified that were not sampled by SEEG (Online Supplemental Data). 3 Figure 2B shows the distribution of QSM kurtosis values across all tubers that were implanted and from the model predictions. This shows the association between higher kurtosis (heavier tails of QSM values) and ictal and interictal tubers. Similar trends were not seen when plotting the median QSM values across all tubers (Fig 2C). interictal tubers (Fig 3). Although there was a modest positive relationship for the ictal model, the regression coefficients were not statistically significant for either model (ictal regression coefficient ¼ 0.42, R2 ¼ 0.23, P ¼ .16; interictal regression coefficient ¼ 0.10, R2 ¼ 0.003, P ¼ .88). FIG 1. Methods summary. A, Illustrative images of T1-, T2-FLAIR, segmented tubers and overlying electrodes, QSM, and R2* maps used in this study. B, Histograms show distributions of QSM and R2* values for the left motor strip tuber identiﬁed in A. FIG 1. Methods summary. A, Illustrative images of T1-, T2-FLAIR, segmented tubers and overlying electrodes, QSM, and R2* maps used in this study. B, Histograms show distributions of QSM and R2* values for the left motor strip tuber identiﬁed in A. FIG 1. Methods summary. Statistical Analysis A, Illustrative images of T1-, T2-FLAIR, segmented tubers and overlying electrodes, QSM, and R2* maps used in this study. B, Histograms show distributions of QSM and R2* values for the left motor strip tuber identiﬁed in A. interictal tubers (Fig 3). Although there was a modest positive relationship for the ictal model, the regression coefficients were not statistically significant for either model (ictal regression coefficient ¼ 0.42, R2 ¼ 0.23, P ¼ .16; interictal regression coefficient ¼ 0.10, R2 ¼ 0.003, P ¼ .88). Figure 2B shows the distribution of QSM kurtosis values across all tubers that were implanted and from the model predictions. This shows the association between higher kurtosis (heavier tails of QSM values) and ictal and interictal tubers. Similar trends were not seen when plotting the median QSM values across all tubers (Fig 2C). 4 Chari 2023 www.ajnr.org Illustrative Example Last, the predictions of the models were used to assess whether there was a linear association between the Engel out- come and the total number of unresected predicted ictal and As an example, we present the case of a 10-year-old child diag- nosed with TSC at 11months of age with 4 different seizure types, Output of GEE models. A, The 2 2 tables illustrate the sensitivity and speciﬁcity of the developed models to ident the implanted tubers B, Violin plots of the kurtosis of the QSM histograms across implanted and nonimplanted tub he SEEG-identiﬁed and model-predicted ictal and interictal tubers, albeit with a degree of overlap. C, Violin plots cross implanted and nonimplanted tubers show higher kurtosis in the SEEG-identiﬁed and model-predicted ictal no difference between groups. FIG 2. Output of GEE models. A, The 2 2 tables illustrate the sensitivity and speciﬁcity of the developed models to identify ictal and interictal tubers in the implanted tubers B, Violin plots of the kurtosis of the QSM histograms across implanted and nonimplanted tubers show higher kur- tosis in the SEEG-identiﬁed and model-predicted ictal and interictal tubers, albeit with a degree of overlap. C, Violin plots of the median QSM values across implanted and nonimplanted tubers show higher kurtosis in the SEEG-identiﬁed and model-predicted ictal and interictal tubers, showing no difference between groups. FIG 2. Output of GEE models. A, The 2 2 tables illustrate the sensitivity and speciﬁcity of the developed models to identify ictal and interictal tubers in the implanted tubers B, Violin plots of the kurtosis of the QSM histograms across implanted and nonimplanted tubers show higher kur- tosis in the SEEG-identiﬁed and model-predicted ictal and interictal tubers, albeit with a degree of overlap. C, Violin plots of the median QSM values across implanted and nonimplanted tubers show higher kurtosis in the SEEG-identiﬁed and model-predicted ictal and interictal tubers, showing no difference between groups. FIG 2. Output of GEE models. A, The 2 2 tables illustrate the sensitivity and speciﬁcity of the developed models to identify ictal and interictal tubers in the implanted tubers B, Violin plots of the kurtosis of the QSM histograms across implanted and nonimplanted tubers show higher kur- tosis in the SEEG-identiﬁed and model-predicted ictal and interictal tubers, albeit with a degree of overlap. Illustrative Example C, Violin plots of the median QSM values across implanted and nonimplanted tubers show higher kurtosis in the SEEG-identiﬁed and model-predicted ictal and interictal tubers, showing no difference between groups. AJNR Am J Neuroradiol : 2023 www.ajnr.org 5 FIG 3. Association between model-predicted unresected tubers and postoperative Engel outcomes. There was no signiﬁcant correlation between outcome and the total number of unresected ictal and interictal tubers predicted by the model (ictal regression coefﬁcient¼ 0.42, R2 ¼ 0.23, P ¼ .16; interictal regression coefﬁcient¼ 0.10, R2¼ 0.003, P ¼ .88). Note that for this analysis, 2 patients who did not undergo subse- quent surgical intervention were classiﬁed as Engel class IV, and the Engel class was considered a linear variable. FIG 3. Association between model-predicted unresected tubers and postoperative Engel outcomes. There was no signiﬁcant correlation between outcome and the total number of unresected ictal and interictal tubers predicted by the model (ictal regression coefﬁcient¼ 0.42, R2 ¼ 0.23, P ¼ .16; interictal regression coefﬁcient¼ 0.10, R2¼ 0.003, P ¼ .88). Note that for this analysis, 2 patients who did not undergo subse- quent surgical intervention were classiﬁed as Engel class IV, and the Engel class was considered a linear variable. On the basis of the ground truth of SEEG interpretation, we found that a model containing QSM and R2* signal characteris- tics may be helpful in identifying putative epileptogenic tubers in TSC with a high level of specificity but low sensitivity (Fig 2A). This finding suggests that preoperative QSM may be a useful adjunct for the selection of tubers for SEEG exploration because ictal lesions nearly always had high kurtosis in our sample. These results warrant prospective assessment. Specifically, epilepsy in TSC can be associated with complex networks; therefore, tubers distant from the regions identified by semiology and video-elec- troencephalography may be involved in seizure generation and warrant sampling.7 Most interesting, most patients in this cohort (8/10) had additional unimplanted tubers identified as potentially epileptogenic by the model, which, combined with the evidence that only 2 patients had an Engel Class I outcome, provides pre- liminary evidence that these additional tubers may have been worthwhile to sample as part of the SEEG exploration. 6 Chari 2023 www.ajnr.org Illustrative Example The poor correlation of unresected predicted ictal tubers with outcome (Fig 3) is explained by the poor sensitivity of the model, and fur- ther prospective work might identify additional features to improve the sensitivity, such as other modalities of MR imaging incorporated into the model or data such as electrical source modeling.20 However, the high specificity suggests that the models may be useful in the prospective identification of puta- tive epileptogenic tubers that could then be targeted for confir- mation through SEEG recordings. Indeed, there is a precedent for using radiologic biomarkers to identify additional areas to explore during SEEG.21 including, most commonly, asymmetric spasms with more right- body involvement than left. Ictal video telemetry recordings for 3 seizure types lateralized to the left hemisphere, while one lateral- ized to the right. Interictal EEG showed bilateral epileptiform activity, but this was more pronounced on the left. MR imaging showed evidence of bilateral tubers with no clear dominant tuber. An interictal magnetoencephalogram was performed, showing interictal activity in the left prefrontal and temporoparietal regions. On the basis of the findings above, a bilateral SEEG im- plantation was planned with more left-sided than right-sided sided electrodes (Fig 4A). This identified 2 tubers involved in sei- zure onset (Fig 4B, red tubers). These tubers were subsequently resected, and 2 years after the operation, the patients had an Engel Class III outcome with significant reduction in seizure fre- quency and duration and associated improvement in cognition and attention. The ictal GEE model using the QSM data identified an addi- tional potential epileptogenic tuber in the right occipital region (Fig 4B, green tuber), which was not sampled by the SEEG elec- trodes. The normalized QSM histograms of all 3 tubers are shown, illustrating a narrower width and, therefore, increased kurtosis compared with all the other 15 tubers in the same patient (Fig 4C). DISCUSSION To our knowledge, this is only the second study to use QSM in TSC and the first to quantitatively analyze the QSM characteris- tics. The previous study showed that QSM was feasible in the context of identifying calcifications in both tubers and subepen- dymal nodules. In our GEE models, we identified the kurtosis of the QSM signal histogram being significantly associated with both ictal FIG 4. Illustrative case example. A, Illustration of bilateral SEEG implantation with a more left-sided electrode. B, Sagittal and axial T1 images with overlying tuber segmentation. The 2 identiﬁed epileptogenic tubers that were subsequently resected are shown in red, while a third tuber in green was not sampled but identiﬁed as potentially epileptogenic by the GEE model. C, Histograms of the QSM values of the 3 tubers shown in B overlaid on the histograms of 15 other tubers from the same patient (in white). Note that they all seem to have a higher kurtosis. FIG 4. Illustrative case example. A, Illustration of bilateral SEEG implantation with a more left-sided electrode. B, Sagittal and axial T1 image with overlying tuber segmentation. The 2 identiﬁed epileptogenic tubers that were subsequently resected are shown in red, while a third tube in green was not sampled but identiﬁed as potentially epileptogenic by the GEE model. C, Histograms of the QSM values of the 3 tubers show in B overlaid on the histograms of 15 other tubers from the same patient (in white). Note that they all seem to have a higher kurtosis. FIG 4. Illustrative case example. A, Illustration of bilateral SEEG implantation with a more left-sided electrode. B, Sagittal and axial T1 images with overlying tuber segmentation. The 2 identiﬁed epileptogenic tubers that were subsequently resected are shown in red, while a third tuber in green was not sampled but identiﬁed as potentially epileptogenic by the GEE model. C, Histograms of the QSM values of the 3 tubers shown in B overlaid on the histograms of 15 other tubers from the same patient (in white). Note that they all seem to have a higher kurtosis. require electrophysiologic and histopathologic correlation, which may give insight into why certain radiologic characteristics (for example, high QSM kurtosis or low T2-FLAIR intensity) are associated with epileptogenicity. This knowledge may aid the targeting of putative epileptogenic tubers in future SEEG implantations. DISCUSSION In addition, our model does not account for the dynamics of epileptogenicity within a tuber, where the core, pe- riphery, and perituberal tissue may contribute differently to sei- zure onset; again, this issue would require further study.9 QSM signal distributions have also been linked to a chronic inflam- matory response and glial activation in MS lesions,22 and it would, therefore, be interesting to correlate longitudinal imag- ing findings with markers of epileptogenicity and, ultimately, histology to understand the potential role of inflammation in tubers and how they contribute to epileptogenesis. (P ¼ .04) and interictal (P ¼ .005) tubers, with increased kurtosis in epileptogenic tubers without significant changes in the me- dian QSM values. This finding suggests that although there may not necessarily be an average increase in the calcium content within epileptogenic tubers, the distribution of QSM values had thicker tails. Increased kurtosis of QSM values being associated with epileptogenicity is a novel finding and requires both a bio- logic explanation and external validation, especially because it does not fully agree with previous studies that indicate calcifica- tion as a marker of epileptogenicity.10,11 For example, Lorio et al14 found decreased QSM values, corresponding to increased calcium and zinc in focal epileptogenic lesions such as in focal cortical dysplasia type IIb. The high kurtosis in this study could be explained by the thicker tails on both sides of the distribution, indicating areas of low susceptibility (eg, from high calcium) and high susceptibility (eg, from areas of increased blood flow or iron deposition) compared with nonepileptogenic tubers. (P ¼ .04) and interictal (P ¼ .005) tubers, with increased kurtosis in epileptogenic tubers without significant changes in the me- dian QSM values. This finding suggests that although there may not necessarily be an average increase in the calcium content within epileptogenic tubers, the distribution of QSM values had thicker tails. Increased kurtosis of QSM values being associated with epileptogenicity is a novel finding and requires both a bio- logic explanation and external validation, especially because it does not fully agree with previous studies that indicate calcifica- tion as a marker of epileptogenicity.10,11 For example, Lorio et al14 found decreased QSM values, corresponding to increased calcium and zinc in focal epileptogenic lesions such as in focal cortical dysplasia type IIb. AJNR Am J Neuroradiol : 2023 www.ajnr.org DISCUSSION The high kurtosis in this study could be explained by the thicker tails on both sides of the distribution, indicating areas of low susceptibility (eg, from high calcium) and high susceptibility (eg, from areas of increased blood flow or iron deposition) compared with nonepileptogenic tubers. The study has a number of limitations. First, it is a small sin- gle-center retrospective series that requires internal prospective and external validation. The cohort was small, and only 18% of subjects were seizure-free at last follow-up, indicating a complex Indeed, alterations of iron deposition have also been described in radiologically classified tubers of focal cortical dysplasia; these 7 2. Arya R, Tenney JR, Horn PS, et al. Long-term outcomes of resective epilepsy surgery after invasive presurgical evaluation in children with tuberous sclerosis complex and bilateral multiple lesions. J Neurosurg Pediatr 2015;15:26–33 CrossRef Medline 2. Arya R, Tenney JR, Horn PS, et al. Long-term outcomes of resective epilepsy surgery after invasive presurgical evaluation in children with tuberous sclerosis complex and bilateral multiple lesions. J Neurosurg Pediatr 2015;15:26–33 CrossRef Medline cohort. QSM is also inherently associated with artifacts, such as from the cortical surface, which may have affected the results; de- spite our attempts to reduce such artifacts, novel postprocessing pipelines may help reduce them further.23 The utility of the model would also be improved by understanding the biologic ba- sis for increased kurtosis leading to epileptogenicity, such as post- operative analysis of tissue mineral content. The tubers were segmented using only the T1- and T2-FLAIR sequences. It is con- ceivable that incorporating the QSM maps into the interpretation of tuber characteristics may aid radiologic interpretation of the nature and extent of tubers. 3. Fallah A, Guyatt GH, Snead OC, et al. Predictors of seizure out- comes in children with tuberous sclerosis complex and intracta- ble epilepsy undergoing resective epilepsy surgery: an individual participant data meta-analysis. PLoS One 2013;8:e53565 CrossRef Medline 3. Fallah A, Guyatt GH, Snead OC, et al. Predictors of seizure out- comes in children with tuberous sclerosis complex and intracta- ble epilepsy undergoing resective epilepsy surgery: an individual participant data meta-analysis. PLoS One 2013;8:e53565 CrossRef Medline 4. Fallah A, Rodgers SD, Weil AG, et al. Resective epilepsy surgery for tuberous sclerosis in children: determining predictors of seizure outcomes in a multicenter retrospective cohort study. Neurosurgery 2015;77:517–24; discussion 524 CrossRef Medline 4. Fallah A, Rodgers SD, Weil AG, et al. Disclosure forms provided by the authors are available with the full text and PDF of this article at www.ajnr.org. DISCUSSION Altman NR, Purser RK, Post MJ. Tuberous sclerosis: characteristics at CT and MR imaging. Radiology 1988;167:527–32 CrossRef Medline 11. Zhang MN, Zou LP, Wang YY, et al. Calcification in cerebral paren- chyma affects pharmacoresistant epilepsy in tuberous sclerosis. Seizure 2018;60:86–90 CrossRef Medline 11. Zhang MN, Zou LP, Wang YY, et al. Calcification in cerebral paren- chyma affects pharmacoresistant epilepsy in tuberous sclerosis. Seizure 2018;60:86–90 CrossRef Medline 12. Chen W, Zhu W, Kovanlikaya I, et al. Intracranial calcifications and hemorrhages: characterization with quantitative susceptibility mapping. Radiology 2014;270:496–505 CrossRef Medline 13. Ruetten PP, Gillard JH, Graves MJ. Introduction to quantitative sus- ceptibility mapping and susceptibility weighted imaging. Br J Radiol 2019;92:20181016 CrossRef Medline DISCUSSION Resective epilepsy surgery for tuberous sclerosis in children: determining predictors of seizure outcomes in a multicenter retrospective cohort study. Neurosurgery 2015;77:517–24; discussion 524 CrossRef Medline We were unable to include quantitative analyses of T1- and T2-FLAIR signal characteristics, CT scan densities, and the SEEG signals in this study, but these may be useful constructs for future studies for quantitatively assessing associations between CT/MR imaging signal characteristics and quantitative markers of SEEG epileptogenicity. Existing SEEG markers of epileptogenicity include cortico-cortical evoked potentials, neuronal spiking activ- ity and fast ripples, and more in-depth analyses could relate QSM characteristics to these.24-26 In addition, there may be a more nuanced interpretation of SEEG in TSC that we have not consid- ered; the ethos at our institution is to consider tubers as individual entities being either involved or not involved in the seizure onset, but we acknowledge that other schools may consider parts of tubers or perituberal tissue to be epileptogenic; therefore, future studies may seek to assess QSM signal characteristic distributions within and around tubers as markers of epileptogenic tissue. There is also a possibility of dynamic changes in QSM signals, which were not captured in this study because the postoperative imaging protocols did not include QSM in our institution. 5. Liu S, Yu T, Guan Y, et al. Resective epilepsy surgery in tuberous sclerosis complex: a nationwide multicentre retrospective study from China. Brain 2020;143:570–81 CrossRef Medline 5. Liu S, Yu T, Guan Y, et al. Resective epilepsy surgery in tuberous sclerosis complex: a nationwide multicentre retrospective study from China. Brain 2020;143:570–81 CrossRef Medline 6. Specchio N, Pepi C, de Palma L, et al. Surgery for drug-resistant tuberous sclerosis complex-associated epilepsy: who, when, and what. Epileptic Disord 2021;23:53–73 CrossRef Medline 6. Specchio N, Pepi C, de Palma L, et al. Surgery for drug-resistant tuberous sclerosis complex-associated epilepsy: who, when, and what. Epileptic Disord 2021;23:53–73 CrossRef Medline 7. Park JT, Vaca GF. Stereo-EEG in tuberous sclerosis complex. Pediatr Neurol Briefs 2020;34:6 CrossRef Medline 8. Gallagher A, Grant EP, Madan N, et al. MRI findings reveal three different types of tubers in patients with tuberous sclerosis com- plex. J Neurol 2010;257:1373–81 CrossRef Medline 9. Neal A, Ostrowsky-Coste K, Jung J, et al. Epileptogenicity in tuber- ous sclerosis complex: a stereoelectroencephalographic study. Epilepsia 2020;61:81–95 CrossRef Medline 10. Altman NR, Purser RK, Post MJ. Tuberous sclerosis: characteristics at CT and MR imaging. Radiology 1988;167:527–32 CrossRef Medline 10. CONCLUSIONS Despite these limitations, this study provides important proof of principle that quantification and assessment of tuber mineral con- tent through QSM may be a useful biomarker in the identification of the putative epileptogenic tubers in TSC. Most important, our results, that high kurtosis is associated with epileptogenicity with a high specificity and low sensitivity, did not support our hypothesis that epileptogenicity would be associated with increased calcium and, therefore, a greater prevalence of lower QSM values. This result does not refute the hypothesis that calcification is an impor- tant marker in tuber epileptogenicity but may indicate a more complex process. Increased QSM kurtosis could involve changes in mineral content (eg, calcium and iron) and perhaps inflamma- tion and blood flow changes, all of which may relate to epileptoge- nicity. Larger cohorts, external validation, and correlation with histologic and tissue mineral analysis are required to further this work. 14. Lorio S, Sedlacik J, So PW, et al. Quantitative MRI susceptibility mapping reveals cortical signatures of changes in iron, calcium and zinc in malformations of cortical development in children with drug-resistant epilepsy. Neuroimage 2021;238:118102 CrossRef Medline 15. Jenkinson M. BET: MR-Based Estimation of Brain, Skull and Scalp Surfaces. In: Proceedings of the Annual Meeting of the Organization for Human Brain Mapping, Toronto, Ontario, Canada. June 12–16, 2005 16. Liu T, Wisnieff C, Lou M, et al. Nonlinear formulation of the magnetic field to source relationship for robust quantitative sus- ceptibility mapping. Magn Reson Med 2013;69:467–76 CrossRef Medline 17. Liu T, Khalidov I, de Rochefort L, et al. A novel background field re- moval method for MRI using projection onto dipole fields (PDF). NMR Biomed 2011;24:1129–36 CrossRef Medline 18. Karsa A, Punwani S, Shmueli K. An optimized and highly repeat- able MRI acquisition and processing pipeline for quantitative sus- ceptibility mapping in the head-and-neck region. Magn Reson Med 2020;84:3206–22 CrossRef Medline 19. Yushkevich PA, Piven J, Hazlett HC, et al. User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability. Neuroimage 2006;31:1116–28 CrossRef Medline 8 Chari 2023 www.ajnr.org 24. Wang Y, Yuan L, Zhang S, et al. Fast ripples as a biomarker of epilepto- genic tuber in tuberous sclerosis complex patients using stereo-electro- encephalograph. Front Hum Neurosci 2021;15:680295 CrossRef Medline g 25. Yu X, Ding P, Yuan L, et al. Cortico-cortical evoked potentials in children with tuberous sclerosis complex using stereo-electroence- phalography. Front Neurol 2019;10:1093 CrossRef Medline AJNR Am J Neuroradiol : 2023 www.ajnr.org 9 p g p y 26. Despouy E, Curot J, Denuelle M, et al. Neuronal spiking activity highlights a gradient of epileptogenicity in human tuberous scle- rosis lesions. Clin Neurophysiol 2019;130:537–47 CrossRef Medline 21. Chari A, Adler S, Wagstyl K, et al. IDEAL approach to the evaluation of machine learning technology in epilepsy surgery: protocol for the MAST trial. BMJ Surg Interv Health Technol 2022;4:e000109 CrossRef Medline 22. Gillen KM, Mubarak M, Park C, et al. QSM is an imaging biomarker for chronic glial activation in multiple sclerosis lesions. Ann Clin Transl Neurol 2021;8:877–86 CrossRef Medline 23. Yaghmaie N, Syeda WT, Wu C, et al. QSMART: quantitative susceptibil- ity mapping artifact reduction technique. Neuroimage 2021;231:117701 CrossRef Medline 23. Yaghmaie N, Syeda WT, Wu C, et al. QSMART: quantitative susceptibil- ity mapping artifact reduction technique. Neuroimage 2021;231:117701 CrossRef Medline AJNR Am J Neuroradiol : 2023 www.ajnr.org 9 REFERENCES 1. Nabbout R, Belousova E, Benedik MP, et al. Historical patterns of diagnosis, treatments, and outcome of epilepsy associated with tuberous sclerosis complex: results from TOSCA Registry. Front Neurol 2021;12:697467 CrossRef Medline 20. Centeno M, Tierney TM, Perani S, et al. Combined electroencepha- lography-functional magnetic resonance imaging and electrical source imaging improves localization of pediatric focal epilepsy. Ann Neurol 2017;82:278–87 CrossRef Medline 21. Chari A, Adler S, Wagstyl K, et al. IDEAL approach to the evaluation of machine learning technology in epilepsy surgery: protocol for the MAST trial. BMJ Surg Interv Health Technol 2022;4:e000109 CrossRef Medline g 22. Gillen KM, Mubarak M, Park C, et al. QSM is an imaging biomarker for chronic glial activation in multiple sclerosis lesions. Ann Clin Transl Neurol 2021;8:877–86 CrossRef Medline 23. Yaghmaie N, Syeda WT, Wu C, et al. QSMART: quantitative susceptibil- ity mapping artifact reduction technique. Neuroimage 2021;231:117701 CrossRef Medline 26. Despouy E, Curot J, Denuelle M, et al. Neuronal spiking activity highlights a gradient of epileptogenicity in human tuberous scle- rosis lesions. Clin Neurophysiol 2019;130:537–47 CrossRef Medline
https://openalex.org/W2041972711	https://escholarship.org/content/qt4tc24599/qt4tc24599.pdf?t=odltb2	English	null	Central power generation versus distributed generation – An air quality assessment in the South Coast Air Basin of California	Atmospheric environment	2,010	cc-by	8,171	UC Irvine UC Irvine Previously Published Works Title Central power generation versus distributed generation – An air quality assessment in the South Coast Air Basin of California Permalink https://escholarship.org/uc/item/4tc24599 Journal Atmospheric Environment, 44(26) ISSN 1352-2310 Authors Carreras-Sospedra, Marc Vutukuru, Satish Brouwer, Jacob et al. Publication Date 2010-08-01 DOI 10.1016/j.atmosenv.2010.05.017 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Central power generation versus distributed generation – An air quality assessment in the South Coast Air Basin of California Permalink https://escholarship.org/uc/item/4tc24599 Journal Atmospheric Environment, 44(26) ISSN 1352-2310 Authors Carreras-Sospedra, Marc Vutukuru, Satish Brouwer, Jacob et al. Publication Date 2010-08-01 DOI 10.1016/j.atmosenv.2010.05.017 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Central power generation versus distributed generation – An air quality assessment i the South Coast Air Basin of California Permalink https://escholarship.org/uc/item/4tc24599 Journal Atmospheric Environment, 44(26) ISSN 1352-2310 Authors Carreras-Sospedra, Marc Vutukuru, Satish Brouwer, Jacob et al. Publication Date 2010-08-01 DOI 10.1016/j.atmosenv.2010.05.017 Copyright Information This work is made available under the terms of a Creative Commons Attribution Lice availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Title Title Central power generation versus distributed generation – An air quality assessment in the South Coast Air Basin of California Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ arc Carreras-Sospedra a, Satish Vutukuru b, Jacob Brouwer c, Donald Dabdub a,* a Computational Environmental Sciences Laboratory, Department of Mechanical & Aerospace Engineering, University of California, Irvin b ICF International, 394 Paciﬁc Ave., Suite 200, San Francisco, CA 94111, USA a Computational Environmental Sciences Laboratory, Department of Mechanical & Aerospace Engineering, University of California, Irvine, Irvine, CA 92697-3975, USA b ICF International, 394 Paciﬁc Ave., Suite 200, San Francisco, CA 94111, USA c Ad d P d E P D f M h i l & A E i i U i i f C lif i I i I i CA 92697 3975 USA r and Energy Program, Department of Mechanical & Aerospace Engineering, University of California, Irvine, Irvine, CA 92697-3975, USA c Advanced Power and Energy Program, Department of Mechanical & Aerospace Engineering, University of California, Irvine, Irvine, CA a r t i c l e i n f o Article history: Received 20 August 2009 Received in revised form 13 April 2010 Accepted 10 May 2010 Keywords: Distributed generation Central generation Air quality modeling Reactivity Article history: Received 20 August 2009 Received in revised form 13 April 2010 Accepted 10 May 2010 Keywords: Distributed generation Central generation Air quality modeling Reactivity Article history: Received 20 August 2009 Received in revised form 13 April 2010 Accepted 10 May 2010 This study assesses the air quality impacts of central power generation and compares them with the impacts of distributed generation (DG). The central power plant emissions factors used are from a newly installed combined cycle gas turbine system. Because location of power plants is a key parameter affecting air quality impacts, this study considers three potential locations for the installation of central power plants. Air quality impacts are evaluated for the South Coast Air Basin of California, in the year 2010, using a three-dimensional air quality model. Results are compared to air quality impacts from two potential DG scenarios to meet the same power demand as that of the central power plant case. Keywords: Distributed generation Central generation Air quality modeling Reactivity Even though emissions from central generation are lower than emissions from the DG technology mix considered herein, central generation concentrates emissions in a small area, whereas DG spreads emissions throughout a larger cross-section of the air basin. As a result, air quality impacts from central generation are more signiﬁcant than those from DG. The study also shows that assessment of air quality impacts from distributed and central generation should not only consider emissions levels, but also the spatial and temporal distribution of emissions and the air quality that results from atmospheric chemistry and trans- port e highly non-linear processes. Finally, analysis of population exposure to ozone and PM2.5 shows that central generation located in coastal areas upwind from populated areas would cause the highest population exposure and even though emissions from central generation are considerably lower than DG emissions spread throughout the basin, results show that central generation causes a higher pollutant exposure than DG. 2010 Elsevier Ltd. All rights reserved. * Corresponding author at: Department of Mechanical & Aerospace Engineering, The Henry Samueli School of Engineering, University of California, Irvine, Irvine, CA 92697-3975, USA. E-mail address: ddabdub@uci.edu (D. Dabdub). Powered by the California Digital Library University of California eScholarship.org Atmospheric Environment 44 (2010) 3215e3223 1352-2310/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.atmosenv.2010.05.017 * Corresponding author at: Department of Mechanical & Aerospace Engineering, The Henry Samueli School of Engineering, University of California, Irvine, Irvine, CA 92697-3975, USA. E-mail address: ddabdub@uci.edu (D. Dabdub). 1352-2310/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.atmosenv.2010.05.017 2.1. Meteorological conditions The Southern California Air Quality Study (SCAQS) was a comprehensive campaign of atmospheric measurements that took place in the SoCAB, during August 27e29, 1987. The study collected an extensive set of meteorological and air quality data that has been used widely to validate air quality models (Meng et al., 1998; Grifﬁn et al., 2002a,b; Pun et al., 2002; Moya et al., 2002). Zeldin et al. (1990) indicated that August 28, 1987 is repre- sentative of the meteorological conditions in the SoCAB, which makes it suitable for modeling an air quality episode. In addition, the August 27e28,1987 episode is statistically within the top 10% of severe ozone-forming meteorological conditions. Hence, meteo- rological conditions for August 28 are used herein as the basis for comparing DG to central generation air quality impacts. Rodriguez et al. (2006) studied the potential air quality impacts of DG in the South Coast Air Basin of California (SoCAB) in the year 2010. Rodriguez et al. presented a series of possible DG implementation scenarios, and estimated their air quality impacts with respect to a baseline 2010 scenario that included no DG or other additional in-basin generation. Air quality impacts of DG reported by Rodriguez et al. were small due to expected low market penetration of DG by 2010. However, that study assumed that if no DG were installed, electricity would be imported from outside the basin, and as a result, no emissions from central generation would be introduced into the SoCAB in the central generation case. Hence, no real comparison between DG and in-basin central generation was shown. The SCAQS episode in August 27e29, 1987 was characterized by a weak onshore pressure gradient and warming temperatures aloft. The wind ﬂow was characterized by a sea breeze during the day and a weak land-mountain breeze at night. The presence of a well-deﬁned diurnal inversion layer at the top of neutral and unstable layers near the surface, along with a slightly stable nocturnal boundary layer, facilitated the accumulation of pollutants throughout the SoCAB, which lead to a high ozone concentration episode. The present study considers the possibility that limitations in the transmission of electricity could require additional in-basin generation. In-basin generation could be met either by DG or by central generation. 1. Introduction a high deployment of renewable electricity production by the year 2020. The intermittent nature of renewable sources like wind and solar power may require additional power generation that is needed to ramp-up and ramp-down electricity production quickly to compensate for the intermittent renewable sources (Porter, 2007). Generally, fast-response dispatchable technologies are based upon fossil fueled power generators that produce an emissions impact. Distributed generation (DG) is characterized by the widespread installation of many stationary power generators close to the point of electricity use within an urban air basin. In contrast, conventional, centralized power plants tend to be located in remote areas from which electricity must be transmitted to end users. However, increasing electricity demand combined with stressed transmission lines, and increasingly challenging right-of-way and environmental obstacles to transmission line additions may force even central power generation back into air basins. Recent power plant applica- tions in southern California conﬁrm this trend (CEC, 2007a). In addition, the Renewable Portfolio Standard for California requires DG has the potential to meet the power demands in the near future. Deployment of DG technologies might provide additional beneﬁts such as increased energy efﬁciency, electrical reliability, power quality, and reductions in production costs. Furthermore, power generation near the place of consumption minimizes elec- tricity transmission losses and can be used for cogeneration of heating and cooling, generally termed as combined cooling, heating and power (CCHP). In addition, DG could provide dispatchable balancing power to compensate for the high deployment of inter- mittent sources of electricity, like wind power (Østergaard, 2005). However, deployment of DG introduces new emissions sources that are spread throughout an air basin, in a manner that is spatially and M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 3216 temporally different from central generation, which concentrates pollutant emissions in a limited number of emitting foci. The UCI-CIT model includes the CalTech Atmospheric Chemistry Mechanism (CACM) (Grifﬁn et al., 2002a,b; Pun et al., 2002). This chemical mechanism is intended for use in three-dimensional urban/ regional atmospheric models, with O3 formation and secondary organic aerosol (SOA) production. CACM includes 191 species and 361 reactions to accurately describe the chemical processes. Previous studies evaluated different DG technologies and sug- gested that only the lowest emitting DG technology (fuel cells) could be competitive with combined cycle power generation from an emissions perspective (Ianucci et al., 2000; Allison and Lents, 2002; Heath et al., 2006). 2.1. Meteorological conditions This work analyzes the air quality impacts of in-basin central generation in the SoCAB in the year 2010, and compares central generation impacts to the air quality impacts of DG. In addition, the present work assesses the variability of human exposure due to central and distributed power generation. 1. Introduction These studies provide valuable insights, but assume outdated emission factors for DG, only consider emissions impacts and do not account for atmospheric chemistry and transport in the airshed, which must be accounted for to determine ambient air quality impacts. 2. Model formulation Baseline emissions for the simulations are based on the emissions inventory developed by the South Coast Air Quality Management District (SCAQMD) for the 2003 Air Quality Management Plan (AQMP) to demonstrate attainment of the 1-h ozone standard (SCAQMD, 2003). This emissions inventory includes current emission controls planned for 2010 and other measures that would reduce baseline emissions to a level at which ozone concentration would not exceed the federal 1-h air quality standard (120 ppb). Emissions from distributed or central generation cases of the current study are esti- mated using a separate methodology described subsequently and added to these baseline emissions. The University of California, Irvine e California Institute of Technology (UCI-CIT) atmospheric chemistry and transport model is used to analyze the air quality in the SoCAB. The UCI-CIT model stems from the CIT model developed by McRae and Seinfeld (1982) and incorporates later developments in chemistry (Harley et al., 1993; Grifﬁn et al., 2002a), in aerosol formation (Meng et al., 1998; Pun et al., 2002; Grifﬁn et al., 2002b, 2003), in new numer- ical solution of advection (Nguyen and Dabdub, 2001). The UCI-CIT model has been applied extensively to study the air quality of the SoCAB (Grifﬁn et al., 2002a; Nguyen and Dabdub, 2002; Carreras- Sospedra et al., 2006). The computational domain for this study, shown in Fig.1, corresponds to an irregular region composed of 994 columns of cells. Each column resolves a 5 km by 5 km region in the x, y plane and extends 1100 m in height. The columns are partitioned into 5 cells in the z direction. 2.2.1. Sample distributed generation scenarios To compare the same central generation capacity with the capacity installed in the DG scenarios, the sample plant considered in this study has ﬁve 240-MW combined cycle turbines, with a total capacity of 1200 MW. Gas-phase and aerosol phase chemical specia- tion of emissions is based upon speciation of a natural gas recipro- cating internal combustion engine (ARB, 2008). In addition, size resolution of particles is based upon measurements of particles emissions from a gas turbine combustor (Brundish et al., 2005). Early projections suggested that the total fraction of energy demand met by DG could be as high as 20% of the electricity load growth by 2020 (Tomashefsky and Marks, 2002). Newer reports show that only 337 MW of generating capacity was installed with support from the Self Generation Incentive Program by the end of 2008 (CPUC, 2009). While this does not account for all DG installations, this level suggests a slower trend. Rodriguez et al. (2006) evaluated the air quality impacts of various DG market penetration levels from 2002 to 2010. Only scenarios that assumed a penetration of 20% or more of the increased electricity demand from 2002 to 2010 produced discernable air quality impacts on ozone and PM2.5. This level of market penetration corresponds to a generating capacity of 1062 MW. To assess the effect of location on the potential air quality impacts of installing a new central power plant, three locations are selected for this study: 1) Huntington Beach, Orange County, 2) Etiwanda, San Bernardino County, and 3) El Segundo, Los Angeles County. These locations are selected because they already have licensed the installation of central power plants, and they could be amenable to installing extra capacity in the future. Huntington Beach represents a location that is generally upwind from River- side, which typically experiences poor air quality during episodes. On the other hand, Etiwanda represents a location that is far downwind from Los Angeles, the main focus of emissions in the SoCAB, and near the area with the poorest air quality conditions. Finally, El Segundo is located upwind from central Los Angeles. The installation of a power plant in El Segundo has the potential to impact a highly populated area just downwind during an episode. Hence, these three locations are illustrative of the variety of air quality impacts that central generation could produce. 2.2.1. Sample distributed generation scenarios 2.2.1. Sample distributed generation scenarios A distributed generation scenario is deﬁned by a set of param- eters that determine which technologies and in what manner (spatially and temporally resolved) DG is deployed in an area of Fig. 1. Computational domain of the UCI-CIT airshed model that represents the South Coast Air Basin of California. Fig. 1. Computational domain of the UCI-CIT airshed model that represents the South Coast Air Basin of California. M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 3217 (CEC, 2007b). In the case of the SoCAB, restrictive emission stan- dards in the South Coast Air Quality Management District (SCAQMD) only allow for use of natural gas power plants. Therefore, this study analyzes the air quality impacts of a prototypical state-of- the-art, low emissions NG combined cycle power plant. interest. This set of parameters includes: (a) DG market penetration: the total capacity of DG installed in the basin, (b) DG technology mix: the set of technologies that are expected to be deployed in DG installations, (c) Emissions associated with each DG unit type: the emissions released by each DG technology based upon existing performance and regulations, (d) Spatial distribution of the DG within the basin: the spatial allocation of emissions from DG, based upon socio-economic factors and land-use data, (e) Operational duty cycle of each DG unit: the temporal variation of emissions from each type and application of DG, and (f) Emissions displaced by DG installation: the potential to remove existing emissions due to combined heat and power (CHP), substituting for boilers or other heating, cooling, or electrical applications. Emission factors are obtained from the High Desert Power Plant Project (CEC, 2000), which was installed in the Mojave Desert, and came on-line in April 2003. The power plant consists of three 240-MW combined cycle gas turbines with selective catalytic reduction systems for oxides of nitrogen (NOx) control. The present study analyzes the air quality impacts of the operation of a plant under two scenarios: 1) continued normal operation during 24 h, and 2) discontinuous oper- ation that includes two start-up (2 h event1) and two shut-down events (1 h event1), and a total of 18 h of normal operation, which is considered herein a ‘worst-day’ scenario in terms of pollutant emis- sions. 2.2.1. Sample distributed generation scenarios g g p y The current study selects two sample DG scenarios presented by Rodriguez et al. that consider a high penetration of DG, namely 20% of the increased demand from 2002 to 2010. The ﬁrst DG scenario, DG scenario 1, corresponds to a case in which DG market pene- tration is developed based upon the methodology developed by Samuelsen et al. (2005) and Medrano et al. (2008). The method- ology employs detailed land-use geographical information systems (GIS) data and market studies for DG implementation that can produce a realistic estimate of the DG technology mix that could be installed in the SoCAB. In addition, the methodology accounts for the potential of CHP applications, assuming that only 30% of the heat can be recovered due to thermodynamic limits, heat losses and temporal mismatch between thermal demand and excess heat production. The technology mix of DG scenario 1 is presented in Table 1. The second DG scenario, DG scenario 2, assumes an alter- native DG technology mix, which is also presented in Table 1. In addition, the spatial distribution of DG installations is proportional to the distribution of population density in the SoCAB in 2010, to assess the impacts of spatial allocation of emissions. Finally, DG scenario 2 does not include emissions displacements due to CHP. The analysis of these two different DG scenarios illustrates a range of potential air quality impacts from DG installations due to the different parameters assumed in the DG scenario development. 2.2.3. Comparison of emissions from central power plants and distributed generation Emissions that result from the scenario development method- ology for the two distributed generation scenarios and from the two central generation scenarios are presented in Table 2. Total emissions from normal operation of a central power plant are signiﬁcantly lower than emissions from DG, except for oxides of nitrogen (NOx) and ammonia (NH3). NOx emissions from DG scenario 1 are lower than NOx emissions from central generation due to the emissions displacement of CHP applications associated with the DG installations. On the other hand, total emissions from central generation under ‘worst-day’ conditions are comparable to 2.2.2. Central power plant scenarios Fossil fuel-based power generation in California mostly uses natural gas (NG), although there are a few coal-based power plants M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 3218 Table 2 Daily emissions from selected distributed generation scenarios and from central generation under normal conditions of operation and under discontinuous opera- tion (‘worst-day’). Fig. 2 shows air quality impacts on peak ground-level ozone concentrations throughout the basin produced by the DG and central generation scenarios, plotted as the difference between scenario concentrations and those of the baseline case. In general, impacts on peak ground-level ozone concentrations are related to NOx emissions. In the SoCAB, NOx concentrations are typically high, leading to volatile organic compounds (VOC)-limited ozone production conditions. Small additions of NOx emissions under VOC-limited conditions tend to decrease ozone concentration. Hence, scenarios with increases in NOx emissions produce reduc- tions in peak ozone concentration in some regions of the basin (Fig. 2(b)e(f)). On the contrary, the DG scenario 1 (Fig. 2(a)) reduces NOx emissions, and hence, produces small increases in peak ozone concentrations. The range of impacts on O3 in the DG scenarios is 1 ppb. Impacts on O3 due to central generation depend upon Pollutant emissions (tons day1) ROG CO NOx NH3 SOx PM10 DG scenario 1 (R3) 0.64 9.06 0.35 0.80 0.12 0.97 DG scenario 2 (PW2010) 0.80 8.19 2.54 0.25 0.10 0.61 Normal Central 0.04 1.05 1.08 1.60 0.00 0.06 Worst-day Central 0.86 20.63 1.99 1.60 0.07 1.09 emissions from the DG scenarios. However, emissions from DG are spread throughout the air basin, whereas emissions from central generation are concentrated as an elevated point source. As a result, air quality impacts from DG are likely to be different from those of central generation due to the spatial distribution of emissions. Fig. 2. Increase in peak O3 concentration with respect to the base case (negative values represent decreases in concentration with respect to base case): (a) DG scenario 1, (b) DG scenario 2, (c) normal operation of Huntington Beach central power plant, (d) ‘worst-day’ operation of Huntington Beach central power plant, (e) normal operation of Etiwanda central power plant, (f) ‘worst-day’ operation of Etiwanda central power plant, (g) normal operation of El Segundo central power plant, (h) ‘worst-day’ operation of El Segundo central power plant. Fig. 2. Table 1 Table 1 Parameters for two sample distributed generation scenarios (from Rodriguez et al., 2006). DG scenarioa Description Penetration (% of increased demand) Technology mixb (%) GT ICE MTG FC PV Hybrid 1 GIS land-use distribution, technology mix depends on activity sector, realistic duty cycles, and CHP 20 48 18 15 10 5 4 2 Population-weighted spatial distribution, DG operated base-loaded 20 30 30 25 7 8 e a DG scenarios 1 and 2 correspond to scenarios R3 and PW2010 presented in Rodriguez et al. (2006). b GT: gas turbines; ICE: natural gas internal combustion engines; MTG: micro-turbine generators; FC: fuel cells; PV: photovoltaic; hybrid: gas turbine þ fuel cell hybrid systems. Table 1 Parameters for two sample distributed generation scenarios (from Rodriguez et al., 2006). DG scenarioa Description Penetration (% of increased demand) Technology mixb (%) GT ICE MTG FC PV Hybrid 1 GIS land-use distribution, technology mix depends on activity sector, realistic duty cycles, and CHP 20 48 18 15 10 5 4 2 Population-weighted spatial distribution, DG operated base-loaded 20 30 30 25 7 8 e a DG scenarios 1 and 2 correspond to scenarios R3 and PW2010 presented in Rodriguez et al. (2006). b GT: gas turbines; ICE: natural gas internal combustion engines; MTG: micro-turbine generators; FC: fuel cells; PV: photovoltaic; hybrid: gas turbine þ fuel cell hybrid systems. Table 2 Increase in peak O3 concentration with respect to the base case (negative values represent decreases in concentration with respect to base case): (a) DG scenario 1, (b) DG scenario 2, (c) normal operation of Huntington Beach central power plant, (d) ‘worst-day’ operation of Huntington Beach central power plant, (e) normal operation of Etiwanda central power plant, (f) ‘worst-day’ operation of Etiwanda central power plant, (g) normal operation of El Segundo central power plant, (h) ‘worst-day’ operation of El Segundo central power plant. M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 3219 to changes in secondary formation of aerosol due to the addition of NOx and sulfur oxides (SOx). Impacts of DG on 24-h average PM2.5 concentrations are smaller than 1 mg m3, whereas central genera- tion under normal conditions increases PM2.5 concentrations by as much as 4 mg m3. Operation of central generation under ‘worst-day’ conditions produces increases in 24-h average PM2.5 concentrations up to 15 mg m3. Impacts on PM2.5 due to direct emissions of particles are localized near the location of the central power plant and correspond to the highest impacts. On the other hand, impacts on secondary PM2.5 occur far downwind from the central plant, leading to increases in 24-h average PM2.5 of less than 3 mg m3. Importantly, these increases occur in locations that are already highly impacted by PM episodes. location and operating conditions. Impacts on O3 due to the plant installed in Etiwanda are signiﬁcantly smaller than the impacts produced by scenarios that install the plant in Huntington Beach and El Segundo. Under normal conditions, the plant in Huntington Beach reduces peak O3 concentrations by 11 ppb in some locations, but, it also increases peak O3 concentrations by 2 ppb in other locations. Operation of the same plant under ‘worst-day’ conditions leads to decreases in peak O3 concentrations of 13 ppb and increases of 6 ppb in various locations as shown in Fig. 2(f). In addition, the geographic area affected by increases in ozone concentration due to ‘worst-day’ operating conditions is larger than the area affected by the same plant operated under normal conditions regardless of installation location. location and operating conditions. Impacts on O3 due to the plant installed in Etiwanda are signiﬁcantly smaller than the impacts produced by scenarios that install the plant in Huntington Beach and El Segundo. 3.1. Spatial sensitivity of air quality impacts As shown in Figs. 2 and 3, the intensity and distribution of air quality impacts from power plants depends greatly upon the loca- tion of the plant. Atmospheric transport due to wind determines the distribution of a plume and emissions from other local sources determines how the emissions from a power plant interact through chemical and physical processes with the surrounding atmosphere. As a result, meteorological conditions are an important factor for determining the air quality impacts. The present article focuses on a meteorological episode that is most representative of the typical meteorology of the SoCAB under high ozone and PM formation conditions. While a single episode cannot fully represent the air quality impacts of point sources, this particular episode reﬂects the most common meteorological conditions for the area. Hence, conclusions from this study can be used to describe the most common impacts expected for the scenarios presented here. PðO3Þ ¼ kCO;OH½CO½OH þ X i ki;OHYi½ROGi½OH; (1) (1) where ki,OH represents the kinetic rate constant for the reaction between OH and species i, Yi represents the stoichiometric yield of peroxy radicals in the reaction between OH and reactive organic gas (ROG) species i, and the bracket notation represents mixing ratios. As a result, meteorological conditions are an important factor for determining the air quality impacts. The present article focuses on a meteorological episode that is most representative of the typical meteorology of the SoCAB under high ozone and PM formation conditions. While a single episode cannot fully represent the air quality impacts of point sources, this particular episode reﬂects the most common meteorological conditions for the area. Hence, conclusions from this study can be used to describe the most common impacts expected for the scenarios presented here. The overall reactivity depends upon the total amount of VOC and the VOC chemical composition, and hence, on baseline emissions. The total daily reactivity estimated for 2010 is presented in Fig. 4. Peak ozone production potential occurs in the northeastern part of the domain, where peak ozone occurs. Overall, high rates of ozone production are located downwind in the same areas as the areas that exhibit high ground-level ozone concentrations. Although ozone precursors are mostly emitted in the central part of the domain e around Los Angeles and Long Beach e ozone production potential peaks downwind, where the sinks of ozone are considerably lower than those near the central part. Table 2 Maximum ozone reactivity in the South Coast Air Basin of California estimated for the base case 2010, without the addit m ozone reactivity in the South Coast Air Basin of California estimated for the base case 2010, without the addition of distributed or ce (Karamchandani et al., 2002; Vijayaraghavan et al., 2006). The plume effect generally retards the reactivity of pollutants from the stack, resulting in air quality impacts of elevated sources using the plume-in-grid model that are smaller in intensity, but more widespread in area, in comparison with the impacts obtained with a conventional air quality model. oxidizes to NO2 and then it photolyzes back to NO, producing a number of ozone molecules for every NOx molecule before NOx is removed by termination reactions. Thus, the presence of VOC is key to the resulting air quality impacts from a point source that emits large quantities of NOx. The capacity of a VOC mixture to provide a reactive environment that yields high concentrations of ozone is determined by ozone reactivity scales (Carter, 1994; Grifﬁn et al., 2004). The ozone-forming potential of a VOC and CO mixture, P(O3), is calculated as follows, based on Grifﬁn et al. (2004): Table 2 Under normal conditions, the plant in Huntington Beach reduces peak O3 concentrations by 11 ppb in some locations, but, it also increases peak O3 concentrations by 2 ppb in other locations. Operation of the same plant under ‘worst-day’ conditions leads to decreases in peak O3 concentrations of 13 ppb and increases of 6 ppb in various locations as shown in Fig. 2(f). In addition, the geographic area affected by increases in ozone concentration due to ‘worst-day’ operating conditions is larger than the area affected by the same plant operated under normal conditions regardless of installation location. Note that other studies have simulated elevated sources using a plume-in-grid model, which incorporates a plume model into a conventional Eulerian model, such as the UCI-CIT model Air quality impacts on 24-h average PM2.5 concentrations are presented in the difference plots of Fig. 3. Changes in PM2.5 are due to changes in direct emissions of particles from the scenarios and due Fig. 3. Increase in 24-h average PM2.5 concentrations with respect to the base case (negative values represent decreases in concentration with respect to base case): (a) DG scenario 1, (b) DG scenario 2, (c) normal operation of Huntington Beach central power plant, (d) ‘worst-day’ operation of Huntington Beach central power plant, (e) normal operation of Etiwanda central power plant, (f) ‘worst-day’ operation of Etiwanda central power plant, (g) normal operation of El Segundo central power plant, (h) ‘worst-day’ operation of El Segundo central power plant. Fig. 3. Increase in 24-h average PM2.5 concentrations with respect to the base case (negative values represent decreases in concentration with respect to base case): (a) DG scenario 1, (b) DG scenario 2, (c) normal operation of Huntington Beach central power plant, (d) ‘worst-day’ operation of Huntington Beach central power plant, (e) normal operation of Etiwanda central power plant, (f) ‘worst-day’ operation of Etiwanda central power plant, (g) normal operation of El Segundo central power plant, (h) ‘worst-day’ operation of El Segundo central power plant. 3220 M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 Fig. 4. Maximum ozone reactivity in the South Coast Air Basin of California estimated for the base case 2010, without the addition of distributed or central generation. y in the South Coast Air Basin of California estimated for the base case 2010, without the addition of distributed or central generation. Fig. 4. 3.1. Spatial sensitivity of air quality impacts (2005) calculated values of ozone production efﬁciency (OPE) as the ratio between the ozone produced (Ox ¼ O3 þ NO2 þ PAN þ NO3 þ 2NO3 þ 3N2O5, where PAN corresponds to peroxyalkyl nitrates) and the ozone oxidized (NOZ ¼ NOY NOx, where NOY ¼ NOx þ HONO þ HNO3 þ N2O5 þ NO3 þ PAN). The OPE values are then obtained by the slope of the plot of Ox concentrations versus NOZ concentrations, as shown in Fig. 5. The OPE values reported by Springston et al. are more than four times smaller than the values obtained for the SoCAB, because those values correspond to an isolated plume in a remote area, far from other anthropogenic emissions. An alternative way to calculate OPE based upon the ratio between O3 production and NOx destruction (following Grifﬁn et al., 2004) provides values that agree better with the ones reported by Springston et al. The values of O3 production, NOx destruction and OPE values are presented in Fig. 6. Qualitatively, both methods to determine ozone reactivity show the same trend in reactivity for the three power plant locations. Namely, O3 production efﬁciency is largest in Etiwanda, and smallest in El Segundo. Hence, if location of a power plant is based upon OPE values alone, then El Segundo would be the preferred power plant location amongst the three. 3.1. Spatial sensitivity of air quality impacts (2005) calculated values of ozone production efﬁciency (OPE) as the ratio between the ozone produced (Ox ¼ O3 þ NO2 þ PAN þ NO3 þ 2NO3 þ 3N2O5, where PAN corresponds to peroxyalkyl nitrates) and the ozone oxidized (NOZ ¼ NOY NOx, where NOY ¼ NOx þ HONO þ HNO3 þ N2O5 þ NO3 þ PAN). The OPE values are then obtained by the slope of the plot of Ox concentrations versus NOZ concentrations, as shown in Fig. 5. The OPE values reported by Springston et al. are more than four times smaller than the values obtained for the SoCAB, because those values correspond to an isolated plume in a remote area, far from other anthropogenic emissions. An alternative way to calculate OPE based upon the ratio between O3 production and NOx destruction (following Grifﬁn et al., 2004) provides values that agree better with the ones reported by Springston et al. The values of O3 production, NOx destruction and OPE values are presented in Fig. 6. Qualitatively, both methods to determine ozone reactivity show the same trend in reactivity for the three power plant locations. Namely, O3 production efﬁciency is largest in Etiwanda, and smallest in El Segundo. Hence, if location of a power plant is based upon OPE values alone, then El Segundo would be the preferred power plant location amongst the three. Nguyen and Dabdub (2002). Hence, the largest increases in PM2.5 occur downstream from Huntington Beach power plant location, because there are high emissions of ammonia released downwind from that location. Overall, whereas the impacts from direct emissions of PM2.5 are not sensitive to location, the impacts of power plant location on secondary aerosol depend strongly on the presence of ammonia downwind from the plume. Fig. 7 shows the concentrations of NOZ, mainly constituted by HNO3, downwind from each of the three power plant locations. Concentrations of NOZ downwind from Etiwanda are signiﬁcantly higher than for the other two power plant locations, because ammonia emissions downwind from the plant in Etiwanda are not as high. plants, as shown in Fig. 2. However, it is interesting to note that the smallest decrease in ozone concentration occurs for the plant in Etiwanda, because it is located in an area with higher ozone production potential compared to the other plants. Springston et al. 3.1. Spatial sensitivity of air quality impacts The three power plants considered in this study are located in areas with moderate-to-low ozone reactivity potential. Only the power plant located in Etiwanda shows high reactivity downwind from the plant. Ozone is formed through the oxidation of VOC and the catalytic cycle of NOeNO2 formation. In the presence of VOC and sunlight, NO Fig. 5. Ozone production potential calculated based on Springston et al. (2005) in the three plumes: Huntington Beach (HB), El Segundo (ES), and Etiwanda (ET). Even though ozone reactivity is positive throughout the basin, the most prominent impacts from power plants result in a reduction in ozone concentration. Previous studies based on ﬁeld measure- ments of plume concentrations determined that concentrations of NOx below 10 ppb enhance ozone formation, whereas concentra- tions above 15 ppb suppress ozone formation (Ryerson et al., 2001; Springston et al., 2005). In contrast, current simulation results show that ozone formation is suppressed even at lower NOx concentrations. Average NOx concentrations in the plume from each power plant are above 15 ppb only until 9:00 am (see Fig. 7(a)). In addition, NOx concentrations in the plumes from Huntington Beach and Etiwanda are below 10 ppb during the afternoon hours. Even under these low NOx concentrations in the plumes, ozone concen- trations decrease in this region due to emissions from the power Fig. 5. Ozone production potential calculated based on Springston et al. (2005) in the three plumes: Huntington Beach (HB), El Segundo (ES), and Etiwanda (ET). M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 3221 Fig. 6. Alternative ozone production potential based on Grifﬁn et al. (2004) in the three plumes: Huntington Beach (HB), El Segundo (ES), and Etiwanda (ET). The ﬁgures present: (a) O3 production during day-light, P(O3), (b) NOx destruction, L(NOx), (c) ozone production efﬁciency, OPE ¼ P(O3)/L(NOx). Fig. 6. Alternative ozone production potential based on Grifﬁn et al. (2004) in the three plumes: Huntington Beach (HB), El Segundo (ES), and Etiwanda (ET). The ﬁgures present: (a) O3 production during day-light, P(O3), (b) NOx destruction, L(NOx), (c) ozone production efﬁciency, OPE ¼ P(O3)/L(NOx). plants, as shown in Fig. 2. However, it is interesting to note that the smallest decrease in ozone concentration occurs for the plant in Etiwanda, because it is located in an area with higher ozone production potential compared to the other plants. Springston et al. 4. Conclusions California Energy Commission (CEC), 2000. Commission Decision: Application for Certiﬁcation for the High Desert Power Project. High Desert Power, LLC. Docket No 97-AFC-1, P800-00-003. Air quality impacts caused by central generation of electricity are contrasted with the effects of distributed generation with compa- rable capacity. Emissions from central generation under “normal” operating conditions are signiﬁcantly lower than emissions from DG to meet the same electricity demands. Only NH3 emissions and NOx emissions from DG scenarios that include the use of CHP are lower in some of the DG cases compared to the central generation cases. Emissions from central generation under ‘worst-day’ condi- tions are comparable to the emissions from DG. Even though emissions from central generation are lower than emissions from the DG scenarios considered herein, central generation concen- trates emissions in a small area, whereas DG spreads emissions throughout a large area of the air basin. As a result, air quality impacts from central generation are greater and more concentrated than the impacts from DG. In addition, impacts of central generation were found to depend strongly upon the location of the power plant. Amongst the three locations explored in this study, the plant located in Huntington Beach e upwind from the areas with high ozone and PM2.5 concentrations e has the greatest negative air quality impact. California Energy Commission (CEC), 2007a. Energy facilities siting and licensing process. Available at. http://www.energy.ca.gov/sitingcases/index.html. California Energy Commission (CEC), 2007b. California’s major sources of energy. Available at. http://www.energy.ca.gov/html/energysources.html. California Public Utilities Commission (CPUC), 2009. California self-generation incentive program. Eight year impacts evaluation report. Submitted to PG&E and the Self-Generation Incentive Program Working Group. Prepared by Itron, Inc., Vancouver, USA. Carreras-Sospedra, M., Rodriguez, M., Brouwer, J., Dabdub, D., 2006. Air quality modeling of the South Coast Air Basin of California: what do numbers really mean? Journal of the Air and Waste Management Association 56, 1184e1195. Carter, W.P.L., 1994. Development of ozone reactivity scales for volatile organic compounds. Journal of the Air and Waste Management Association 44, 881e899. Grifﬁn, R.J., Dabdub, D., Seinfeld, J.H., 2002a. Secondary organic aerosol. 1. Atmo- spheric chemical mechanism for production of molecular constituents. Journal of Geophysical Research 107 (D17), 4332. p y ( ) Grifﬁn, R.J., Dabdub, D., Kleeman, M.J., Fraser, M.P., Cass, G.R., Seinfeld, J.H., 2002b. Secondary organic aerosol. 3. Urban/regional scale model of size- and compo- sition-resolved aerosols. Journal of Geophysical Research 107 (D17), 4334. Table 3 emissions, and the solution of atmospheric chemistry and transport in an airshed model to determine air quality impacts. Table 3 Increase in pollutant exposure due to changes in peak ozone and daily average PM2.5 concentrations estimated for distributed and central electricity generation scenarios. The ranking in exposure of each scenario is included in parentheses. Table 3 Increase in pollutant exposure due to changes in peak ozone and daily average PM2.5 concentrations estimated for distributed and central electricity generation scenarios. The ranking in exposure of each scenario is included in parentheses. Analysis of population exposure to ozone and PM2.5 shows that central generation located in coastal areas upwind from populated areas would cause the most adverse effects, even if emissions from central generation are considerably lower than DG emissions spread throughout the basin. Conversely, human exposure from central generation located downwind from the central area of the SoCAB would be comparable to the effects from DG. In conclusion, exposure to pollutants is not strictly related to total pollution emissions, but rather is affected by the spatial and temporal distribution of emis- sions and resulting atmospheric chemistry and transport that leads to high ground-level concentrations near population centers. Increase in pollutant exposure O3 106 person ppb PM2.5 106 person mg m3 DG scenario 1 (R3) 0.72 (1) 1.50 (8) DG scenario 2 (PW2010) 1.71 (5) 1.22 (7) Huntington Beach normal 1.39 (4) 4.19 (3) Huntington Beach worst-day 3.64 (7) 10.77 (1) El Segundo normal 1.01 (3) 2.17 (4) El Segundo worst-day 4.61 (8) 6.96 (2) Etiwanda normal 0.78 (2) 0.02 (6) Etiwanda worst-day 2.48 (6) 1.07 (5) Increase in pollutant exposure References Allison, J.E., Lents, J., 2002. Encouraging distributed generation of power that improves air quality: can we have our cake and eat it too? Energy Policy 30, 737e752. ARB, 2008. Speciation Proﬁles Used in Air Resources Board Modeling. California Air Resources Board. Available at. http://www.arb.ca.gov/ei/speciate/speciate.htm (last accessed in December 2008.). Brundish, K.D., Miller, M.N., Wilson, C.W., Jefferies, M., Hilton, M., Johnson, M.P., 2005. Measurement of smoke particle size and distribution within a gas turbine combustor. Journal of Engineering for Gas Turbines and Power e Transactions of the ASME 127, 286e294. Acknowledgements scenarios increase exposure to PM2.5, except for the plant in Eti- wanda when it is operating under normal conditions. Moreover, DG scenarios cause the overall lowest pollutant exposure to PM2.5. Any beneﬁts from ozone reductions are largely offset by the deleterious effects from particles released or produced in the atmosphere as a result of emissions from the central plants located in Huntington Beach and El Segundo. Finally, both the normal-operation case for Etiwanda and DG scenario 2 produce beneﬁts in ozone and PM2.5 exposure, separately. Beneﬁts for both pollutants occur despite the fact that these scenarios introduce net increased emissions. These results show the non-direct relationship between human exposure and direct emissions. In particular, exposure depends upon the spatial and temporal distribution of emissions and how these emissions interact with atmospheric chemistry and transport to form secondary pollutants and disperse species of concern to human health in relation to population. We graciously acknowledge the ﬁnancial support of the Cal- ifornia Energy Commission (CEC) and the program management of Marla Mueller. Any opinions, ﬁndings, and conclusions or recom- mendations expressed in this material are those of the authors and do not necessarily reﬂect the views of the CEC. We thank the Cal- ifornia Air Resources Board and South Coast Air Quality Manage- ment District for their provision of the emissions inventory. 3.2. Spatial sensitivity of human exposure The spatial distribution of air quality impacts is not necessarily correlated with population density. To assess human exposure to air quality impacts analysis of population-weighted concentrations was accomplished. This analysis considers the change in peak ozone and daily average PM2.5 concentration due to central and distributed generation multiplied by local population density to quantify the change in exposure expressed as person$ppb for ozone and person$(mg m3) for PM2.5 for a given area. The basin-wide increases in population exposure for each case are presented in Table 3. Interestingly, all scenarios except DG scenario 1 introduce additional NOx emissions that reduce peak ozone concentrations and exposure. Only DG scenario 1 causes a net reduction in NOx emissions, which leads to a slight increase in ozone exposure. In the case of particulate matter, central generation The increase in NOx, which produces a dip in ozone concen- trations in the plumes, leads to an increase in nitric acid that is available to form nitrates in the aerosol phase, increasing the concentration of PM2.5. However, formation of nitrates in the SoCAB is directly related to the availability of ammonia, as suggested by Fig. 7. Hourly average concentrations of (a) NOx and NOY, and (b) NOZ in the plumes of the three power plants: Huntington Beach (HB), El Segundo (ES), and Etiwanda (ET). g. 7. Hourly average concentrations of (a) NOx and NOY, and (b) NOZ in the plumes of the three power plants: Huntington Beach (HB), of (a) NOx and NOY, and (b) NOZ in the plumes of the three power plants: Huntington Beach (HB), El Segundo (ES), and Etiwanda (ET) Fig. 7. Hourly average concentrations of (a) NOx and NOY, and (b) NOZ in the plumes of the three power plants: Huntington Beach (HB), El Segundo (ES), and Etiwanda (ET). M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 3222 4. Conclusions Grifﬁn, R.J., Nguyen, K., Dabdub, D., Seinfeld, J.H., 2003. A coupled hydro- phobicehydrophilic model for predicting secondary organic aerosol formation. Journal of Atmospheric Chemistry 44, 171e190. Grifﬁn, R.J., Revelle, M.K., Dabdub, D., 2004. Modeling the oxidative capacity of the atmosphere of the South Coast Air Basin of California: 1. Ozone formation metrics. Environmental Science and Technology 38, 746e752. Harley, R.A., Russell, A.G., McRae, G.J., Cass, G.R., Seinfeld, J.H., 1993. Photochemical modeling of the Southern California air quality study. Environmental Science and Technology 27, 378e388. Air quality simulations show that implementation of DG would potentially cause smaller air quality impacts than central generation located in the SoCAB, even though DG installations release more emissions than central generation. This is especially evident if the central power plant is installed near the coast, upwind from areas with typically poor air quality. This study shows that assessment of air quality impacts from distributed and central generation should not only consider the magnitude of air pollutant emissions. Assessment of air quality impacts requires detailed understanding of the implementation scenarios, temporally and spatially resolved Heath, A.G., Hoats, A.S., Nazaroff, W.W., 2006. Intake fraction assessment of the air pollutant exposure implications of a shift toward distributed electricity generation. Atmospheric Environment 40, 7164e7177. Ianucci, J., Horgan, S., Eyer, J., Cibulka, L., 2000. Air Pollution Emissions Impacts Associated with the Economic Market Potential of Distributed Generation in California. Distributed Utility Associates. Prepared for The California Air Resources Board. Contract #97e326. Karamchandani, P., Seigneur, C., Vijayaraghavan, K., Wu, S.-Y., 2002. Development and application of a state-of the-science plume-in-grid model. Journal of Geophysical Research 107 (D19), 4403. M. Carreras-Sospedra et al. / Atmospheric Environment 44 (2010) 3215e3223 3223 McRae, G.J., Seinfeld, J.H., 1982. Development of a second-generation mathematical- model for urban air-pollution. 1. Model formulation. Atmospheric Environment 16, 679e696. Rodriguez, M.A., Carreras-Sospedra, M., Medrano, M., Brouwer, J., Samuelsen, G.S., Dabdub, D., 2006. Air quality impacts of distributed power generation in the South Coast Air Basin of California 1: scenario development and modeling analysis. Atmospheric Environment 40, 5508e5521. Medrano, M., Brouwer, J., Carreras-Sospedra, M., Rodriguez, M.A., Dabdub, D., Samuelsen, G.S., 2008. A methodology for developing distributed generation scenarios in urban areas using geographical information systems. International Journal of Energy Technology and Policy 6, 413e434. 4. Conclusions Ryerson, T.B., Trainer, M., Holloway, J.S., Parrish, D.D., Huey, L.G., Sueper, D.T., Frost, G.J., Donnelly, S.G., Schaufßer, S., Atlas, E.L., Kuster, W.C., Goldan, P.D., Hubler, G., Meagher, J.F., Fehsenfeld, F.C., 2001. Observations of ozone formation in power plant plumes and implications for ozone control strategies. Science 292, 719e723. Journal of Energy Technology and Policy 6, 413e434. Meng, Z., Dabdub, D., Seinfeld, J.H., 1998. Size-resolved and chemically resolved model for aerosol dynamics. Journal of Geophysical Research e Atmospheres 103, 3419e3435. SCAQMD, Air Quality Management Plan (AQMP), 2003. Air Quality Management District (AQMD). Available at. http://www.aqmd.gov/aqmp/AQMD03AQMP.htm. Moya, M., Pandis, S.N., Jacobson, M.Z., 2002. Is the size distribution of urban aerosols determined by thermodynamic equilibrium? An application to Southern California. Atmospheric Environment 36, 2349e2365. Samuelsen, S., Dabdub, D., Brouwer, J., Medrano, M., Rodriguez, M., Carreras- Sospedra, M., 2005. Air Quality Impacts of Distributed Generation. California Energy Commission. PIER Energy-Related Environmental Research. CEC-500-2005-069-F. Nguyen, K., Dabdub, D., 2001. Two-level time-marching scheme using splines for solving the advection equation. Atmospheric Environment 35, 1627e1637. Springston, S.R., Kleinman, L.I., Brechtel, F., Lee, Y.-N., Nunnermacker, L.J., Wang, J., 2005. Chemical evolution of an isolated power plant plume during the TexAQS 2000 study. Atmospheric Environment 39, 3431e3443. g q p Nguyen, K., Dabdub, D., 2002. NOx and VOC control and its effects on the formation of aerosols. Aerosol Science and Technology 36, 560e572. Østergaard, P.A., 2005. Modeling grid losses and the geographic distribution of electricity generation. Renewable Energy 30, 977e987. Tomashefsky, S., Marks, M., 2002. Distributed Generation Strategic Plan. California Energy Commission. Vijayaraghavan, K., Karamchandani, P., Seigneur, C., 2006. Plume-in-grid modeling of summer air pollution in Central California. Atmospheric Environment 40, 5097e5109. Porter, K., Intermittency Analysis Project Team,, 2007. Intermittency Analysis Project: Summary of Final Results. California Energy Commission. PIER Research Development & Demonstration Program. CEC-500-2007-081. Development & Demonstration Program. CEC-500-2007-0 Zeldin, M.D., Bregman, L.D., Horie Y., 1990. A meteorological and air quality assessment of the representativeness of the 1987 SCAQS intensive days. Final report to the South Coast Air Quality Management District. Pun, B.K., Grifﬁn, R.J., Seigneur, C., Seinfeld, J.H., 2002. Secondary organic aerosol. 2. Thermodynamic model for gas/particle partitioning of molecular constituents. Journal of Geophysical Research 107 (D17), 4333.
https://openalex.org/W4361883478	https://figshare.com/articles/journal_contribution/Data_Supplement_from_An_Integrative_Analysis_of_the_Tumorigenic_Role_of_TAZ_in_Human_Non_Small_Cell_Lung_Cancer/22454655/1/files/39905769.pdf	English	null	Data Supplement from An Integrative Analysis of the Tumorigenic Role of TAZ in Human Non–Small Cell Lung Cancer	null	2,023	cc-by	1,101	Supplementary Figure S7 A B Gene set: gefitinib sensitive genes TAZ-high vs. TAZ-low, P = 0.020 Erlotinib (EGFR-TKI) ZD6474 (EGFR-TKI) TAE684 (ALK inhibitor) Crizotinib Paclitaxel Irinotecan 11 4 TAZ expression TAZ expression TAZ expression TAZ expression TAZ expression TAZ expression P = 0.078 P = 0.783 P = 0.084 P = 0.885 P = 0.646 P = 0.934 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 resistant sensitive resistant sensitive resistant sensitive resistant sensitive resistant sensitive resistant sensitive TAZ -high TAZ-low Supplementary Figure S7. TAZ expression level is associated with EGFR-TKI sensitivity. A, the association between TAZ expression and sensitivity to selected drugs in NSCLC cell lines. NSCLC cell lines were divided into two groups (resistant or sensitive), according to half maximal inhibitory concentration (IC50). Data about gene expression and IC50 values were obtained from Cancer Cell Line Encyclopedia database. P values were calculated by the Wilcoxon rank-sum test. The middle bar, median; box, inter-quartile range. B, GSEA was performed in the Uppsala cohort using the set of 139 gefitinib sensitive genes, the expressional dynamics of which are altered by addition of gefitinib in EGF-treated human primary lung epithelial cells (Yamauchi et al.). The enrichment of gefitinib sensitive genes is shown schematically with the genes that correlated best with high TAZ expression on the left and those that correlated best with low TAZ expression on the right. Supplementary Figure S7 A B Gene set: gefitinib sensitive genes TAZ-high vs. TAZ-low, P = 0.020 Erlotinib (EGFR-TKI) ZD6474 (EGFR-TKI) TAE684 (ALK inhibitor) Crizotinib Paclitaxel Irinotecan 11 4 TAZ expression TAZ expression TAZ expression TAZ expression TAZ expression TAZ expression P = 0.078 P = 0.783 P = 0.084 P = 0.885 P = 0.646 P = 0.934 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 resistant sensitive resistant sensitive resistant sensitive resistant sensitive resistant sensitive resistant sensitive TAZ -high TAZ-low Supplementary Figure S7. TAZ expression level is associated with EGFR-TKI sensitivity. A, the association between TAZ expression and sensitivity to selected drugs in NSCLC cell lines. NSCLC cell lines were divided into two groups (resistant or sensitive), according to half maximal inhibitory concentration (IC50). Data about gene expression and IC50 values were obtained from Cancer Cell Line Encyclopedia database. P values were calculated by the Wilcoxon rank-sum test. The middle bar, median; box, inter-quartile range. B, GSEA was performed in the Uppsala cohort using the set of 139 gefitinib sensitive genes, the expressional dynamics of which are altered by addition of gefitinib in EGF-treated human primary lung epithelial cells (Yamauchi et al.). The enrichment of gefitinib sensitive genes is shown schematically with the genes that correlated best with high TAZ expression on the left and those that correlated best with low TAZ expression on the right. Supplementary Figure S7 A B Gene set: gefitinib sensitive genes TAZ-high vs. TAZ-low, P = 0.020 Erlotinib (EGFR-TKI) ZD6474 (EGFR-TKI) TAE684 (ALK inhibitor) Crizotinib Paclitaxel Irinotecan 11 4 TAZ expression TAZ expression TAZ expression TAZ expression TAZ expression TAZ expression P = 0.078 P = 0.783 P = 0.084 P = 0.885 P = 0.646 P = 0.934 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 resistant sensitive resistant sensitive resistant sensitive resistant sensitive resistant sensitive resistant sensitive A Erlotinib (EGFR-TKI) ZD6474 (EGFR-TKI) Paclitaxel 11 4 TAZ expression TAZ expression TAZ expression P = 0.078 P = 0.783 P = 0.084 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 resistant sensitive resistant sensitive resistant sensitive A Erlotinib (EGFR-TKI) TAZ expression P 11 4 5 6 7 8 9 10 resistant sensitive A TAE684 (ALK inhibitor) Crizotinib Irinotecan TAZ expression TAZ expression TAZ expression P = 0.885 P = 0.646 P = 0.934 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 resistant sensitive resistant sensitive resistant sensitive B Gene set: gefitinib sensitive genes TAZ-high vs. TAZ-low, P = 0.020 TAZ -high TAZ-low Supplementary Figure S7. TAZ expression level is associated with EGFR-TKI sensitivity. B Gene set: gefitinib sensitive genes TAZ-high vs. TAZ-low, P = 0.020 TAZ -high TAZ-low B Supplementary Figure S7. TAZ expression level is associated with EGFR-TKI sensitivity. A, the association between TAZ expression and sensitivity to selected drugs in NSCLC cell lines. NSCLC cell lines were divided into two groups (resistant or sensitive), according to half maximal inhibitory concentration (IC50). Data about gene expression and IC50 values were obtained from Cancer Cell Line Encyclopedia database. P values were calculated by the Wilcoxon rank-sum test. The middle bar, median; box, inter-quartile range. B, GSEA was performed in the Uppsala cohort using the set of 139 gefitinib sensitive genes, the expressional dynamics of which are altered by addition of gefitinib in EGF-treated human primary lung epithelial cells (Yamauchi et al.). Supplementary Figure S7 The enrichment of gefitinib sensitive genes is shown schematically with the genes that correlated best with high TAZ expression on the left and those that correlated best with low TAZ expression on the right. Supplementary Figure S7. TAZ expression level is associated with EGFR-TKI sensitivity. A, the association between TAZ expression and sensitivity to selected drugs in NSCLC cell lines. NSCLC cell lines were divided into two groups (resistant or sensitive), according to half maximal inhibitory concentration (IC50). Data about gene expression and IC50 values were obtained from Cancer Cell Line Encyclopedia database. P values were calculated by the Wilcoxon rank-sum test. The middle bar, median; box, inter-quartile range. B, GSEA was performed in the Uppsala cohort using the set of 139 gefitinib sensitive genes, the expressional dynamics of which are altered by addition of gefitinib in EGF-treated human primary lung epithelial cells (Yamauchi et al.). The enrichment of gefitinib sensitive genes is shown schematically with the genes that correlated best with high TAZ expression on the left and those that correlated best with low TAZ expression on the right.
https://openalex.org/W3004991897	http://pure-oai.bham.ac.uk/ws/files/94063974/infa.12325.pdf	English	null	Infant screen exposure links to toddlers' inhibition, but not other EF constructs: A propensity score study	Infancy	2,020	cc-by	9,572	Infant screen exposure links to toddlers’ inhibition, but not other EF constructs The NewFAMS Study Team DOI: 10.1111/infa.12325 10.1111/infa.12325 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): The NewFAMS Study Team 2020, 'Infant screen exposure links to toddlers’ inhibition, but not other EF constructs: a propensity score study', Infancy, vol. 25, no. 2, pp. 205-222. https://doi.org/10.1111/infa.12325, https://doi.org/10.1111/infa.12325 Link to publication on Research at Birmingham portal University of Birmingham University of Birmingham General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. 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R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Funding information National Science Foundation, Grant/Award Number: BCS-1429152; Economic and Social Research Council, Grant/Award Number: ES/L010648/1; Nederlandse Organisatie voor Wetenschappelijk Onderzoek, Grant/Award Number: 464- 13-141 Correspondence Correspondence Gabrielle McHarg, Centre for Family Research, Free School Lane, University of Cambridge, Cambridge CB2 3RQ, UK. Email: ggm25@cam.ac.uk Gabrielle McHarg1 \| Andrew D. Ribner2 Claire Hughes1 \| The NewFAMS Study Team Gabrielle McHarg1 \| Andrew D. Ribner2 Claire Hughes1 \| The NewFAMS Study Team 1Centre for Family Research, University of Cambridge, Cambridge, UK 2Department of Applied Psychology, New York University, New York, NY, USA 3Department of Psychology, University of Birmingham, Birmingham, UK Infant screen exposure links to toddlers' inhibition, but not other EF constructs: A propensity score study Gabrielle McHarg1 \| Andrew D. Ribner2 \| Rory T. Devine3 \| Claire Hughes1 \| The NewFAMS Study Team y p g p p y © 2020 The Authors. Infancy published by Wiley Periodicals, Inc. on behalf of International Congress of Infant Studies Abstract Abstract Technology is pervasive in homes of families with young children, despite evidence for negative associations be- tween infant exposure to screen-based media and cogni- tive development that has led the American Academy of Pediatrics (AAP) to discourage parents from exposing chil- dren under the age of 18 months to any kind of screen time (AAP, 2016). Here, we apply a propensity score matching approach to estimate relations between electronic screen- based media use in infancy and executive function in early toddlerhood. In an international sample of 416 firstborn in- fants, parental report of regular exposure to screen-based media at 4 months predicted poorer performance on a test of inhibition at 14 months, but was unrelated to either cogni- tive flexibility or working memory at 14 months. Results of this study are therefore consistent with the view that early exposure to screen-based media adversely affects the devel- opment of executive function. Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 DOI: 10.1111/infa.12325 electronic screen-based media is pervasive. Nearly 97% of households in the United States report hav- ing at least one television (Nielsen, 2017), and 95% of households with children between the ages of 0 and 8 have at least one smartphone (Common Sense Media, 2017). In addition, a recent UK study of 131 children aged 6–36 months found that 82% of the sample watched television daily, and 49% used mobile touchscreen devices daily (Taylor, Monaghan, & Westermann, 2018). The evidence behind the AAP recommendation for very young children is limited, and potential long-term effects of very early electronic screen-based media use remain unknown. However, there is growing interest in the association between very early screen use and children's executive function (e.g., Lillard & Peterson, 2011). Executive function (EF; a multidimensional construct that broadly encompasses the ability to inhibit prepotent responses, keep multiple pieces of information in mind and manipulate them in working memory, and flexibly shift attention between multiple stimuli in pursuit of goals) develops throughout the lifespan and can be reliably measured as early as toddler- hood (e.g., Devine, Ribner, & Hughes, 2019; Johansson, Marciszko, Brocki, & Bohlin, 2016; Mulder, Hoofs, Verhagen, van der Veen, & Leseman, 2014). Supporting EF is critical, as it is implicated in the development of academic and interpersonal skills (Blair & Razza, 2007; Brock, Rimm-Kaufman, Nathanson, & Grimm, 2009; Devine & Hughes, 2014). Exposure to electronic screen-based media might be particularly detrimental to the development of EF; however, evidence to date is mixed and differences in results might depend upon context or platform of viewing, media content, or child age. In groups of older children, increased time spent watching television is negatively associated with EF, perhaps due to its impact on attentional capac- ities (Nathanson, Alade, Sharp, Rasmussen, & Christy, 2014; Ribner, Fitzpatrick, & Blair, 2017). This has been shown experimentally: Lillard and Peterson (2011) found that 4-year-old children who watched a fast-paced cartoon, rather than either an educational cartoon or no television, performed significantly worse on EF tasks immediately after watching. 1 \| INTRODUCTION Parents and teachers often discuss how much television young children should watch, but the answer to this question remains unclear. Despite recommendations from the American Academy of Pediatrics that children under 2 years of age should have very limited screen exposure (AAP, 2016), access to 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. \| 205 wileyonlinelibrary.com/journal/infa Infancy. 2020;25:205–222. 206 MCHARG et al. 2.1 \| Participants Participants were recruited as a part of a larger longitudinal study of parents and their firstborn chil- dren in the United Kingdom (UK), the United States (US), and the Netherlands (NL). To be eligible for the current study, potential participants had to (a) be first-time parents, (b) be expecting to deliver a healthy singleton baby, (c) be planning to speak the native language of the recruiting country (i.e., English or Dutch) as the child's primary language, and (d) have no history of severe mental illness (e.g., psychosis) or substance misuse. We recruited 474 expectant couples attending prenatal classes and appointments at local hospitals in the East of England and in New York City, and at maternity events in the Netherlands. An additional 10 families were recruited, but these families were not eligible for follow-up when the infants were 4 months old due to birth complications or having left the area. All remaining participants were born full term (after 36 weeks) and without birth complications. Of fami- lies recruited, 416 (93.3%; NEngland = 194; NNYC = 100; NNetherlands = 122) families agreed to partici- pate in a home visit when their infants (212 boys, 204 girls) were both 4 months (MAge = 4.26 months, SD = 0.45 months, range: 2.97–6.23 months) and 14 months (MAge = 14.42 months, SD = 0.57 months, range: 9.47–18.40 months of age). Of note, despite the seemingly large age range, age was not con- trolled for in analyses because it is not appropriate to control for post-treatment variables in propensity score analyses (described below) and there was no relation between age and EF variables at 14 months in the UK sample (see Devine et al., 2019). All procedures performed were in accordance with the ethical standards of the institutional and/or national research committees involved and were acceptable according to the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The National Health Service (NHS UK) Research Ethics Committee (London Bloomsbury), and the University Committee on Activities Involving Human Subjects at New York University approved the study protocol (REF: 14/LO/1113). EF. We use a propensity score modeling approach in a large, longitudinal, international sample of firstborn children to estimate effects of exposure to screen-based media on EF at 14 months. Because we could not ethically ask some parents to expose their children to screens, propensity score matching was used to create post hoc experimental groups. Based on what we know about television and its immediate effect on EF in older children, it is possible there are detrimental long-term effects of very early regular screen exposure on inhibition, working memory, and set-shifting. These findings suggest that there could be temporary “state” effects on EF, but say nothing about effects on individual differences in chronic or lasting “traits.” Results from a follow-up study identified that it was not pace, but on-screen fan- tastical elements, contrasted with on-screen real-life events, that seemed to negatively affect EF in 4- to 6-year-old children (Lillard, Drell, Richey, Boguszewski, & Smith, 2015). Huber, Yeates, Meyer, Fleckhammer, and Kaufman (2018) found that children were less likely to delay gratification after viewing a cartoon than after playing an educational app, suggesting not only content, but interactivity of screen time affects EF. Indeed, playing the app was associated with increased working memory. What is even less clear is how children's EF is affected by screen exposure in the first 6 months of life. Young infants only view screens for 3–5 s at a time (for summary, see Kirkorian, Pempek, & Choi, 2017), and child-directed programming does not seem to be understood by until age two (Anderson & Subrahmanyam, 2017; Hipp et al., 2017). Before this, all television content can be understood as background television, especially in the very early months when parents are likely to be watching adult-directed content around their infants (Anderson & Subrahmanyam, 2017). For infants and toddlers 12 months and older, research suggests that adult-content and background screen exposure is detrimental for EF development (Linebarger, Barr, Lapierre, & Piotrowski, 2014). Indeed, Barr, Lauricella, Zack, and Calvert (2010) found that higher levels of exposure to adult screen content at 12–14 months of age were related to lower inhibitory self-control and metacognition skills at age four. However, it is unclear how adult-directed background television may affect young infants' cogni- tive development. In addition, 4-month-old infants are almost certainly not interacting with apps and games directly and therefore are not likely getting the EF benefits of interactive screen use. In order to estimate the relations between screen exposure at 4 months of age and EF at 14 months, the current study exploits natural variation in whether parents exposed their young children to elec- tronic screen-based media. We expect that screen exposure early in life will have adverse effects on MCHARG et al. 207 correlated (all rs > .64; ps < .001); therefore, a single variable representing regular exposure to elec- tronic screen-based media was computed according to whether the value for a typical weekday or weekend day was >0. If parents reported more than 0 hr on either a typical weekday or weekend day (i.e., the parent reported any regular screen use; n = 276), that child received a value of “1”; if both typical weekday and weekend day were exactly 0 (i.e., the parent reported the child did not use screen time regularly), that child received a value of “0” (N = 111). correlated (all rs > .64; ps < .001); therefore, a single variable representing regular exposure to elec- tronic screen-based media was computed according to whether the value for a typical weekday or weekend day was >0. If parents reported more than 0 hr on either a typical weekday or weekend day (i.e., the parent reported any regular screen use; n = 276), that child received a value of “1”; if both typical weekday and weekend day were exactly 0 (i.e., the parent reported the child did not use screen time regularly), that child received a value of “0” (N = 111). 2.2.2 \| Outcome variables At 14 months, toddlers completed a short series of tasks based on tasks which are reported and elabo- rated on in Devine et al., 2019. Infants were seated on a parent's lap across a table from the examiner, and parents were asked to remain silent during each task and not to influence their infant's behavior. Infants were monitored and provided with breaks between tasks if necessary, and were praised at the end of each task regardless of performance. Inhibition In the Prohibition task (Friedman, Miyake, Robinson, & Hewitt, 2011), toddlers were shown a glittery wand by the experimenter while the examiner verbally engaged the infant. Next, the examiner looked the toddler in the eye, raised their index finger, and said: “No, don't touch!”. The wand was then placed within reaching distance of the toddler, and the examiner turned around for 30 s. The latency to the first time the toddler touched the wand was recorded (possible range: 0–30 s). Double coding of 60 of videos revealed high inter-rater agreement, ICC = 0.99, p < .001. 2.2.1 \| Independent variable Parents (mostly mothers) were asked to report the amount of time infants spent exposed to electronic screen-based media at 4 months of age. As a part of a comprehensive questionnaire completed either during or before the home visit, parents reported “Number of hours watching TV/DVDs or looking at iPADs or computer” on a typical weekday, weekend day, and on the day prior to completion of the questionnaire (adapted from Thompson, Adair, & Bentley, 2013). Responses were allowed to be given and were recorded in any unit (e.g., minutes, parts of hours) and were later converted to hours. Screen time on a typical weekday, weekend day, and the day prior to survey completion was highly 208 MCHARG et al. MCHARG et al. Factor score estimation A model was estimated in which each of the task indicators loaded onto separate latent factors repre- senting working memory and cognitive flexibility. As the inhibition task was comprised of just one indicator, we did not estimate a latent factor for this task. The two latent factors for each working memory and cognitive flexibility, as well as the indicator for inhibition, were allowed to correlate. This model provided a good fit to the data, χ2 (100) = 195.320, p < .0001, RMSEA = 0.047, 90% CI [0.038, 0.057], CFI = 0.977, TLI = 0.973. Detailed information about item-level task performance and latent variable estimation and reliability is reported elsewhere (Devine et al., 2019). To reduce model complexity and capitalize on the benefits of latent variable modeling, Bayesian plausible latent factor scores were estimated using multiple imputations (Asparouhov & Muthen, 2010). MCHARG et al. 209 Cognitive flexibility Cognitive flexibility The Ball Run task was based on the Trucks task developed by Hughes and Ensor (2005). A toy that had three circular holes on the top running from left to right (i.e., green, yellow, red) and a metal chute that allowed a ball to roll down through the toy was introduced to the infant. The middle hole (yellow) was sealed for the whole task, and the two holes on either end could be sealed or opened. The bottom of the toy was fitted with a pressure-activated speaker programmed to play 5 s of a nursery song (“The Wheels on the Bus”) when pressed by the ball. There were three phases: In the rule learning phase, the examiner introduced the toy to the toddler and illustrated how to play, either by green ball in the green hole (on the left-hand side of the toy) or the red ball in the red hole (on the right-hand side of the toy; counterbalanced across children). In the second phase, the examiner handed the ball to the toddler directly over the middle of the toy and looking at the infant and said, “Now you try!”. Toddlers were praised for each correct placement and reinforced through activation of the musical switch, and incorrect tries were met with “Oh, it didn't work!” and were followed by the next of six trials. For those children who placed the ball correctly four or more times (n = 159), a rule-reversal phase was carried out. To begin, the researcher placed the ball that had been used (e.g., the green ball) and conspicuously put it away. Next, the examiner got the other ball (e.g., the red ball) and swapped the brackets to open the corresponding hole and closing the hole for the ball that was put away. One ex- ample was shown to the child, and the examiner then repeated the experiment in the same way as the second phase with the new ball. Scoring took place offline, and double coding of 60 videos revealed perfect inter-rater reliability for each trial, Kappa = 1.00. Children received one point for each correct placement. Toddlers who did not place 4 or more balls correctly in the learning phase received a score of 0 on each trial of the reversal phase. Working memory In the Three Boxes task (Miller & Marcovitch, 2015), toddlers were asked to find red, yellow, and blue plastic cars hidden in matched-color garages. The toy was placed just out of reach of the toddler, and the examiner called attention to the process of putting the cars in the garages while doing so. Children were then given the chance to find cars in a series of searches, and, after each success, the child was allowed to play with the car briefly before it was very obviously placed behind the examiner. The door to the garage was then closed conspicuously. Between searches, there was a short delay in which the view of the garages was obstructed and the experimenter counted to 5. Since all garages contained a car, the toddler was always successful on the first trial. If toddlers pointed to an empty garage, the examiner opened the garage, looked inside, and said “Oh, it's not there. Let's have another go” and closed the door before starting the next trial. This continued until all cars were found or until the child made three consecutive errors. Scoring took place offline and double coding of 60 videos revealed perfect inter-rater reliability for each trial, Kappa = 1.00. Scoring was adapted from Garon, Smith, and Bryson (2014) in a similar multi-location search task and created two scores: total number of searches to find the (a) second and (b) third cars (i.e., 0 = did not find; 1 = 3 searches; 2 = 2 searches; 3 = 1 search). A third score based on adult research on self-ordered search tasks (Owen, Downes, Sahakian, Polkey, & Robbins, 1990) was also created for efficiency. The strategy score recorded the approach used to search for the hidden cars with higher scores indicating a more efficient search strategy (i.e., 0 = starts in the middle, 1 = starts at either edge, 2 = starts at either edge and then selects middle but then repeats a search, 3 = starts at edge, then middle, then other edge). 2.2.3 To control for possible selection effects that might be associated with child electronic screen-based media use, a range of control variables (all measured before 4 months) were entered into a logit model predicting regular screen use at 4 months. For both parents, the following variables were included from report when mothers were in their last month of pregnancy: parent age at time of childbirth, educational attainment, general well-being, anxiety, depression, life satisfaction, couple's satisfaction, self-efficacy in the nurturing role, and social support. Child attention and temperament, both collected at 4 months of age, were also included as propensity score covariates. Additional variables were in- cluded for child gender and country of recruitment with the UK as reference group. 210 MCHARG et al. Social support Parents completed the Multidimensional Scale of Perceived Social Support (Zimet, Dahlem, Zimet, & Farley, 1988); higher values indicate higher levels of perceived social support from family and friends. Items showed adequate internal consistency for mothers (α = .94) and fathers (α = .92). Subjective social status Parents completed the Subjective Social Status Ladder (Singh-Manoux, Adler, & Marmot, 2003). Higher values indicate participants view themselves as having higher social status. Self-efficacy in the nurturing role Self-efficacy in the nurturing role Parents completed the Self-Efficacy in the Nurturing Role scale (Pederson, Bryan, Huffman, & Del Carmen, 1989); higher values indicate higher levels of comfort and confidence as a parent of a young child. Items showed adequate internal consistency for mothers (α = .87) and fathers (α = .87). Parent well-being Parent well being Parents completed the General Health Questionnaire (Goldberg et al., 1997); higher values indicate more concerns with general well-being. Questionnaire items showed adequate internal consistency for mothers (α = .74) and fathers (α = .79). Parents completed the State-Trait Anxiety Inventory (Spielberger, Gorsuch, Lushene, Vagg, & Jacobs, 1983); higher values indicate higher levels of anxi- ety symptomatology. Items showed adequate internal consistency for mothers (α = .77) and fathers (α = .73). Parents completed the Center for Epidemiological Study-Depression Scale (Radloff, 1977); higher values indicate higher levels of depression symptomatology. Items showed adequate internal consistency for mothers (α = .80) and fathers (α = .83). Parents completed the Satisfaction with Life scale (Diener, Emmons, Larsen, & Griffin, 1985); higher values indicate lower levels of satisfaction (higher levels of dissatisfaction) with the respondent's own life. Items showed adequate internal con- sistency for mothers (α = .89) and fathers (α = .88). p f Parents completed the Couple's Satisfaction Inventory (Funk & Rogge, 2007); higher values indicate higher levels of satisfaction in the respondent's partnership. Items showed adequate internal consist- ency for mothers (α = .96) and fathers (α = .94). Parent well-being Attention We measured infant visual attention at 4 months (Cuevas & Bell, 2014). Infants were seated on a parent's lap facing the examiner (seated approximately 1 m from the infant). The examiner rattled an attractive toy three times and held it up to his/her right or left (counterbalanced across infants). The examiner held the stimulus in position until the infant looked away for at least 3 s. At this point, the examiner lowered the toy and repeated the procedure for three further trials. Child gaze was recorded using a camera placed on a tripod behind the examiner. The footage was coded offline using JHab Java Habituation Software (version 1.0.0) (Casstevens, 2007). Amount of time spent looking at the stimulus on each trial was recorded. Inter-rater reliability based on 45 cases was ac- ceptable, for all four trials 0.77 < ICC < 0.98. Median looking duration across the four trials of the task was used. Child temperament Mothers completed the Infant Behavior Questionnaire-Very Short Form (IBQ) (Putnam, Helbig, Garstein, Rothbart, & Leerkes, 2014), a widely used parent-report assessment to characterize infant 2.2.4 For ethical reasons, it was not feasible to use randomized control trials—the gold standard for estimat- ing causal effects—when studying effects of screen-based media use for young children. Thus, one increasingly common method to address this and estimate causal effects by identifying comparable groups is propensity score analysis (Schneider, Carnoy, Kilpatrick, Schmidt, & Shavelson, 2007). Propensity score analysis relies on a model of treatment assignment to match individuals on the basis of the probability of receiving “treatment” (i.e., regular exposure to screen-based media). Such mod- els are estimated using standard logistic regression, where the outcome is the treatment indicator and the predictors are covariates. A match for each observation who received “treatment” is then assigned by choosing the control observation with the closest propensity score. Propensity score matching may be practically useful because it does not hold as strict assumptions about the linearity of the data as required for traditional regression models. Because this method uses a matching procedure, it is also helpful for identifying clear treatment and counterfactual groups about whom to interpret findings (Gelman & Hill, 2007). We sought to examine the relation between regular electronic screen-based media use in infancy with each inhibition, working memory, and cognitive flexibility in early toddlerhood. To do this, we estimated weights representing each infant's propensity to be exposed to screen-based media using a logit model with regular screen use as the outcome variable and covariates specified above as predic- tors. Based on these predicted probabilities, we applied a genetic-matching algorithm to match infants who did not regularly use screens with those who did based on similar propensity to use screens ac- cording to covariates. Each individual EF measure was then regressed on the treatment variable and covariates using propensity scores as sample weights (Rosenbaum & Rubin, 1983). All analyses were performed in RStudio using the matchit package (Ho, Imai, King, & Stuart, 2011). Replacements for missing data were estimated with multiple imputation using chained equations prior to matching. All cases for whom regular media use was reported (n = 387) were included in analysis; missing data for cases for whom regular media use was reported ranged from 17.4 (n = 67) to 0%; and on average, variables were missing <5% of data (4.3%, n = 16.81 data points missing). Five datasets were estimated using the mi package (Su, Gelman, Hill, & Yajima, 2011). 2.2.4 Propensity scores were estimated for each dataset sequentially, and regression analyses were performed on each dataset. Coefficients were pooled manually using Rubin's (1987) rules. temperament. Temperament was represented by three scale scores, all of which demonstrated ad- equate internal consistency: Distress to Limitations (α = .83), Duration of Orienting (α = .75), and Distress to Approach (α = .77). Child temperament p Mothers completed the Infant Behavior Questionnaire-Very Short Form (IBQ) (Putnam, Helbig, Garstein, Rothbart, & Leerkes, 2014), a widely used parent-report assessment to characterize infant \| 211 MCHARG et al. temperament. Temperament was represented by three scale scores, all of which demonstrated ad- equate internal consistency: Distress to Limitations (α = .83), Duration of Orienting (α = .75), and Distress to Approach (α = .77). 211 MCHARG et al. 3 \| RESULTS Tables 1 and 2 show descriptive statistics and the extent to which data were missing for each variable. Regular use of electronic screen-based media differed by country (F(2, 384) = 10.10, p < .001) such that children from the UK (81.9%, n = 145) were more likely to be regularly exposed to electronic screen-based media than were children in either the United States (65.8%, n = 75; p < .001) or the Netherlands (58.3%, p = 56; p = .008). Pre-matching balance and post-matching balance for one data- set are shown in Table 2. Propensity score balance for the no-screen time group and the regular screen time group is shown in Figure 1 (Table 3). Abbreviations: CES-D, Center for Epidemiological Studies-Depression Scale; CSI, Couple's Satisfaction Index; SEN the Nurturing Role; SLS, Satisfaction with Life Scale; SSS, Subjective Social Status; STAI, State-Trait Anxiety Inv 212 \| 212 \| MCHARG et al. 212 TABLE 1 Descriptive statistics of continuous variables included in regressions TABLE 1 Descriptive statistics of continuous variables included in regressions TABLE 1 Descriptive statistics of continuous variables included in regressions N Mean SD Range Infant age 4 months 414 4.24 0.45 2.97–6.23 Infant attention 4 months 400 7.29 4.79 0.00–26.89 Mom SSS 404 7.32 1.09 3.75–10.00 Dad SSS 404 7.29 1.16 2.84–10.00 Mom well-being 396 1.98 2.18 0–12 Dad well-being 378 1.39 2.03 0–11 Mom STAI 396 10.40 2.99 3–22 Dad STAI 376 10.85 2.81 6–20 Mom CES-D 396 29.66 5.83 20–54 Dad CES-D 376 27.31 6.06 19–54 Mom SLS 396 10.20 4.71 5–35 Dad SLS 376 12.18 5.75 5–35 Mom CSI 396 88.73 7.62 52–97 Dad CSI 376 87.61 8.39 52–97 Mom SEN 395 86.47 12.55 46–112 Dad SEN 375 84.89 13.11 38–112 Mom social support 395 70.99 11.83 12–84 Dad social support 374 65.99 13.04 12–84 Media use per day (hours) 387 0.88 1.25 0.00–8.61 Inhibition 342 14.14 12.42 0–30 Working memory 402 0.12 0.74 −0.79 to 1.74 Cognitive flexibility 402 0.01 0.58 −0.79 to 1.57 Distress to limitations 424 3.41 0.92 1.50–6.29 Duration of orienting 424 4.21 0.93 1.83–7.00 Approach 424 2.01 0.76 1.00–7.00 Abbreviations: CES-D, Center for Epidemiological Studies-Depression Scale; CSI, Couple's Satisfaction Index; SEN, Self-efficacy in the Nurturing Role; SLS, Satisfaction with Life Scale; SSS, Subjective Social Status; STAI, State-Trait Anxiety Inventory. Media use was associated with lower inhibition, such that infants who were regularly exposed to electronic screen-based media touched the wand over 5 s earlier in the delay task than did those were not regularly exposed to electronic screen-based media (b = −4.26, p = .008). This associa- tion between propensity for media use and inhibition is shown graphically in Figure 2. Media use was not significantly associated with working memory (as measured by performance on the Multi- location Search task; p = .905) or cognitive flexibility (as measured by the Ball Run task; p = .917). Unweighted regressions with the same covariates were also estimated using a continuous measure of screen-based media use on an average day to examine whether the relations between exposure to electronic screen-based media and EF were linear in nature. Time spent watching media was not linearly associated with inhibition (b = −0.02, p = .437). Table 4 reports the results of the weighted regressions predicting EF. 213 MCHARG et al. 212 \| TABLE 2 Frequencies of categorical variables Frequency % χ2 (media vs. non-media) non-matched χ2 matched Child female 204 49.0 0.25 0.14 Mom employment 397 1.73 3.15 Full time 302 76.1 Part time 62 15.6 Home maker 9 2.3 Student 13 3.3 Seeking employment 11 2.8 Dad employment 397 2.90 1.79 Full time 369 92.9 Part time 15 3.8 Student 8 2 Seeking employment 5 1.3 Mom history of depression/anxiety 396 2.25** 3.58* No 327 82.6 Depression 32 8.1 Anxiety 22 5.6 Depression and anxiety 15 3.8 Dad history of depression/anxiety 397 11.80 6.46 No 353 88.9 Depression 22 5.5 Anxiety 14 3.5 Depression and anxiety 8 2 Mom Age at enrollment 404 1.70 1.61 21–24 15 3.7 25–29 116 28.7 30–34 186 46 35+ 87 21.5 Dad age at enrollment 404 0.50 1.61 21–24 4 1 25–29 77 19.1 30–34 187 46.3 35+ 136 33.7 Mom education 398 15.89 14.74 Upper secondary 20 5 Post-secondary not tertiary 13 3.3 Short-cycle tertiary 32 8 Bachelors 146 36.7 Masters 126 31.7 (Continues) TABLE 2 Frequencies of categorical variables (Continues) 214 \| MCHARG et al. Frequency % χ2 (media vs. non-media) non-matched χ2 matched Doctoral 52 13.1 Other 9 2.3 Dad education 379 27.17 19.00 Primary 1 0.3 Lower secondary 8 2.1 Upper secondary 29 7.7 Post-secondary not tertiary 22 5.8 Short-cycle tertiary 33 8.7 Bachelors 140 36.9 Masters 90 23.7 Doctoral 46 12.1 Other 10 2.7 p < .05; p < .01. TABLE 2 (Continued) 214 MCHARG et al. 4 \| DISCUSSION Inhibition at 14-months of age was negatively associated with screen exposure at 4 months. The in- verse relation between screen time and inhibition mirrors previous findings (Barr et al., 2010; Lillard & Peterson, 2011), but in a much younger sample over a longer period of time. Our results therefore extend prior findings by suggesting a longitudinal association between screen exposure and inhibi- tion, rather than a simple short-term effect. Notably, 4-month-old infants are not mobile and their screen time is at the discretion of their parents and caregivers. Additionally, the current study investigated the associations between overall screen exposure and EF, rather than interest in or attention to screens. Therefore, it is not likely that the inverse relation between screen exposure and inhibition is reflective of infants with lower inhibition being drawn to screens at this early age. Notably, the association between screen exposure and inhibition was found over and above parent characteristics. In other words, even if parents who are struggling to cope are especially likely to expose their infants to screens, this early exposure to screens has a specific and negative association with toddler EF even when mental health problems or couple conflict is considered. This is particu- larly important when considering the directionality of findings; it is not likely that parents who were unable to cope with their impulsive infants at 4 months of age were exposing those infants to more screens, but that infants who were exposed to more screen time had lower inhibition 10 months later. Importantly, however, this relation may reflect a greater exposure to screens among more impulsive children, perhaps because their parents are struggling to find ways to soothe them, even if it did not impact those parents' mental well-being. In addition, the quality of parental attention is related to EF (Hughes & Devine, 2017). Screens may take parents' attention away from their children, which in turn may decrease positive parent–child in- teractions and impact later EF. Another possibility is that parents with low levels of inhibitory control themselves might be more likely to leave screens on or watch television themselves, such that genetic factors might mediate the apparently environmental influence of screens. Indeed, parent exposure to 215 MCHARG et al. \| 215 MCHARG et al. FIGURE 1 Propensity score overlap for regular screen exposure and no-screen exposure groups. 4 \| DISCUSSION The goal of the propensity score approach is to match participants as closely as possible to one another across groups such that biggest difference is whether or not children used screens, and good propensity score matching was achieved FIGURE 1 Propensity score overlap for regular screen exposure and no-screen exposure groups. The goal of the propensity score approach is to match participants as closely as possible to one another across groups such that biggest difference is whether or not children used screens, and good propensity score matching was achieved screens is a strong predictor of child screen exposure (Beyens & Eggermont, 2014) and EF is related inter-generationally (Cuevas et al., 2014; Ellefson, Ng, Wang, & Hughes, 2017). Future research using cross-lagged designs and inter-generational measures of EF is needed to test these hypotheses. Whether screen time is directly inversely related to inhibition or indirectly related due to parental attention or both, this finding is striking. Even when infants are likely getting very little entertainment or content out of their screen exposure, this screen time is related to less positive outcomes in cogni- tive development. Notably, there was not a linear relationship between screen exposure and latency to touch, suggesting that it is not massed amounts of screen exposure, but instead any regular exposure that seems to have a detrimental effect. Note that the present study contrasted with the findings reported by Barr et al. (2010) in showing no relation between media use and either working memory or cognitive flexibility. However, this null effect could reflect infants' lack of active viewing; the mechanics behind negative effects of television on EF might be content specific or about engagement with on-screen interactions. Importantly, our results with regard to inhibition are in line with Huber et al. (2018), who found that passive viewing was related to lower inhibition more so than active engagement with an app, and who found a positive association between playing on the app and working memory. Thus, our findings support the idea that interactivity might make an impact. Content may matter as well; indeed, screen exposure in early in- fancy is likely to involve television for adults and hence show more life-like events than the fantastical events that Lillard et al. (2015) found to be especially detrimental. \| \| 216 TABLE 3 Pre- and post-matching mean differences Unmatched Matched No screens Screens t p-value No screens Screens t p-value Infant Age 4.27 4.24 0.60 .546 4.21 4.24 −0.43 .670 Mom well-being 1.33 1.51 −0.88 .382 1.10 1.51 −2.06 .040 Mom SSS 7.38 7.26 0.82 .415 7.53 7.26 1.97 .049 Mom STAI 10.18 10.42 −0.59 .554 9.80 10.42 −1.54 .125 Mom CES-D 29.21 29.39 −0.23 .815 28.07 29.39 −1.81 .071 Mom SLS 9.78 10.18 −0.64 .522 9.35 10.18 −1.36 .176 Mom CSI 87.28 88.79 −1.30 .194 88.65 88.79 −0.12 .902 Mom SEN 87.39 84.54 1.42 .157 85.30 84.54 0.38 .704 Mom social support 71.33 70.54 0.48 .634 72.68 70.54 1.29 .198 Dad well-being 1.07 1.12 −0.21 .835 1.05 1.12 −0.31 .760 Dad STAI 10.64 11.35 −1.60 .111 10.76 11.35 −1.35 .177 Dad CES-D 28.19 27.13 1.27 .206 27.81 27.13 0.81 .419 Dad SSS 7.46 7.22 1.59 .114 7.49 7.22 1.86 .064 US (vs. UK) 1.28 1.20 1.30 .193 1.20 1.20 −0.13 .894 NL (vs. UK) 1.33 1.27 1.01 .312 1.30 1.27 0.42 .675 Dad SLS 11.77 12.74 −1.17 .243 11.59 12.74 −1.36 .173 Dad CSI 88.59 88.20 0.34 .731 88.78 88.20 0.51 .611 Dad SEN 82.92 85.29 −1.33 .186 82.26 85.29 −1.71 .089 Dad social support 66.08 66.03 0.03 .978 67.93 66.03 1.07 .287 Infant attention 7.83 7.10 1.10 .273 7.34 7.10 0.37 .709 Distress to limitations 3.37 3.45 −0.66 .507 3.45 3.45 0.04 .966 Duration of orienting 4.14 4.25 −0.90 .367 4.22 4.25 −0.30 .761 Approach 1.99 2.05 −0.59 .558 1.92 2.05 −1.25 .211 Abbreviations: CES-D, Center for Epidemiological Studies-Depression Scale; CSI, Couple's Satisfaction Index; NL, Netherlands; SEN, Self-efficacy in the Nurturing Role; SLS, Satisfaction with Life Scale; SSS, Subjective Social Status; STAI, State-Trait Anxiety Inventory; UK, United Kingdom; US, United States. TABLE 3 Pre- and post-matching mean differences Abbreviations: CES-D, Center for Epidemiological Studies-Depression Scale; CSI, Couple's Satisfaction Index; NL, Netherlands; SEN, Self-efficacy in the Nurturing Role; SLS, Satisfaction with Life Scale; SSS, Subjective Social Status; STAI, State-Trait Anxiety Inventory; UK, United Kingdom; US, United States. UK are regularly granted longer parental leave than parents in the USA and therefore may have screens on for more hours of the day with their infants than parents in the USA. However, since there is generous parental leave in the Netherlands, as well, this cannot be the only explanation. 4 \| DISCUSSION In addition, these findings suggest that the effects of screen exposure may not be global, affecting all aspects of cognition, but specific, affecting selected domains. Importantly, there were differences in screen exposure by country, whereby children in the UK were more likely to be regularly exposed to electronic screen-based media than children in the Netherlands or in the USA. One possible explanation for this difference may be that parents in the MCHARG et al. \| It may be that there are characteristics of the locations of each of the samples that contribute to varied screen time or that parents who are likely to participate in longitudinal studies have different characteristics in each country. There are likely several cultural differences at play across the three countries that lend themselves to using more or less screens, especially with their infants present, and this should be examined in future research. Notably, these differences were accounted for in propensity score matching, such that, where possible, children were matched with a child in the other group with someone in the same country. 217 MCHARG et al. \| 218 \| TABLE 4 (Continued) Latency to touch Multi-location search task Ball run b SE t p b SE t p b SE t p Dad well-being −0.73 0.47 −0.98 .326 −0.04 0.03 −0.25 .800 0.03 0.02 0.22 .823 Mom CES-D −0.17 0.19 −0.36 .721 0.00 0.01 −0.02 .981 0.00 0.01 0.03 .976 Mom SLS −0.09 0.19 −0.19 .849 0.01 0.01 0.11 .909 0.01 0.01 0.06 .956 Mom CSI 0.02 0.10 0.05 .961 0.00 0.01 0.04 .968 0.00 0.00 −0.02 .982 Mom SEN 0.07 0.06 0.30 .765 0.00 0.00 −0.07 .945 0.00 0.00 −0.04 .965 Mom social support −0.04 0.07 −0.13 .894 0.00 0.00 −0.02 .986 0.00 0.00 0.05 .963 Dad SSS 0.92 0.77 0.98 .330 0.01 0.04 0.06 .951 0.00 0.04 0.01 .996 US (vs. UK) −2.27 2.28 −0.78 .436 −0.12 0.13 −0.35 .730 0.17 0.10 0.53 .598 NL (vs. UK) −8.93 2.29 −3.65 .000 0.06 0.13 0.16 .871 0.09 0.10 0.27 .784 Dad SLS 0.25 0.13 0.68 .499 0.00 0.01 0.04 .969 −0.01 0.01 −0.07 .946 Dad CSI 0.07 0.11 0.20 .844 −0.01 0.01 −0.09 .931 0.00 0.00 0.03 .973 Dad SEN 0.04 0.06 0.16 .876 0.01 0.00 0.11 .915 0.00 0.00 −0.07 .946 Dad social support 0.03 0.06 0.13 .895 0.00 0.00 0.01 .990 0.00 0.00 0.00 .999 Infant attention −0.03 0.16 −0.07 .944 −0.02 0.01 −0.19 .848 0.02 0.01 0.18 .854 Distress to limitations −0.86 0.93 −0.76 .449 −0.04 0.05 −0.17 .867 −0.05 0.04 −0.23 .818 Duration of orienting −0.99 0.84 −0.95 .343 0.03 0.05 0.14 .888 −0.01 0.04 −0.03 .977 Approach 0.34 1.00 0.26 .796 0.09 0.06 0.39 .697 −0.03 0.05 −0.16 .874 Abbreviations: CSI, Couple's Satisfaction Index; Dep & Anx, History of Anxiety or Depression; NL, Netherlands; PT, Part Time; SEN, Self-efficacy in the Nurturing Role; SLS, Satisfaction with Life Scale; SSS, Subjective Social Status; UK, United Kingdom; US, United States. TABLE 4 (Continued) \| TABLE 4 Results of weighted regressions predicting EF g g p g Latency to touch Multi-location search task Ball run b SE t p b SE t p b SE t p (Intercept) 0.80 20.16 0.04 .972 0.22 1.15 0.20 .839 −0.23 0.92 −0.23 .818 Media use −4.26 1.80 −2.69 .008 0.04 0.10 0.12 .905 −0.03 0.08 −0.10 .917 Infant age 2.93 1.90 1.63 .103 −0.10 0.11 −0.32 .752 0.09 0.09 0.30 .762 Infant female 1.54 1.51 0.75 .453 0.00 0.09 0.02 .988 −0.13 0.07 −0.48 .635 Mom employed PT 1.89 4.07 0.33 .739 −0.04 0.23 −0.08 .938 −0.21 0.19 −0.46 .646 Mom homemaker −8.14 4.58 −2.20 .029* −0.22 0.26 −0.42 .676 −0.04 0.21 −0.09 .925 Mom student −7.47 3.78 −2.19 .029* −0.08 0.22 −0.16 .873 0.01 0.17 0.01 .988 Mom unemployed −1.99 4.81 −0.62 .534 0.34 0.27 0.66 .512 −0.13 0.22 −0.28 .783 Mom anxiety 1.49 2.72 0.44 .660 −0.30 0.16 −0.77 .443 −0.09 0.12 −0.26 .796 Mom depression 1.29 2.76 0.55 .580 −0.11 0.16 −0.27 .786 −0.03 0.13 −0.08 .940 Mom Dep & Anx −1.51 2.92 −0.75 .455 −0.12 0.17 −0.29 .771 0.17 0.13 0.47 .639 Mom well-being 0.91 0.61 1.01 .316 0.02 0.03 0.12 .907 −0.03 0.03 −0.19 .847 Mom SSS −1.13 0.95 −1.06 .289 0.00 0.05 0.01 .991 0.00 0.04 0.00 .997 Mom age 25–29 −8.30 3.91 −2.68 .008 0.04 0.22 0.09 .932 0.02 0.18 0.05 .962 Mom age 30–34 3.37 2.71 1.35 .178 −0.05 0.15 −0.13 .898 −0.08 0.12 −0.24 .812 Mom age 35+ −1.49 1.60 −0.83 .405 −0.08 0.09 −0.28 .783 0.09 0.07 0.32 .748 Dad age 25–29 11.96 5.74 3.43 .001 −0.31 0.33 −0.53 .598 0.27 0.26 0.53 .600 Dad age 30–34 −5.19 4.13 −2.38 .018* −0.06 0.24 −0.13 .896 −0.02 0.19 −0.05 .957 Dad age 35+ 3.40 2.23 1.60 .110 0.11 0.13 0.31 .758 −0.03 0.10 −0.08 .936 Mom education 0.01 0.70 0.01 .991 0.08 0.04 0.42 .677 0.03 0.03 0.16 .870 Mom STAI −0.05 0.29 −0.08 .936 −0.02 0.02 −0.13 .896 −0.01 0.01 −0.07 .941 Dad employed PT −4.14 4.15 −1.38 .168 0.06 0.24 0.12 .902 −0.05 0.19 −0.11 .913 Dad student −3.94 4.95 −1.24 .217 0.21 0.28 0.37 .708 −0.34 0.23 −0.70 .485 Dad unemployed −3.48 5.79 −1.20 .230 0.25 0.33 0.41 .682 0.12 0.26 0.23 .820 Dad anxiety −1.29 3.74 −0.61 .545 0.16 0.21 0.35 .729 −0.30 0.17 −0.72 .473 Dad depression 0.93 3.56 0.28 .778 −0.10 0.20 −0.22 .827 −0.23 0.16 −0.55 .586 Dad Dep & Anx 1.57 3.53 0.42 .676 0.07 0.20 0.16 .874 0.04 0.16 0.10 .918 (Continues) MCHARG et al. TABLE 4 (Continued) Several key limitations to this study deserve note. Although propensity score methods provide a basis for causal inference, the design of the study is not experimental, and many household variables, such as why infants are exposed to screens in the first place, could not be controlled. In addition, all assumptions made about content are strictly speculative. Additionally, there are a number of other unmeasured parent and child characteristics that might be associated with either child screen exposure or child EF (or both), including parent media habits and parent EF. As these data are a part of a larger longitudinal study, there are limitations to the amount of data that could be collected from parents of very young children without placing undue burden upon the family; further, for data that are available at later timepoints in the same dataset (e.g., parent EF is available when children are 14 months of age), including these in models would be in violation of the assumptions of propensity score modeling, specifically that all control variables be from prior to “treatment” (i.e., media exposure). In sum, screen exposure at 4 months of age appears to be negatively associated with inhibition 10 months later (over and above early levels of attention and individual differences in temperament). \| 219 MCHARG et al. FIGURE 2 Relations between propensity for media use and inhibition. There was a statistically significant difference in inhibition at 14 months between the screen use and no-screen use groups at 4 months, such that infants exposed to screens were more likely to be less inhibited than infants not exposed to screens MCHARG et al. 219 FIGURE 2 Relations between propensity for media use and inhibition. There was a statistically significant difference in inhibition at 14 months between the screen use and no-screen use groups at 4 months, such that infants exposed to screens were more likely to be less inhibited than infants not exposed to screens This suggests there may be further long-term negative effects of very early screen exposure. In all, the current study expands the research on screen time and executive functioning by investigating lon- gitudinal relations from a very young age. There appear to be specific relations between early screen exposure and later inhibition; these relations are not linear, but suggest that any regular exposure to screens may be detrimental. ACKNOWLEDGMENTS We would like to thank the families for allowing us into their lives and their homes. The New Fathers and Mothers (NewFAMs) Study Team includes Clancy Blair, Claire Hughes, Judi Mesman, Rory Devine, Lenneke Alink, Wendy Browne, Marjolein Branger, Rosanneke Emmen, Sarah Foley, Lara Kyriakou, Anja Lindberg, Gabrielle McHarg, Andrew Ribner, and Mi-Lan Woudstra. Support for this research was provided by ESRC ES/L010648/1 to CH, NSF BCS-1429152 to CB, and NWO 464-13- 141 to JM. TABLE 4 (Continued) In contrast, there were no relations between screen time and set-shifting or working memory, contrary to previous findings in older children. Parents and other caregivers should take care when making decisions about screen time for infants, as the current research suggests there could be detrimental effects on later EF. CONFLICT OF INTEREST CONFLICT OF INTEREST The authors declare no conflicts of interest in relation to the funding bodies involved in the current study. O C The authors declare no conflicts of interest in relation to the funding bodies involved in the current study. REFERENCES American Academy of Pediatrics (2016). Media and Young Minds. Pediatrics, 138(5), e20162591. https://doi. org/10.1542/peds.2016-2591 American Academy of Pediatrics (2016). Media and Young Minds. Pediatrics, 138(5), e20162591. https://doi. org/10.1542/peds.2016-2591 Anderson, D. R., & Subrahmanyam, K. (2017). Digital screen media and cognitive development. Ped S57–S61. https://doi.org/10.1542/peds.2016-1758C Asparouhov, T., & Muthen, B. (2010). Weighted least squares estimation with missing data. Retrieved from http:// ww.statmodel2.com/download/GstrucMissingRevision.pdf Barr, R., Lauricella, A., Zack, E., & Calvert, S. L. (2010). Infant and early childhood exposure to adult-directed and child-directed television programming: Relations with cognitive skills at age four. Merrill-Palmer Quarterly, 21–48. https://doi.org/10.1353/mpq.0.0038 Beyens, I., & Eggermont, S. (2014). Putting young children in front of the television: Antecedents and outcomes of parents' use of television as a babysitter. Communication Quarterly, 62(1), 57–74. https://doi.org/10.1080/01463 373.2013.860904 Blair, C., & Razza, R. P. (2007). Relating effortful control, executive function, and false belief understand- ing to emerging math and literacy ability in kindergarten. Child Development, 78(2), 647–663. https://doi. org/10.1111/j.1467-8624.2007.01019.x Brock, L. L., Rimm-Kaufman, S. E., Nathanson, L., & Grimm, K. J. (2009). The contributions of ‘hot’ and ‘cool’ execu- tive function to children's academic achievement, learning-related behaviors, and engagement in kindergarten. Early Childhood Research Quarterly, 24(3), 337–349. https://doi.org/10.1016/j.ecresq.2009.06.001 d Research Quarterly, 24(3), 337–349. https://doi.org/10.1016/j.ecresq.2009.06.001 Casstevens, R. M. (2007). JHab: Java Habituation Software (Version 1.0.0) [Computer Softw Casstevens, R. M. (2007). JHab: Java Habituation Software (Version 1.0.0) [Computer Software]. Chevy Chase, MD. Common Sense Media (2017). New research by Common Sense finds major spike in mobile media use and device own- ership by children age 0 to 8. Retrieved from https://www.commonsensemedia.org/about-us/news/press-releases/ new-research-by-common-sense-finds-major-spike-in-mobile-media-use-and Cuevas, K., & Bell, M. A. (2014). Infant attention and early childhood executive function. Child Development, 85(2), 397–404. https://doi.org/10.1111/cdev.12126 Cuevas, K., Deater-Deckard, K., Kim-Spoon, J., Watson, A. J., Morasch, K. C., & Bell, M. A. (2014). What's mom got to do with it? Contributions of maternal executive function and caregiving to the development of executive function across early childhood. Developmental Science, 17(2), 224–238. https://doi.org/10.1111/desc.12073 Devine, R. T., & Hughes, C. (2014). Relations between false belief understanding and executive function in early child- hood: A meta-analysis. Child Development, 85(5), 1777–1794. https://doi.org/10.1111/cdev.12237 Devine, R. T., Ribner, A., & Hughes, C. (2019). Measuring and predicting individual differences in executive functions at 14 months: A longitudinal study. Child Development, 90(5), e618–e636. Diener, E. D., Emmons, R. A., Larsen, R. J., & Griffin, S. (1985). The satisfaction with life scale. Journal of Personality Assessment, 49(1), 71–75. ORCID ORCID Gabrielle McHarg https://orcid.org/0000-0002-2239-7294 Andrew D. Ribner https://orcid.org/0000-0001-7188-223X MCHARG et al. 220 Ho, D. E., Imai, K., King, G., & Stuart, E. A. (2011). MatchIt: Nonparametric preprocessing for parametric causal in- ference. Journal of Statistical Software, 42(8). Ho, D. E., Imai, K., King, G., & Stuart, E. A. (2011). MatchIt: Nonparametric preprocessing for parametric causal in- ference. Journal of Statistical Software, 42(8). Huber, B., Yeates, M., Meyer, D., Fleckhammer, L., & Kaufman, J. (2018). The effects of screen media content on young children's executive functioning. Journal of Experimental Child Psychology, 170, 72–85. https://doi.org/10.1016/j. jecp.2018.01.006 Huber, B., Yeates, M., Meyer, D., Fleckhammer, L., & Kaufman, J. (2018). The effects of screen media content on young children's executive functioning. Journal of Experimental Child Psychology, 170, 72–85. https://doi.org/10.1016/j. jecp.2018.01.006 Hughes, C., & Devine, R. T. (2017). For better or for worse? Positive and negative parental influences on young chil- dren's executive function. Child Development, 90(2), 593–609. https://doi.org/10.1111/cdev.12915 Hughes, C., & Devine, R. T. (2017). For better or for worse? Positive and negative parental influences on young chil- dren's executive function. Child Development, 90(2), 593–609. https://doi.org/10.1111/cdev.12915 Hughes, C., & Ensor, R. (2005). Executive function and theory of mind in 2 year olds: A family affair? Developmental Neuropsychology, 28, 645–668. https://doi.org/10.1207/s15326942dn2802_5 Johansson, M., Marciszko, C., Brocki, K., & Bohlin, G. (2016). Individual differences in early executive functions: A longitudinal study from 12 to 13 months. Infant and Child Development, 25, 533–549. https://doi.org/10.1002/ icd.1952 Kirkorian, H., Pempek, T., & Choi, K. (2017). The role of online processing in young children's learning from inter- active and noninteractive digital media. In R. Barr, & D. N. Linebarger (Eds.), Media exposure during infancy and early childhood (pp. 65–90). Cham, Switzerland: Springer International Publishing. Lillard, A. S., Drell, M. B., Richey, E. M., Boguszewski, K., & Smith, E. D. (2015). Further examination of the imme- diate impact of television on children's executive function. Developmental Psychology, 51(6), 792–805. https://doi. org/10.1037/a0039097 Lillard, A. S., & Peterson, J. (2011). The immediate impact of different types of television on young children's executive function. Pediatrics, 128(4), 644–649. https://doi.org/10.1542/peds.2010-1919 Linebarger, D. L., Barr, R., Lapierre, M. A., & Piotrowski, J. T. (2014). Associations between parenting, media use, cumulative risk, and children's executive functioning. Journal of Developmental & Behavioral Pediatrics, 35(6), 367–377. https://doi.org/10.1097/DBP.0000000000000069 77. https://doi.org/10.1097/DBP.0000000000000069 Miller, S. E., & Marcovitch, S. (2015). Examining executive function in the second year of life: Coherence, stability, and relations to joint attention and language. Developmental Psychology, 51, 101–114. REFERENCES https://doi.org/10.1207/s15327752jpa4901_13 Ellefson, M. R., Ng, F. F., Wang, Q., & Hughes, C. (2017). Efficiency of executive function: A two-generation cross-cul- tural comparison of samples from Hong Kong and the United Kingdom. Psychological Science, 28(5), 555–566. https://doi.org/10.1177/0956797616687812 Friedman, N. P., Miyake, A., Robinson, J. L., & Hewitt, J. K. (2011). Developmental trajectories in toddlers' self-restraint predict individual differences in executive functions 14 years later: A behavioral genetic analysis. Developmental Psychology, 47, 1410–1430. https://doi.org/10.1037/a0023750 Funk, J. L., & Rogge, R. D. (2007). Testing the ruler with item response theory: Increasing precision of measurement for relationship satisfaction with the Couples Satisfaction Index. Journal of Family Psychology, 21(4), 572–583. https ://doi.org/10.1037/0893-3200.21.4.572 Garon, N., Smith, I. M., & Bryson, S. E. (2014). A novel executive function battery for pre-schoolers: Sensitivity to age differences. Child Neuropsychology, 20, 713–736. https://doi.org/10.1080/09297049.2013.857650 Gelman, A., & Hill, J. (2007). Data analysis using regression and multilevelhierarchical models, Vol. 1. New York, NY: Cambridge University Press. Goldberg, D., Gater, R., Sartorius, N., Ustun, T., Piccinelli, M., Gureje, O., & Rutter, C. (1997). The validity of two versions of the GHQ in the WHO study of mental illness in general health care. Psychological Medicine, 27(1), 191–197. https://doi.org/10.1017/S0033291796004242 Hipp, D., Gerhardstein, P., Zimmermann, L., Moser, A., Taylor, G., & Barr, R. (2017). The dimensional divide: Learning from TV and touchscreens during early childhood. In R. Barr, & D. N. Linebarger (Eds.), Media exposure during infancy and early childhood (pp. 33–54). Cham, Switzerland: Springer International Publishing. MCHARG et al. 221 https://doi.org/10.1037/ a0038359 Mulder, H., Hoofs, H., Verhagen, J., Van der Veen, I., & Leseman, P. P. M. (2014). Psychometric properties and conver- gent and predictive validity of an executive function test battery for two-year-olds. Frontiers in Psychology, 5(733). https://doi.org/10.3389/fpsyg.2014.00733 Nathanson, A. I., Alade, F., Sharp, M. L., Rasmussen, E. E., & Christy, K. (2014). The relation between television expo- sure and executive function among preschoolers. Developmental Psychology, 50(5), 1497. https://doi.org/10.1037/ a0035714 Nielsen (2017). Nielsen estimates 119.6 million TV homes in the U.S. for the 2017–2018 TV season. Retrieved from https://www.nielsen.com/us/en/insights/news/2017/nielsen-estimates-119-6-million-us-tv-homes-2017-2018-tv- season.html Owen, A. M., Downes, J. J., Sahakian, B. J., Polkey, C. E., & Robbins, T. W. (1990). Planning and spa- tial working memory following frontal lobe lesions in man. Neuropsychologia, 28, 1021–1034. https://doi. org/10.1016/0028-3932(90)90137-D Pederson, F. A., Bryan, Y. E., Huffman, L. C., & Del Carmen, R. (1989). Construction of self and offspring in the preg- nancy and early infancy period. Society for Research in Child Development. Putnam, S., Helbig, A., Garstein, M., Rothbart, M., & Leerkes, E. (2014). Development and assessment of short and very short forms of the infant behavior questionnaire-revised. Journal of Personality Assessment, 96(4), 445–458. https://doi.org/10.1080/00223891.2013.841171 g Radloff, L. (1977). The CES-D scale: A self report depression scale for research in the general population. Applied P h l i l M 1(3) 385 401 h //d i /10 1177/014662167700100306 Radloff, L. (1977). The CES-D scale: A self report depression scale for research in the general population. Applied Psychological Measurements, 1(3), 385–401. https://doi.org/10.1177/014662167700100306 Ribner, A., Fitzpatrick, C., & Blair, C. (2017). Family socioeconomic status moderates associations between television viewing and school readiness skills. Journal of Developmental & Behavioral Pediatrics, 38(3), 233–239. https://doi. org/10.1097/DBP.0000000000000425 Rosenbaum, P. R., & Rubin, D. B. (1983). The central role of the propensity score in observational studies for causal effects. Biometrika, 70(1), 41–55. bin, D. B. (1987). Multiple imputation for nonresponse in surveys. New York, NY: John Wiley and Son Schneider, B., Carnoy, M., Kilpatrick, J., Schmidt, W. H., & Shavelson, R. J. (2007). Estimating causal effects using experimental and observational design. Washington, D.C. : American Educational & Reseach Association. 222 How to cite this article: McHarg G, Ribner AD, Devine RT, Hughes C; The NewFAMS Study Team. Infant screen exposure links to toddlers' inhibition, but not other EF constructs: A propensity score study. Infancy. 2020;25:205–222. https://doi.org/10.1111/infa.12325 MCHARG et al. Singh-Manoux, A., Adler, N., & Marmot, M. (2003). Subjective social status: Its determinants and its association with measures of ill-health in the Whitehall II study. Social Science & Medice, 56, 1321–1333. https://doi.org/10.1016/ S0277-9536(02)00131-4 Spielberger, C., Gorsuch, R., Lushene, R., Vagg, P., & Jacobs, G. (1983). Manual for the State-Trait Anxiety Inventory. Palo Alto, CA: Consulting Psychologists Press. Su, Y. S., Gelman, A. E., Hill, J., & Yajima, M. (2011). Multiple imputation with diagnostics (mi) in R: Opening win- dows into the black box. Journal of Statistical Software, 45(2). Taylor, G., Monaghan, P., & Westermann, G. (2018). Investigating the association between children's screen media ex- posure and vocabulary size in the UK. Journal of Children and Media, 12(1), 51–65. https://doi.org/10.1080/17482 798.2017.1365737 Thompson, A., Adair, L. S., & Bentley, M. E. (2013). Maternal characteristics and perception of temperament associated with infant TV exposure. Pediatrics, 131(2), e390–e397. https://doi.org/10.1542/peds.2012-1224 Zimet, G. D., Dahlem, N. W., Zimet, S. G., & Farley, G. K. (1988). The multidimensional scale of perceived social support. Journal of Personality Assessment, 52(1), 30–41. https://doi.org/10.1207/s15327752jpa5201_2
https://openalex.org/W4377291405	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/F3908E54825ED0C2AD0F96F13DBFFC8E/S2071832223000275a.pdf/div-class-title-does-digitalization-reshape-the-principle-of-non-intervention-div.pdf	English	null	Does Digitalization Reshape the Principle of Non-Intervention?	German law journal	2,023	cc-by	12,043	© The Author(s), 2023. Published by Cambridge University Press on behalf of the German Law Journal. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. 2This neglect had extended to Western academia, the only more prominent article dealing substantively with the principle appears to be the one by Maziar Jamnejad & Michael Wood, The Principle of Non-intervention, 22 LEIDEN J. INT’L L. 345 (2009). Does Digitalization Reshape the Principle of Non-Intervention? Lukas Willmer1, 2 1Humboldt University of Berlin, Berlin, Germany and 2Berlin Potsdam Research Group “The International Rule of Law – Rise or Decline?”, Berlin, Germany Email: lukas.willmer@kfg-intlaw.de (Received 10 April 2023; accepted 11 April 2023) 1TOM GINSBURG, DEMOCRACIES AND INTERNATIONAL LAW 5 (2021). Abstract While digitalization has led to renewed attention to the principle of non-intervention, not the least by Western states rediscovering the protective dimension of sovereignty, it remains plagued by a certain vagueness. Attempts by academics to fill the gaps lead to starkly different results, ranging from the inser- tion of democratic values to the inadvertent reinforcement of protectionist tendencies. Overall, digitaliza- tion has so far had less of an effect on the principle of non-intervention than its renewed importance may have on the type of international law more generally. Keywords: Non-intervention; cyberspace; election interferences; regime neutrality https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press German Law Journal (2023), 24, pp. 508–521 doi:10.1017/glj.2023.27 ARTICLE Special Issue: International Law and Digitalization ewed Attention: A Broadened Scope of State Actors Invoking Non-interventio Digitalization has led to renewed attention to the principle of non-intervention and, more impor- tantly, broadened the scope of states invoking it. Basic assumptions about which states are likely to rely on the principle become invalid. It is no longer invoked primarily by comparatively weak (I) or authoritarian states (II). I. Power Asymmetries First, the principle of non-intervention has in the past mostly been invoked by weaker states against more powerful states.3 When it found expression in the Monroe doctrine, it was an attempt by the US, at that point young and relatively weak, to fend off influence from former colonial powers in the Americas.4 When US foreign policy became more and more dominant in the region, Latin American states embraced the principle to oppose just that.5 After the decolo- nization process, newly independent states guarded their hard-won sovereignty.6 Even in the rare instance in which a Western state alludes to the principle, such as when Germany objected to US sanctions against the North Stream II pipeline as an interference in its internal affairs,7 it takes place in a situation of clear power asymmetry. Digitalization, at least for now, changes the assumption that a powerful actor has relatively little to fear from weaker adversaries. Cyber weapons are comparatively cheap and easy to acquire compared to conventional capabilities. The US, for example, is not only concerned about Russian and Chinese cyber activities, but also about cyber operations in Iran and North Korea.8 Germany has warned that “[i]n cyberspace, only limited resources are often needed to cause significant harm.”9 It remains to be seen, though, whether this development will consolidate in the long run or whether more technologically advanced states will eventually be able to adapt and protect themselves against cyber-attacks while states with less cyber capabilities remain vulnerable. 3STEPHEN D. KRASNER, SOVEREIGNTY: ORGANIZED HYPOCRISY 25 (1999). 4HANSPETER NEUHOLD, THE LAW OF INTERNATIONAL CONFLICTS: FORCE, INTERVENTION AND PEACEFUL DISPUTE SETTLEMENT 163 (2015). 5ARNULF BECKER LORCA, MESTIZO INTERNATIONAL LAW: A GLOBAL INTELLECTUAL HISTORY 1842–1933 341–352 (2014). 6Neuhold, supra note 4, at 165. 7German Government Press Release 432, German Government Notes Sanctions Against Nordstream 2 and Turkstream with Regret (Dec. 21, 2019) https://www.bundesregierung.de/breg-de/suche/bundesregierung-nimmt-sanktionen-gegen- nordstream2-und-turkstream-mit-bedauern-zur-kenntnis-1708962 [hereinafter Germany]. 8OFF. OF THE DIR. OF NAT’L INTEL., ANNUAL THREAT ASSESSMENT OF THE US INTELLIGENCE COMMUNITY 20 (2021). 9Germany, On the Application of International Law in Cyberspace 1 (2021) https://www.auswaertiges-amt.de/blob/ 2446304/32e7b2498e10b74fb17204c54665bdf0/on-the-application-of-international-law-in-cyberspace-data.pdf. A. Of Moats and Drawbridges For the utopia of an interconnected, globalized world, where information freely flows across borders and states are massively interdependent, the principle of non-intervention seems like a Westphalian castle with moats and drawbridges in between glitzy, translucent glass towers. p g g y g Since the end of the Cold War, a “cosmopolitan paradigm” has supposedly developed that “celebrates the international level as “domesticating” sovereignty and its attendant risks.”1 The expansion of international law, especially human rights law, has diminished the domaine réservé and, so goes the argument, with it the scope and importance of the principle of non- intervention. Indeed, Western states, arguably the most ardent supporters of this development, have largely refrained from even mentioning the principle at the UN.2 Yet the principle of non-intervention is back on the agenda, in particular within the cyber context. Interconnectedness also creates vulnerabilities and Western states especially have realized that open and pluralistic societies may be less resilient against foreign interferences than assumed. Of course, not all states share this utopia of interconnectedness in the first place, and for many non-Western states, the principle of non-intervention has never been off the agenda. Many states have continuously been warier of foreign interferences even before the digital age. https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 509 German Law Journal Does digitalization reshape the principle of non-intervention? This Article will offer some observations. Most importantly, digitalization has led to renewed attention for the principle broadening the scope of state actors that invoke it (B). This renewed attention has, however, not led to further clarity regarding its application (C). Finally, it is worthwhile to note the possible effect of certain academic proposals on the values underlying the principle of non-intervention (D). It will be argued that, at least so far, digitalization has had less of an effect on the principle of non-intervention itself, than the principle’s renewed importance may have on the type of international law more generally (E). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press II. The “Authoritarian Stain” of the Principle of Non-intervention Second, the principle of non-intervention tended to be invoked by more authoritarian states to fend off foreign criticism, or, as they perceive it, foreign interferences. It is especially important but 3STEPHEN D. KRASNER, SOVEREIGNTY: ORGANIZED HYPOCRISY 25 (1999). R NEUHOLD, THE LAW OF INTERNATIONAL CONFLICTS: FORCE, INTERVENTION AND PEACEFUL DISPUTE 63 (2015). ECKER LORCA, MESTIZO INTERNATIONAL LAW: A GLOBAL INTELLECTUAL HISTORY 1842–1933 341–352 (2014). upra note 4, at 165. 7German Government Press Release 432, German Government Notes Sanctions Against Nordstream 2 and Turkstream with Regret (Dec. 21, 2019) https://www.bundesregierung.de/breg-de/suche/bundesregierung-nimmt-sanktionen-gegen- nordstream2-und-turkstream-mit-bedauern-zur-kenntnis-1708962 [hereinafter Germany]. y 8OFF. OF THE DIR. OF NAT’L INTEL., ANNUAL THREAT ASSESSMENT OF THE US INTELLIGENCE COMMUNITY 20 (2021). 9Germany, On the Application of International Law in Cyberspace 1 (2021) https://www.auswaertiges-amt.de/blob/ 2446304/32e7b2498e10b74fb17204c54665bdf0/on-the-application-of-international-law-in-cyberspace-data.pdf. y 8OFF. OF THE DIR. OF NAT’L INTEL., ANNUAL THREAT ASSESSMENT OF THE US INTELLIGENCE COMMUNITY 20 (2021). 9Germany, On the Application of International Law in Cyberspace 1 (2021) https://www.auswaertiges-amt.de/blob/ 2446304/32e7b2498e10b74fb17204c54665bdf0/on-the-application-of-international-law-in-cyberspace-data.pdf. https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press Lukas Willmer 510 also difficult in this regard to distinguish between political and legal invocations of the principle of non-intervention.10 For example, the concerted effort by China and a significant number of other states to defend Chinese treatment of Uighurs in Xinjiang as a domestic matter in the Third Committee of the General Assembly extends beyond political rhetoric.11 Similarly, the increasing enactment of domestic legislation against foreign funding for NGOs backs up a legal claim against political interference through “democracy promotion” that some states consider to be a violation of the principle of non-intervention.12 The preference by authoritarian states for the principle of non-intervention may explain why China, despite its rise to global power status, continues to embrace the principle as one of the basic pillars of its foreign policy, at least on the rhetorical level.13 Meanwhile, Western states have rarely invoked the principle of non-intervention in the past, they have largely avoided even mentioning it in the context of the UN. At least for some states, the principle of non-intervention appeared to have an “authoritarian stain”. p p pp Effects of digitalization challenge this assumption as well. Western states have stopped avoiding the principle and started to refer to it in the cyber context, although this did take some time. 12Heike Krieger, Populist Governments and International Law, 30 EUR. J. INT’L L. 971, 991–994 (2019). 10See Jamnejad & Wood, supra note 2, at 347. 11U.N. GAOR, 74th Sess., at 12, UN Doc A/C.3/74/SR.37 (Nov.26, 2018). 12Heike Krieger, Populist Governments and International Law, 30 EUR. J. INT’L L. 971, 991–994 (2019). 13See, e.g., The Declaration of the Russian Federation and the People’s Republic of China on the Promotion of International Law, U.N. Doc A/70/982, para. 4 (Jul. 8, 2016); see also CONGYAN CAI, THE RISE OF CHINA AND INTERNATIONAL LAW 94 (2019). 14G.A. Res 53/70 (Jan. 4, 1999); see also Permanent Rep. of the Russian Federation, Letter dated 23 September 1998 from the Permanent Representative of the Russian Federation to the United Nations addressed to the Secretary-General, U.N. Doc A/C.1/53/3 (Sept. 30, 1998). 15Liisi Adamson, International Law and International Cyber Norms: A Continuum?, in GOVERNING CYBERSPACE: BEHAVIOR, POWER AND DIPLOMACY 21 (Dennis Broeders & Bibi van den Berg eds., 2020). 16Rep. of the Group of Governmental Experts on Developments in the Field of Information and Telecommunications in the Context of International Security, para. 19, U.N. Doc A/68/98, (June 24, 2013) [hereinafter GGE Report 2013]. 17Rep. of the Group of Governmental Experts on Developments in the Field of Information and Telecommunications in the Context of International Security, para. 28(b), U.N. Doc A/70/174 (July 22, 2015) [hereinafte GGE Report 2015]. 18Rep. of the Group of Governmental Experts on Advancing Responsible State Behaviour in Cyberspace in the Context of International Security, para. 2 (May 28, 2021) https://front.un-arm.org/wp-content/uploads/2021/06/final-report-2019-2021- gge-1-advance-copy.pdf (forthcoming) [hereinafter GGE Report 2021]. 19Open-ended Working Group on Developments in the Field of Information and Telecommunications in the Context of International Security – Final Substantive Report, para. 8, U.N. Doc A/AC.290/2021/CRP.2 (Mar. 10, 2021) [hereinafter OEWG Report]. 20G.A. Res. 75/240 (Dec. 31, 2020); G.A. Res 75/32 (Dec. 7, 2020); G.A. Res 73/266 (Dec. 22, 2018); see also G.A. Res 70/237 (Dec. 23, 2015). 21See Mary Ellen O'Connell Cyber Security without Cyber War 17 J CONFLICT & SEC L 187 199 (2012) j p 11U.N. GAOR, 74th Sess., at 12, UN Doc A/C.3/74/SR.37 (Nov.26, 2018). 13See, e.g., The Declaration of the Russian Federation and the People’s Republic of China on the Promotion of International Law, U.N. Doc A/70/982, para. 4 (Jul. 8, 2016); see also CONGYAN CAI, THE RISE OF CHINA AND INTERNATIONAL LAW 94 (2019). II. The “Authoritarian Stain” of the Principle of Non-intervention The debate over international law in cyberspace was, at least within the UN, kickstarted by Russia, which in 1998 brought the topic to the UN’s agenda.14 Yet the discussions did not produce any substantial outcomes, primarily due to a lack of interest among Western states in cyber regu- lation. This attitude changed after the 2007 cyber-attack against Estonia.15 In 2013, the UN Governmental Group of Experts (GGE) reached a consensus that international law, and in particular the UN Charter, applies to cyberspace.16 In 2015, the GGE went further and specifically identified, inter alia, the principle of non-intervention as being applicable in cyberspace.17 This consensus has subsequently been confirmed in the 2021 GGE report18 as well as by an additionally established Open-Ended Working Group (OEWG)19 and by the General Assembly20. All states that have put forward their views on how international law applies in cyberspace engaged, at least to some extent, with the principle of non-intervention. This is not surprising because non-inter- vention is likely the most obvious norm to regulate violations of sovereignty below the threshold of uses of force. It also became increasingly clear that “cyberwarfare” could not be regulated solely through the prism of Article 2(4) UN Charter.21 Within statements from Western states, reluc- tance to rely on the principle of non-intervention because of an “authoritarian stain” was no 14G.A. Res 53/70 (Jan. 4, 1999); see also Permanent Rep. of the Russian Federation, Letter dated 23 September 1998 from the Permanent Representative of the Russian Federation to the United Nations addressed to the Secretary-General, U.N. Doc A/C.1/53/3 (Sept. 30, 1998). 14G.A. Res 53/70 (Jan. 4, 1999); see also Permanent Rep. of the Russian Federation, Letter dated 23 September 1998 from the Permanent Representative of the Russian Federation to the United Nations addressed to the Secretary-General, U.N. Doc A/C.1/53/3 (Sept. 30, 1998). 15Liisi Adamson, International Law and International Cyber Norms: A Continuum?, in GOVERNING CYBERSPACE: BEHAVIOR, POWER AND DIPLOMACY 21 (Dennis Broeders & Bibi van den Berg eds., 2020). 18Rep. of the Group of Governmental Experts on Advancing Responsible State Behaviour in Cyberspace in the Context of International Security, para. 2 (May 28, 2021) https://front.un-arm.org/wp-content/uploads/2021/06/final-report-2019-2021- gge-1-advance-copy.pdf (forthcoming) [hereinafter GGE Report 2021]. 18Rep. of the Group of Governmental Experts on Advancing Responsible State Behaviour in Cyberspace in the Context of International Security, para. 2 (May 28, 2021) https://front.un-arm.org/wp-content/uploads/2021/06/final-report-2019-2021- gge-1-advance-copy.pdf (forthcoming) [hereinafter GGE Report 2021]. 14G.A. Res 53/70 (Jan. 4, 1999); see also Permanent Rep. of the Russian Federation, Letter dated 23 September 1998 from the Permanent Representative of the Russian Federation to the United Nations addressed to the Secretary-General, U.N. Doc A/C.1/53/3 (Sept. 30, 1998). 0See Jamnejad & Wood, supra note 2, at 347. 10See Jamnejad & Wood, supra note 2, at 347. 11U.N. GAOR, 74th Sess., at 12, UN Doc A/C.3/74/SR.37 (Nov.26, 2018). 12Heike Krieger, Populist Governments and International Law, 30 EUR. J. INT’L L. 971, 991–994 (2019). 13See, e.g., The Declaration of the Russian Federation and the People’s Republic of China on the Promotion of International Law, U.N. Doc A/70/982, para. 4 (Jul. 8, 2016); see also CONGYAN CAI, THE RISE OF CHINA AND INTERNATIONAL LAW 94 (2019). 14 II. The “Authoritarian Stain” of the Principle of Non-intervention gg py p ( g) [ p ] 19Open-ended Working Group on Developments in the Field of Information and Telecommunications in the Context of International Security – Final Substantive Report, para. 8, U.N. Doc A/AC.290/2021/CRP.2 (Mar. 10, 2021) [hereinafter OEWG Report]. gg py p g p 19Open-ended Working Group on Developments in the Field of Information and Telecommunications in the Context of International Security – Final Substantive Report, para. 8, U.N. Doc A/AC.290/2021/CRP.2 (Mar. 10, 2021) [hereinafter OEWG Report]. p 20G.A. Res. 75/240 (Dec. 31, 2020); G.A. Res 75/32 (Dec. 7, 2020); G.A. Res 73/266 (Dec. 22, 2018); see also G.A. Res 70/237 (Dec. 23, 2015). p 20G.A. Res. 75/240 (Dec. 31, 2020); G.A. Res 75/32 (Dec. 7, 2020); G.A. Res 73/266 (Dec. 22, 2018); see also G.A. Res 70/237 (Dec. 23, 2015). 1See Mary Ellen O'Connell, Cyber Security without Cyber War, 17 J. CONFLICT & SEC. L. 187, 199 (2012). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 511 German Law Journal longer perceivable. On the contrary, Western states provided the most detailed assessments of how the principle of non-intervention applies in cyberspace.22 Back in 1981, Western states unani- mously rejected the General Assembly Declaration on Intervention23 because they opposed the concept of a “New International Information Order”, which, inter alia, held the dissemination of false or distorted news as unlawful.24 Now, under increasing pressure from cyber election inter- ferences and certain forms of destabilization of public discourses more generally, some Western states appear to embrace the idea that certain disinformation campaigns can violate the principle of non-intervention.25 Regulatory Vagueness: Which Standards of Non-intervention in Cyberspace? Despite the renewed interest that digitalization has sparked in the principle of non-intervention, its regulatory vagueness persists. States have so far primarily affirmed its applicability to cyber- space without reaching any consensus on how it applies (I). While the proposal by some states to adopt an entirely new cyber treaty has not garnered sufficient support, consensus seems to be more easily reachable with regard to non-binding rules. Those rules may, however, threaten to informalize even accepted international law (II). A fragmentation of regulatory processes at the UN is further complicating the issue (III). 22Australian Mission to the United Nations, Open Ended Working Group on Developments in the Field of Information and Telecommunications in the Context of International Security (2019) https://unoda-web.s3.amazonaws.com/wp-content/ uploads/2019/09/fin-australian-oewg-national-paper-Sept-2019.pdf [hereinafter Australia]; Government of Canada, International Law Applicable in Cyberspace (2022) https://www.international.gc.ca/world-monde/issues_development- enjeux_developpement/peace_security-paix_securite/cyberspace_law-cyberespace_droit.aspx?lang=eng#a3 [hereinafter Canada]; Kersti Kaljulaid, President, President of the Republic at the opening of CyCon 2019 (2019) [hereinafter Estonia]; French Ministry of Defense, Droit International Appliqué aux Opérations dans le Cyberespace (2019), droit- internat-appliqué-aux-opérations-cyberespace-france.pdf (justsecurity.org) [hereinafter France]; Government of Finland, International Law and Cyberspace (2020) https://um.fi/documents/35732/0/KyberkannatPDF_EN.pdf/12bbbbde-623b- 9f86-b254-07d5af3c6d85?t=1603097522727 [hereinafter Finland]; Germany, supra note 7; Roy Schondorf, Israel’s Perspective on Key Legal and Practical Issues Concerning the Application of International Law to Cyber Operations, EJIL: TALK!: BLOG OF THE EUR. J. OF INT’L L. (Dec. 8, 2020) https://www.ejiltalk.org/israels-perspective-on-key-legal-and- practical-issues-concerning-the-application-of-international-law-to-cyber-operations/; Appendix to Letter of 5 July 2019 from the Minister of Foreign Affairs to the President of the House of Representatives on the international legal order in cyber- space (July 1, 2019) https://www.government.nl/ministries/ministry-of-foreign-affairs/documents/parliamentary-documents/ 2019/09/26/letter-to-the-parliament-on-the-international-legal-order-in-cyberspace [hereinafter Netherlands]; New Zealand Foreign Affairs anThe Application of International Law to State Activity in Cyberspace, NEW ZEALAND (2020) https://www. mfat.govt.nz/assets/Peace-Rights-and-Security/International-security/International-Cyber-statement.pdf [hereinafter New Zealand]; United Kingdom Foreign, Commonwealth and Development Office, Application of international law to states’ conduct in cyberspace: UK statement (2021) https://www.gov.uk/government/publications/application-of-international-law- to-states-conduct-in-cyberspace-uk-statement/application-of-international-law-to-states-conduct-in-cyberspace-uk-statement [hereinafter United Kingdom]; Brian J. Egan, Legal Advisor, Remarks on International Law and Stability in Cyberspace (Nov. 10, 2016) https://2009-2017.state.gov/s/l/releases/remarks/264303.htm [hereinafter United States]. I. Affirming Applicability, not Clarifying the Application The principle of non-intervention was always plagued by a certain vagueness. In the early 1920s, Winfield remarked that a “reader, after perusing Phillimore's chapter upon intervention, might close the book with the impression that intervention may be anything from a speech of Lord Palmerston's in the House of Commons to the partition of Poland.”26 According to the ICJ’s Nicaragua judgment, an intervention is prohibited if it bears on matters in which each state 22Australian Mission to the United Nations, Open Ended Working Group on Developments in the Field of Information and Telecommunications in the Context of International Security (2019) https://unoda-web.s3.amazonaws.com/wp-content/ uploads/2019/09/fin-australian-oewg-national-paper-Sept-2019.pdf [hereinafter Australia]; Government of Canada, International Law Applicable in Cyberspace (2022) https://www.international.gc.ca/world-monde/issues_development- enjeux_developpement/peace_security-paix_securite/cyberspace_law-cyberespace_droit.aspx?lang=eng#a3 [hereinafter Canada]; Kersti Kaljulaid, President, President of the Republic at the opening of CyCon 2019 (2019) [hereinafter Estonia]; French Ministry of Defense, Droit International Appliqué aux Opérations dans le Cyberespace (2019), droit- internat-appliqué-aux-opérations-cyberespace-france.pdf (justsecurity.org) [hereinafter France]; Government of Finland, International Law and Cyberspace (2020) https://um.fi/documents/35732/0/KyberkannatPDF_EN.pdf/12bbbbde-623b- 9f86-b254-07d5af3c6d85?t=1603097522727 [hereinafter Finland]; Germany, supra note 7; Roy Schondorf, Israel’s Perspective on Key Legal and Practical Issues Concerning the Application of International Law to Cyber Operations, EJIL: TALK!: BLOG OF THE EUR. J. OF INT’L L. (Dec. 8, 2020) https://www.ejiltalk.org/israels-perspective-on-key-legal-and- practical-issues-concerning-the-application-of-international-law-to-cyber-operations/; Appendix to Letter of 5 July 2019 from the Minister of Foreign Affairs to the President of the House of Representatives on the international legal order in cyber- space (July 1, 2019) https://www.government.nl/ministries/ministry-of-foreign-affairs/documents/parliamentary-documents/ 2019/09/26/letter-to-the-parliament-on-the-international-legal-order-in-cyberspace [hereinafter Netherlands]; New Zealand Foreign Affairs anThe Application of International Law to State Activity in Cyberspace, NEW ZEALAND (2020) https://www. mfat.govt.nz/assets/Peace-Rights-and-Security/International-security/International-Cyber-statement.pdf [hereinafter New Zealand]; United Kingdom Foreign, Commonwealth and Development Office, Application of international law to states’ conduct in cyberspace: UK statement (2021) https://www.gov.uk/government/publications/application-of-international-law- to-states-conduct-in-cyberspace-uk-statement/application-of-international-law-to-states-conduct-in-cyberspace-uk-statement [hereinafter United Kingdom]; Brian J. Egan, Legal Advisor, Remarks on International Law and Stability in Cyberspace (Nov. 10, 2016) https://2009-2017.state.gov/s/l/releases/remarks/264303.htm [hereinafter United States]. p g 23G.A. Res. A/RES/36/103, Declaration on the Inadmissibility of Intervention and Interference in the Internal Affairs of States, (1981). Finland and Greece abstained. 23G.A. Res. A/RES/36/103, Declaration on the Inadmissibility of Intervention and Interference in the Internal Affairs of States, (1981). Finland and Greece abstained. 24Id. at 2. III. lit. d. 25See, e.g., New Zealand, supra note 22, para. 10; Germany supra note 7, at 5; see also France, supra note 22, at 7. 26Percy Henry Winfield, The History of Intervention in International Law, 3 BRIT. YEARBOOK INT’L L. 130 (1922-23). I. Affirming Applicability, not Clarifying the Application g., New Zealand, supra note 22, para. 10; Germany supra note 7, at 5; see also France, supra note 22, at 7. Henry Winfield, The History of Intervention in International Law, 3 BRIT. YEARBOOK INT’L L. 130 (1922-23) https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press Lukas Willmer 512 is, by the principle of state sovereignty, permitted to decide freely while employing methods of coercion.27 Yet, what exactly constitutes coercion has always remained unclear, with Western states tending to propose a rather narrow understanding while many non-Western states advocate including, for example, unilateral economic sanctions as a form of economic coercion as well.28 g p The debates in the GGE and OEWG have not shed much further light on this topic. While many states and several of the final reports have affirmed the applicability of the principle of non-intervention to cyberspace, they did not provide much guidance as to its interpretation. For example, the Chinese submission to the OEWG is limited to the statement that the “principles enshrined in the UN Charter, including sovereign equality, refraining from the use of force, settle- ment of disputes by peaceful means and non-intervention in the internal affairs of other states, apply in cyberspace.”29 Japan submits that “[w]ith respect to the principle of non-intervention, cyber operations may constitute unlawful intervention when requirements including the element of coercion, which are clarified in the Nicaragua judgement (1986), are met.”30 g j g Statements by states that did go into more detail almost all came from Western states.31 Within those statements, different standards of coercion become particularly apparent regarding cyber election interferences. Some states appear to adhere to a narrower interpretation. 27Military and Paramilitary Activities in and Against Nicaragua (Nicar. v. U.S.), Judgment, 1986 I.C.J. Rep. 14, para. 190 (June 27, 1986). 28See, e.g., G.A. Res 74/200 (Jan. 13, 2020). 29OEWG, China’s Submissions to the Open-ended Working Group on Developments in the Field of Information and Telecommunications in the Context of International Security 6 (2019) https://unoda-web.s3.amazonaws.com/wp-content/ uploads/2019/09/china-submissions-oewg-en.pdf; see also Colombia, Informe Recoluci´on A/RES/75/32 16 (2021) https:// front.un-arm.org/wp-content/uploads/2022/03/colombia-ict-security-2021.pdf. 30OEWG, Basic Position of the Government of Japan on International Law Applicable to Cyber Operations 2 (May 28, 2021) https://www.mofa.go.jp/files/100200935.pdf [hereinafter Japan]. 31See Australia, supra note 22; Canada, supra note 22; Estonia, supra note 22; France, supra note 22; Finland, supra note 22; Germany, supra note 22; Israel, supra note 22; The Netherlands, supra note 22; New Zealand, supra note 22; United Kingdom, supra note 22; United States, supra note 22. 32Australia, supra note 22, at 8; Canada, supra note 22, para. 24; Schondorf, supra note 22; United Kingdom, supra note 22, para. 9; United States, supra note 22. 33Suella Braverman, Sec’y of State for the Home Dept. Address Concerning International Law in Future Frontiers (2022) https://www.gov.uk/government/speeches/international-law-in-future-frontiers. 34Netherlands, supra note 22, at 3; New Zealand, supra note 22, para. 10. 35Germany, supra note 7, at 5. 36France, supra note 22, at 7. 27Military and Paramilitary Activities in and Against Nicaragua (Nicar. v. U.S.), Judgment, 1986 I.C.J. Rep. 14, para. 190 (June 27, 1986). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press I. Affirming Applicability, not Clarifying the Application When giving specific examples, these states only refer to interferences manipulating the actual vote tally or, at least partly, preventing the holding of the election at all as a form of coercive behavior.32 Although the UK, in more elaborate remarks on non-intervention, stated that it considers coer- cion to be broader than forcing a state into specific conduct also encompassing acts that “depriv[e] a State of its freedom of control,” it still only gave interferences with the technical election infra- structure as examples.33 Other states seem to consider disinformation campaigns at least as possibly being coercive.34 Germany, for example, is more elaborate in its position, considering online disinformation campaigns as coercive if they “deliberately incite violent political upheaval [ : : : ] significantly impeding the orderly conduct of an election” as they “may be comparable in scale and effect to the support of insurgents.”35 The broadest—and most vague—interpretation is offered by France which suggests that a digital “interference which causes or may cause harm to France’s political, economic, social and cultural system, may constitute a violation of the principle of non-intervention.”36 Thus, there is no consensus whether activities such as the alleged Russian interferences in the 2016 US Presidential elections, which did not affect the technical election infrastructure but rather targeted the public discourse, is prohibited by the principle of non-intervention. It is possible, that interfering with the technical election infrastructure consti- tutes the de minimis-threshold for those states that provide it as a sole example. It may also be the 34Netherlands, supra note 22, at 3; New Zealand, supra note 22, para. 10. 35Germany, supra note 7, at 5. 36France, supra note 22, at 7. https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 513 German Law Journal case, that these states remain strategically silent regarding lower-level interferences for the time being.37 In any event, the debate is an almost entirely intra-Western debate. Even if this debate eventually produces a consensus position within Western states, it would not be sufficiently wide- spread and representative to further develop custom. I. Affirming Applicability, not Clarifying the Application The only exception is a detailed statement released by the General Staff of the Iranian Armed Forces, which mentions “[m]easures like cyber manipulation of elections or engineering the public opinions on the eve of the elections [as] exam- ples of gross intervention.”38 The large majority of states have opted not to provide their opinion at all, despite manifold invitation to do so.39 There are various reasons for this. An important factor is certainly a lack of capacity.40 For many states, foreign election interferences may also not be the most pressing concern. And those states that, in the long term, envision the adoption of an entirely new cyber treaty may not provide their substantive views as their central argument is that existing international law is not sufficiently precise in regulating cyberspace. Developing custom would close the gap which is supposed to be filled by a treaty. g p pp y y Thus, the GGE and OEWG reports accurately reflect the current consensus: The principle of non-intervention applies in cyberspace—but how it applies is unclear. The approach offered by Germany, namely that a cyber operation is coercive if its scale and effect are comparable to an operation in the non-cyber context appears convincing at first, as it purports to simply transpose the existing principle to the cyber context. A similar test is also advanced by many states regarding the use of force, known as “kinetic effect”.41 But how the principle of non-intervention applies in the non-cyber context, specifically where the threshold of coercion lies, is equally unclear. The last attempt to further clarify the content of the principle dates back to 1981 and was not universally accepted.42 Germany references the support for insurgents as an example. This has indeed been considered coercive by the ICJ in Nicaragua, but as a “particularly obvious” form of coercion,43 not as de minimis-threshold. Yet it is precisely the question of where this threshold lies that makes the application of the principle of non-intervention so difficult in practice. Despite renewed atten- tion, the principle of non-intervention remains vague. States thereby risk retaining a principle that will be often invoked, but sufficient consensus on whether it has actually been breached will be seldomly reached. Circumventing the difficult debate about coercion, some states,44 as well as scholars,45 appear to have shifted the focus to a different norm. 37This at least used to be the UK position, see Doug Wilson, Introductory Remarks on the Promise and Limits of Cyber Power in International Law (2020); but see United Kingdom, supra note 22; see also Sue Robertson, Introductory Remarks on the Promise and Limits of Cyber Power in International Law (2020). y ( ) 38Press Release, General Staff of Iranian Armed Forces Warns of Tough Reaction to Any Cyber Threat (2020) https:// nournews.ir/En/News/53144/General-Staff-of-Iranian-Armed-Forces-Warns-of-Tough-Reaction-to-Any-Cyber-Threat [hereinafter Iran]. 39See, e.g., GGE, G.A. Res. A/RES/73/266, para. 2 (Dec. 22, 2018); OEWG, G.A. Res. A/RES/73/27, para. 4 (Dec. 5, 2018); Duncan B. Hollis, Presentation for the Inter-American Judicial Committee, Improving Transparency: International Law and State Cyber Operations (Aug. 7, 2020). 40Hollis, supra note 39, at 6. 41See, e.g., Iran, supra note 38, at art IV; Netherlands, supra note 22, at 3; New Zealand, supra note 22, para. 7; United States, supra note 22. 42Declaration on the Inadmissibility of Intervention and Interference in the Internal Affairs of States, supra note 23. 43Nicar. v. U.S., 1986 I.C.J. para. 205. 44France, supra note 22, at 7; Japan, supra note 30, at 3; Netherlands, supra note 22, at 2; Germany, supra note 7, at 3; Hollis, supra note 39, at 30. 45MICHAEL N. SCHMITT, TALLINN MANUAL 2.0 ON THE INTERNATIONAL LAW APPLICABLE TO CYBER OPERATIONS 17 (2017). 46Henning Lahmann, On the Politics and Ideologies of the Sovereignty Discourse in Cyberspace, 32 DUKE J. COMPAR. & INT’L L. 61, 90 (2021). 38Press Release, General Staff of Iranian Armed Forces Warns of Tough Reaction to Any Cyber Threat (2020) https:// nournews.ir/En/News/53144/General-Staff-of-Iranian-Armed-Forces-Warns-of-Tough-Reaction-to-Any-Cyber-Threat [hereinafter Iran]. 39See, e.g., GGE, G.A. Res. A/RES/73/266, para. 2 (Dec. 22, 2018); OEWG, G.A. Res. A/RES/73/27, para. 4 (Dec. 5, 2018); Duncan B. Hollis, Presentation for the Inter-American Judicial Committee, Improving Transparency: International Law and State Cyber Operations (Aug. 7, 2020). 40Hollis, supra note 39, at 6. 41See, e.g., Iran, supra note 38, at art IV; Netherlands, supra note 22, at 3; New Zealand, supra note 22, para. 7; United States, supra note 22. 42Declaration on the Inadmissibility of Intervention and Interference in the Internal Affairs of States supra note 23 47See submissions by China, Cuba, Egypt, India, Malaysia, Russia at the OEWG, United Nations, OEWG, 5th Meeting of the First Substantive Session (Sept. 11, 2019) https://media.un.org/asset/k1f/k1fbpdsxqt; see also China, supra note 29, para. 6. 48Ma Xinmin, What Kind of Internet Order Do We Need?, 14 CHINESE J. INT’L L. 399, 401 (2015).The author was, at the time of writing, a member of the Department of Treaty and Law at the Chinese Ministry of Foreign Affairs. 49Id. at 400–401. 50United Nations, supra note 47, at 1:04:00 hrs. 51OEWG, Comments on the Pre-Draft Report of the Open Ended Working Group – ICT 2 (Mar. 31, 2020) https://front.un- arm.org/wp-content/uploads/2020/04/comments-by-austria.pdf [hereinafter Austria]. 52OEWG, Draft Substantive Report, UN Doc A/AC.290/2021/L.2, para. 32 (Jan. 19, 2021). 53OEWG, Initial “Pre-Draft” of the Report of the OEWG on Developments in the Field of Information and Telecommunications in the Context of International Security, para. 27 (Apr. 27, 2020). 54GGE Report 2021, supra note 18, at 16. 55Id. paras. 15–73. 56Kubo Mačák, From Cyber Norms to Cyber Rules: Re-engaging States as Law-makers, 30 LEIDEN J. INT’L L. 877, 892-893 (2017); Michael N. Schmitt, The Sixth United Nations GGE and International Law in Cyberspace, JUST SEC. (2021), https:// www.justsecurity.org/76864/the-sixth-united-nations-gge-and-international-law-in-cyberspace/. 57GGE Report 2015, supra note 17, para. 13 (c). 58United Kingdom, supra note 22, para. 12. II. Calls for a Cyber Treaty vs. Trends Towards Informalization II. Calls for a Cyber Treaty vs. Trends Towards Informalization A different response to the lack of clarity of the principle of non-intervention—and international law in cyberspace more generally—are calls to adopt a cyber treaty.47 It is claimed that from cyber- space emerge “unique problems without ready solutions in the existing legal framework.”48 The application of general international law such as the principle of non-intervention is only a first step, on top of which a “framework charter for cyberspace activities” should be drafted.49 Yet, Western states in particular reject the idea of a treaty. Since it has already been confirmed that international law as a whole applies to cyberspace, it is feared that a convention might lead to picking and choosing of certain rules50 and that “it opens the gate for an argumentum e contrario for putting in question the applicability and legally binding character of customary international law, general principles of law and treaty obligations with regard to ICTs.”51 In the 2021 Report of the OEWG, all references to a new binding framework that still existed in the Zero Draft52 as well as the Pre-Draft53 have vanished. Yet the 2021 GGE report “notes the possibility of future elaboration of additional binding obligations, if appropriate.”54 Given this fundamental impasse with some states advocating to further develop existing custom and thus resisting a new treaty, and other states favoring a treaty and thus not providing their views on how to develop existing custom, it is maybe not surprising that the current consensus at the UN does not extend far beyond confirming the application of international law to cyberspace. How specific norms and principles and in particular the principle of non-intervention apply in concrete circumstances remains, at least for now, uncertain. pp y What can be achieved more easily seems to be a consensus on voluntary, non-binding norms or responsible state behavior. The 2021 GGE report, for example, deals with international law on one and a half pages while elaborating on eight and a half pages on non-binding norms.55 While it is often argued that non-binding norms may eventually become custom,56 some may advance adop- tion as a non-binding norm as an argument for the opposite. One example is the UK’s 2021 state- ment on the application of international law to cyberspace. I. Affirming Applicability, not Clarifying the Application The discussion whether sovereignty itself is merely a principle from which specific rules are drawn or a rule itself seems to be motivated, at least in part, by uncertainties in which cyber operations cross the coercion threshold.46 But, being ill-defined J p 44France, supra note 22, at 7; Japan, supra note 30, at 3; Netherlands, supra note 22, at 2; Germany, supra note 7, at 3; Hollis, supra note 39, at 30. p 45MICHAEL N. SCHMITT, TALLINN MANUAL 2.0 ON THE INTERNATIONAL LAW APPLICABLE TO CYBER 17 (2017). ( ) 46Henning Lahmann, On the Politics and Ideologies of the Sovereignty Discourse in Cyberspace, 32 DUKE J. COMPAR. & INT’L L. 61, 90 (2021). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 514 Lukas Willmer and at least as politically charged as the principle of non-intervention, one might wonder whether this simply leads to the replacement of one empty container with another one. p 56Kubo Mačák, From Cyber Norms to Cyber Rules: Re-engaging States as Law-makers, 30 LEIDEN J. INT’L L. 877, 892-893 (2017); Michael N. Schmitt, The Sixth United Nations GGE and International Law in Cyberspace, JUST SEC. (2021), https:// www.justsecurity.org/76864/the-sixth-united-nations-gge-and-international-law-in-cyberspace/. 59Corfu Channel Case (U.K. v. Alb.), Merits, Judgment, 1949 I.C.J. 4, 22 (Apr. 9). 60See, e.g., Eric Talbot Jensen, Due Diligence in Cyber Activities, in DUE DILIGENCE IN THE INTERNATIONAL LEGAL ORDER (Heike Krieger, Anne Peters & Leonhard Kreuzer eds., 2020). 61GGE Report 2013, supra note 16, at 8. 62GGE Report 2015, supra note 17, at 7, 12; GGE Report 2021, supra note 18, at 4, 13. 63GGE Report 2015, supra note 17, at 7. This is also reaffirmed in OEWG Report 2021, supra note 18, para. 31. 64China, Kazakhstan, Kyrgyzstan, Russian Federation, Tajikistan, Uzbekistan, International code of conduct for informa- tion security, Permanent Representative of China, Letter dated 9 January 2015 from the Permanent Representative of China, Kazakhstan, Kyrgyzstan, the Russian Federation, Tajikistan, and Uzbekistan to the United Nations addressed to the Secretary- General, para. 2(3), U.N. Doc A/69/723 (Jan. 13, 2015). 65G.A. Res 60/45 (Jan. 6, 2006). 66François Delerue, From Multilateral to Multistakeholder? New Developments in UN Processes on Cybersecurity, COUNCIL ON FOREIGN RELATIONS BLOG (2020), https://www.cfr.org/blog/multilateral-multistakeholder-new-developments-un- processes-cybersecurity. 67G.A. Res 60/45 para. 4 (Jan. 6, 2006). 68Xymena Kurowska, What Does Russia Want in Cyber Diplomacy? A Primer, in GOVERNING CYBERSPACE: BEHAVIOR, POWER AND DIPLOMACY 94-95 (Dennis Broeders & Bibi van den Berg eds., 2020). 69G.A. Res 73/27 para. 5 (Dec. 11, 2018). 70Kurowska, supra note 68, at 87. II. Calls for a Cyber Treaty vs. Trends Towards Informalization According to the non-binding norm 13(c) adopted by the GGE in 2015, states “should not knowingly allow their territory to be used for internationally wrongful acts using information and telecommunications technology.”57 The UK stresses that “the fact that States have referred to this as a non-binding norm indicates that there is not yet state practice sufficient to establish a specific customary international law rule of ‘due diligence’ applicable to activities in cyberspace.”58 Although norm 13(c) reflects what the ICJ 57GGE Report 2015, supra note 17, para. 13 (c). 58United Kingdom, supra note 22, para. 12. https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 515 German Law Journal has accepted as law in Corfu Channel,59 it is indeed disputed whether and how due diligence applies in cyberspace.60 The merits of this debate are beyond the scope of this paper, but the example illustrates that the formulation of non-binding norms within an area that is already —partly—regulated by international law may be a double-edged sword. pp y p y p p example illustrates that the formulation of non-binding norms within an area t p y g y y g Instead, this approach risks informalizing even accepted rules of international law. The 2013 GGE Report combined recommendations on norms, rules, and principles of responsible behavior by states into one category.61 The 2015 and 2021 GGE Reports clearly distinguish again between binding and non-binding norms.62 But some of the norms that the GGE lists within its report as non-binding closely replicate existing international obligations. This is most obvious in the case of norm 13(f) adopted by the GGE in its 2015 report according to which a “State should not conduct or knowingly support ICT activity contrary to its obligations under international law that inten- tionally damages critical infrastructure [ : : : ]”63 Norm 13(f) thus establishes a non-binding, volun- tary norm to obey the law. The problem is not limited to the GGE outcome. The non-binding SCO Draft Code of Conduct includes a pledge “[n]ot to use information and communications tech- nologies and information and communications networks to interfere in the internal affairs of other states or with the aim of undermining their political, economic and social stability.”64 Compliance with the principle of non-intervention is phrased here as a voluntary choice. p p p p y Both developments would likely cause spill-over effects in the non-cyber context. II. Calls for a Cyber Treaty vs. Trends Towards Informalization Provisions made in a cyber treaty may very well affect what is deemed as acceptable “analog” interference. Increasing informalization could further erode the principle of non-intervention in general. GE Report 2015, supra note 17, at 7, 12; GGE Report 2021, supra note 18, at 4, 13. Eric Talbot Jensen, Due Diligence in Cyber Activities, in DUE DILIGENCE IN THE INTERNATIONAL LEGAL ORDER r, Anne Peters & Leonhard Kreuzer eds., 2020). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press p p p p Report 2015, supra note 17, at 7. This is also reaffirmed in OEWG Report 2021, supra note 18, para. 31. Kurowska, What Does Russia Want in Cyber Diplomacy? A Primer, in GOVERNING CYBERSPACE: BEHAVIOR, DIPLOMACY 94-95 (Dennis Broeders & Bibi van den Berg eds., 2020). III. Fragmentation of Regulatory Processes: from GGE and OEWG to PoA? What has further complicated the issue is the fragmentation of the processes in which cyber issues are discussed at the UN. The primary venues were several GGEs that had been convened, with interruptions, since 2004.65 While their outcomes, especially those in 2013 and 2015, have been lauded, the groups have also been criticized for being non-transparent, exclusive, and unable to engage in a multi-stakeholder dialogue.66 The GGEs are only open to a limited number of govern- mental experts appointed by the Secretary-General.67 It was, at least by some states, perceived to be dominated by Western experts and ended up deadlocked in 2017.68 Thus, in 2019 Russia initi- ated the OEWG, which is open to all member states and was presented as an attempt to make the process “more democratic, inclusive and transparent.”69 It was also perceived as a potential forum to negotiate a cyber treaty,70 although in the end, it was the GGE report where a reference to the https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press Lukas Willmer 516 development of additional norms was retained while it disappeared in the OEWG report.71 To reunite the dual-tracked process, a diverse group of states led by Egypt and France have suggested continuing the debate at a permanent “Programme of Action for advancing responsible State behavior in cyberspace” (PoA).72 According to the proposal, the PoA would create a frame- work and political commitment based on the GGE/OEWG acquis, and have regular working-level meetings focused on implementation as well as review conferences.73 It would also step up capacity building and create an institutional dialogue with other stakeholders.74 Establishing a PoA could, first of all, offer a way out of the geostrategic rivalries that are associated with the GGE/OEWG. Another advantage that has been noted is that it would allow for dissociating different subjects.75 But judging from the outcome of the GGE/OEWG so far, there is at least the risk that international law questions, which have so far been difficult to answer, would be sidelined. For example, while the PoA on small arms and light weapons (SALW PoA) also led to politically binding commitments, the Small Arms Treaty was negotiated through the First Committee, albeit in parallel with the SALW PoA.76 A Cyber PoA might thus at least inspire other initiatives, but the PoA itself will likely continue the focus on non-binding norms. Whether it will be established remains to be seen, though. p p p 79G.A. Res 75/240, para. 1 (Dec. 31, 2020) (Yes: 92/No: 50/Abstentions: 21). 81See, e.g., Russell Buchan, Cyber Attacks: Unlawful Uses of Force or Prohibited Interventions?, 17 J. CONFLICT & SEC. L. 212 (2012); Duncan Hollis, The Influence of War; The War for Influence, 32 TEMPLE INT’L & COMPAR. L.J. 31 (2018); Ido Kilovaty, Doxfare: Politically Motivated Leaks and the Future of the Norm on Non-Intervention in the Era of Weaponized Information, 9 HARVARD NAT’L SEC. J. 146 (2018); Henning Lahmann, Information Operations and the Question of Illegitimate Interference under International Law, 53 ISRAEL L. REV. 189 (2020); Harriet Moynihan, The Application of International Law to State Cyberattacks: Sovereignty and Non-intervention, CHATHAM HOUSE (2019); JENS DAVID OHLIN, ELECTION INTERFERENCE - INTERNATIONAL LAW AND THE FUTURE OF DEMOCRACY (2020); Nicholas Tsagourias, Electoral Cyber Interference, Self-Determination and the Principle of Non-intervention in Cyberspace, in GOVERNING CYBERSPACE: BEHAVIOR, POWER AND DIPLOMACY (Dennis Broeders & Bibi van den Berg eds., 2020); Sean M Watts, Low-intensity Cyber Operations and the Principle of Non-intervention, in CYBER WAR: LAW AND ETHICS FOR VIRTUAL CONFLICTS (Jens David Ohlin, Kevin Goven & Claire Finkelstein eds., 2015). 77GGE Report 2021, supra note 18, para. 97 78OEWG Report, supra note 19, para. 77. 71See OEWG Comments, supra note 51; OEWG Draft Substantive, supra note 52; OEWG Fifth Session, supra note 47. 72Joint Proposal, The Future of Discussions on ICTs and Cyberspace at the UN (Oct. 10, 2020) https://ceipfiles.s3. amazonaws.com/pdf/CyberNorms/UNGGE/JointProposal_TheFutureofDiscussionsonICTsandCyberspace attheUN.pdf. 73Id. 74Id. 75Aude Géry & François Delerue, A New UN Path to Cyber Security, DIRECTIONS BLOG (2020), https://directionsblog.eu/ a-new-un-path-to-cyber-stability/. 76Informal Australian Research Paper, What Next for Advancing Responsible State Behaviour at the United Nations? 9 (Oct. 12, 2020) https://www.internationalcybertech.gov.au/sites/default/files/2020-12/australian-research-paper-what- next-advancing-responsible-state-behaviour-united-nations.pdf. 77GGE Report 2021, supra note 18, para. 97. 78OEWG Report, supra note 19, para. 77. 79G.A. Res 75/240, para. 1 (Dec. 31, 2020) (Yes: 92/No: 50/Abstentions: 21). 80Valentin Weber, How to Strengthen the Program of Action of Advancing Responsible State Behavior in Cyberspace, JUST SEC. (2022). 81See, e.g., Russell Buchan, Cyber Attacks: Unlawful Uses of Force or Prohibited Interventions?, 17 J. CONFLICT & SEC. L. 212 (2012); Duncan Hollis, The Influence of War; The War for Influence, 32 TEMPLE INT’L & COMPAR. L.J. 31 (2018); Ido Kilovaty, Doxfare: Politically Motivated Leaks and the Future of the Norm on Non-Intervention in the Era of Weaponized Information, 9 HARVARD NAT’L SEC. J. 146 (2018); Henning Lahmann, Information Operations and the Question of Illegitimate Interference under International Law, 53 ISRAEL L. REV. 189 (2020); Harriet Moynihan, The Application of International Law to State Cyberattacks: Sovereignty and Non-intervention, CHATHAM HOUSE (2019); JENS DAVID OHLIN, ELECTION INTERFERENCE - INTERNATIONAL LAW AND THE FUTURE OF DEMOCRACY (2020); Nicholas Tsagourias, Electoral Cyber Interference, Self-Determination and the Principle of Non-intervention in Cyberspace, in GOVERNING CYBERSPACE: BEHAVIOR, POWER AND DIPLOMACY (Dennis Broeders & Bibi van den Berg eds., 2020); Sean M Watts, Low-intensity Cyber Operations and the Principle of Non-intervention, in CYBER WAR: LAW AND ETHICS FOR VIRTUAL CONFLICTS (Jens David Ohlin, Kevin Goven & Claire Finkelstein eds., 2015). p 80Valentin Weber, How to Strengthen the Program of Action of Advancing Responsible State Behavior in Cyberspace, JUST SEC. (2022). III. Fragmentation of Regulatory Processes: from GGE and OEWG to PoA? The proposed PoA has been noted as a potential way forward in both the 2021 GGE77 and the OEWG78 reports, but the General Assembly has already adopted—against significant opposition—a resolution introduced by Russia convening a second OEWG from 2021-2025.79 As of early 2022, the PoA has not been set up although its proponents remain committed to it.80 Even if a Cyber PoA is adopted, it might simply replace the GGE, thus failing to achieve its primary goal of ending the dual-track process. D. Addressing Cyber Election Interferences – Altering Values 77GGE Report 2021, supra note 18, para. 97. 78OEWG R p t p t 19 p 77 82G.A. Res. 2665(XXV), principle 6 (Oct. 24 1970) [hereinafter Friendly Relations Declaration]. 83Nicar. v. U.S., 1986 I.C.J. para. 258. 84Thomas M. Franck, The Emerging Right to Democratic Governance, 86 AM. J. INT’L L. 46 (1992). 85See, e.g., African Charter on Democracy and Good Governance, art. 2(1); Inter-American Democratic Charter, art. 1; Treaty on European Union, art. 2. 86Gregory H. Fox, Democracy, Right to, International Protection, MAX PLANCK ENCYCLOPEDIA OF PUBLIC INTERNATIONAL LAW, para. 6 (2008). 87Gregory H. Fox & Brad R. Roth, The Dual Lives of “The Emerging Right to Democratic Governance”, 112 AM J. INT’L L. 67, 69 (2018). 88Friendly Relations Declaration, supra note 82, principle 3, para. 2. 89See supra at notes 31–36 and accompanying text. 90Germany, supra note 7, at 5. I. Introducing Democratic Values International law is formally neutral among regime types. Each state is to be treated equally regardless of its political system82 and every state may decide autonomously how to organize itself politically. This constitutes a central feature of an international legal system based on the sovereign equality of states.83 By protecting each state’s choice of a political system, the principle of non- intervention embodies the regime neutrality of international law. This regime neutrality has been challenged, in particular during the 1990s,84 and it may have been abrogated from on a regional level.85 But a universal entitlement to democratic governance suffers not only from a lack of an accepted definition of democracy,86 it is also hardly reconcilable with a reality in which plenty of evidently non-democratic regimes represent states.87 y g p The principle of non-intervention, of course, also protects a state’s choice in favor of demo- cratic governance. Protecting democratic discourse through the principle of non-intervention has, however, turned out to be a significant challenge. The principle of non-intervention protects the “exercise of [ : : : ] sovereign rights”88 from undue interference. These sovereign rights are primarily exercised by the government as a representative of the state, or at least by state officials. The principle of non-intervention thus focuses on protecting state officials from direct interfer- ences or, in cases of indirect intervention, from interferences that may not target state infrastruc- ture but have repercussions on the agency of the state. This understanding makes it difficult to engage with election interferences that target voters in their decision-making process, as they are traditionally not understood to form part of the state. This is reflected in the differing statements by Western states that have been laid out above regarding which forms of election interference would be considered coercive.89 Some states limit the scope to manipulations of election infra- structure, in other words, infrastructure that is administered by the state, while only a few states would also consider disinformation campaigns as possibly coercive. Those campaigns do not target state infrastructure, but the decision-making process of voters before they cast their ballot. And even in this case, Germany, for example, links the coercive effect of disinformation campaigns to the incitement of “violent political upheaval”90 and thus the potential loss of authority of the executive. D. Addressing Cyber Election Interferences – Altering Values D. Addressing Cyber Election Interferences – Altering Values The academic debate that has been triggered by digitalization and in particular by foreign election interferences has generated a significant number of contributions.81 Some of these proposals, 71See OEWG Comments, supra note 51; OEWG Draft Substantive, supra note 52; OEWG Fifth Session, supra note 47. 72Joint Proposal, The Future of Discussions on ICTs and Cyberspace at the UN (Oct. 10, 2020) https://ceipfiles.s3. amazonaws.com/pdf/CyberNorms/UNGGE/JointProposal_TheFutureofDiscussionsonICTsandCyberspace attheUN.pdf. 75Aude Géry & François Delerue, A New UN Path to Cyber Security, DIRECTIONS BLOG (2020), https://directionsblog.eu/ a-new-un-path-to-cyber-stability/. y y Australian Research Paper, What Next for Advancing Responsible State Behaviour at the United Nations? 2020) https://www.internationalcybertech.gov.au/sites/default/files/2020-12/australian-research-paper-what- 76Informal Australian Research Paper, What Next for Advancing Responsible State Behaviour at the United Nations? 9 (Oct. 12, 2020) https://www.internationalcybertech.gov.au/sites/default/files/2020-12/australian-research-paper-what- next-advancing-responsible-state-behaviour-united-nations.pdf. 81See, e.g., Russell Buchan, Cyber Attacks: Unlawful Uses of Force or Prohibited Interventions?, 17 J. CONFLICT & SEC. L. 212 (2012); Duncan Hollis, The Influence of War; The War for Influence, 32 TEMPLE INT’L & COMPAR. L.J. 31 (2018); Ido Kilovaty, Doxfare: Politically Motivated Leaks and the Future of the Norm on Non-Intervention in the Era of Weaponized Information, 9 HARVARD NAT’L SEC. J. 146 (2018); Henning Lahmann, Information Operations and the Question of Illegitimate Interference under International Law, 53 ISRAEL L. REV. 189 (2020); Harriet Moynihan, The Application of International Law to State Cyberattacks: Sovereignty and Non-intervention, CHATHAM HOUSE (2019); JENS DAVID OHLIN, ELECTION INTERFERENCE - INTERNATIONAL LAW AND THE FUTURE OF DEMOCRACY (2020); Nicholas Tsagourias, Electoral Cyber Interference, Self-Determination and the Principle of Non-intervention in Cyberspace, in GOVERNING CYBERSPACE: BEHAVIOR, POWER AND DIPLOMACY (Dennis Broeders & Bibi van den Berg eds., 2020); Sean M Watts, Low-intensity Cyber Operations and the Principle of Non-intervention, in CYBER WAR: LAW AND ETHICS FOR VIRTUAL CONFLICTS (Jens David Ohlin, Kevin Goven & Claire Finkelstein eds., 2015). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 517 German Law Journal if implemented, would alter the values that are underlying the principle of non-intervention in its current form by introducing democratic values (I). Other proposals would indirectly advance a more robust understanding of sovereignty and non-intervention by aiming to reduce foreign participation in domestic discourses (II). The challenge posed by cyber election interferences thus finds diametrically opposed answers (III). I. Introducing Democratic Values The fact that the principle of non-intervention is structurally blind to formations of the sovereign will outside of state structures may put democratic states at a disadvantage. g Nicolas Tsagourias has therefore suggested reappraising the principle of non-intervention in light of the principle of self-determination.91 He argues that the right to self-determination https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press Lukas Willmer 518 does not cease after the creation of a state but continues to exist as a “right to authentic self-government, that is, the right of a people really and freely to choose its own political and economic regime”,92 which is protected by the principle of non-intervention.93 According to this view, sovereign authority is primarily vested with the people, while the authority of the govern- ment is only derived. Consequently, “a government’s authority and will remain free only when its sourcing is also free.”94 The primary object of protection is no longer the government, but “the people and the process of authority and will formation.”95 Thus, the process of derivation of authority—the election—falls within the scope of the principle of non-intervention. Although this is not made explicit, Tsagourias’ proposal would insert democratic values into the principle of non-intervention as it suggests that the authority of a government must be traced back to the people. This would substantially alter the underlying values of the norm which was supposed to preserve the free choice between different political systems. But to protect this choice, the outcome cannot be predetermined, which is why the source of a government’s authority has so far not been considered. As a consequence, a cyber operation to overthrow a non-democratic govern- ment may become lawful.96 This would be similar to Lori Fisler Damrosch’s suggestion that nonfor- cible political influence is lawful when governments suppress the political rights of their peoples.97 Damrosch wrote her article at the dawn of the cold war, contributing to a debate that culminated in the stipulation of the “emerging right to democratic governance.”98 Tsagourias, although seemingly taking up this debate, is not primarily concerned with spreading democratic values. His proposal is rather meant to protect those democracies that are already established. But even though it is primarily meant as a defensive concept, it is capable of being employed more offensively. 92ANTONIO CASSESE, SELF-DETERMINATION OF PEOPLES: A LEGAL REAPPRAISAL 137 (1995). 93Tsagourias, supra note 81, at 51. 94Id. 95Id. at 52. 96Tsagourias raises this question but ultimately leaves it unanswered, see id. at 57. 97Lori Fisler Damrosch, Politics Across Borders: Non-Intervention and Non-Forcible Influence over Domestic Affairs, 83 AM. J. INT’L L. 1, 37 (1989). 98See Franck, supra note 84. 99Ohlin, supra note 81, at 102. 100Id. at 127. 101Id. at 136. 102Id. Ohlin trusts that social media companies are technically capable of doing this. 103Id. at 97. 104DAVID L. SLOSS, TYRANTS ON TWITTER: PROTECTING DEMOCRACIES FROM CHINESE AND RUSSIAN INFORMATION WARFARE 17 (2022). 92ANTONIO CASSESE, SELF-DETERMINATION OF PEOPLES: A LEGAL REAPPRAISAL 137 (1995). 93Tsagourias, supra note 81, at 51. 94 d at 52. agourias raises this question but ultimately leaves it unanswered, see id. at 57. https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press II. Shielding Domestic Discourses from Foreign Interference The challenge of foreign election interferences has led other authors to propose at least indirect limitations to foreign participation in domestic political processes. Jens David Ohlin considers the Russian interferences in the 2016 US Presidential elections to have been a violation of the right to self-determination since outside actors “masquerading as inside members of the polity” participated in the political process.99 He argues that not only the vote itself but also the preceding deliberative process should remain internal.100 The participation of outside voices is not outright prohibited, but their origin, or at least the fact that they are not members of the polity, must be transparent.101 Social media companies should therefore be required to label postings of a foreign origin.102 But, distinguishing himself from the universalist claim made by Tsagourias, Ohlin explicitly argues that his understanding of self-determination only applies if a state is organized as an electoral democracy thus preserving a state’s freedom to freely choose its political system.103 David Sloss similarly turns his attention from the content of harmful speech to the identity of the speaker.104 But he takes the argument one step further suggesting to completely ban “Chinese Id. at 97. 104DAVID L. SLOSS, TYRANTS ON TWITTER: PROTECTING DEMOCRACIES FROM CHINESE AND RUSSIAN INFORMATION WARFARE 17 (2022). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 519 German Law Journal and Russian agents” from social media platforms.105 To prevent these actors from relying on ficti- tious identities, he proposes that social media users must declare their nationality.106 Member states of an “alliance of democratic states” will verify the declarations of their respective citizens.107 In contrast to Ohlin, Sloss fears that social media companies are not capable of flagging foreign content on their own.108 In addition, election-related content by social media users from “non- democratic states”—all states that are not members of the “Alliance”—will be flagged as being posted by a citizen of a non-democratic state.109 Ohlin and Sloss sketch out a system in which certain speakers are excluded or limited solely based on their origin, not the content of their speech. While only Sloss goes as far as completely banning certain actors, both would require labeling foreign or “non-democratic” content. This constitutes an indirect limitation as it would stigmatize speech. p 113GGE Report 2013, supra note 16, para. 19; G.A. Res 75/240 (Dec. 31, 2020). II. Shielding Domestic Discourses from Foreign Interference The proposals, if implemented, would risk tipping the balance towards a much stronger understanding of sovereignty and the principle of non-intervention. The fact that “State sovereignty and international norms and prin- ciples that flow from sovereignty” apply to cyberspace constitutes by now established consensus.113 As has been pointed out above, Western states have re-embraced the principle of non-interven- tion114 and the protective dimension of sovereignty more generally.115 Yet, this renewed focus on sovereignty is balanced with an emphasis on the free flow of information. Austria has explicitly highlighted that “State sovereignty must not serve as a pretext for tightening control over a State’s citizens, which undermines their basic human rights such as the right to privacy and the freedom of The proposals by Ohlin and Sloss do not directly concern the principle of non-intervention. Sloss does not even rely on international law, but his ideas also rest on the premise that a political community may govern itself without participation from foreigners.111 Ohlin rejects non- intervention as a useful framework for addressing election interferences.112 The potential effect on the principle of non-intervention is rather an indirect one. The proposals, if implemented, would risk tipping the balance towards a much stronger understanding of sovereignty and the principle of non-intervention. The fact that “State sovereignty and international norms and prin- ciples that flow from sovereignty” apply to cyberspace constitutes by now established consensus.113 A h b i d b W h b d h i i l f i 105Id. at 159. 106Id. at 168. 107Id. At 169. 108Id. at 165. 109Id. at 156–157. 110See Franck, supra note 84. 111Sloss, supra note 104, at 11. 112Ohlin, supra note 81, at 88. 113GGE Report 2013, supra note 16, para. 19; G.A. Res 75/240 (Dec. 31, 2020). 114See, e.g., New Zealand, supra note 22, para. 10; Germany supra note 7, at 5; see also France, supra note 22, at 7. 115Lahmann, supra note 46, at 90. 116Pre-Draft Report of the Open Ended Working Group Comments by Austria 3 (Mar. 31, 2020) https://front.un-arm.o wp-content/uploads/2020/04/comments-by-austria.pdf. 117International Covenant on Civil and Political Rights, art. 19, Dec. 16, 1966, 999 U.N.T.S. 171. 118Patryk Pawlak, Operational Guidance for the EU’s International Cooperation on Cyber Capacity Building, EUROPE COMMISSION 39 (2018). 119International Strategy of Cooperation on Cyberspace, Chapter III.1 (2017) http://www.xinhuanet.com//english/chin 2017-03/01/c_136094371.htm; see also Zhang Xinbao, China’s Strategy for International Cooperation on Cyberspa 16 CHINESE J. INT’L L. 377, 380 (2017). 110See Franck, supra note 84. II. Shielding Domestic Discourses from Foreign Interference It is meant to signal to the domestic audience that these forms of speech are less legitimate because they are foreign, in line with the premise that the deliberative process before an election should remain internal.110 The proposals by Ohlin and Sloss do not directly concern the principle of non-intervention. Sloss does not even rely on international law, but his ideas also rest on the premise that a political community may govern itself without participation from foreigners.111 Ohlin rejects non- intervention as a useful framework for addressing election interferences.112 The potential effect on the principle of non-intervention is rather an indirect one. The proposals, if implemented, would risk tipping the balance towards a much stronger understanding of sovereignty and the principle of non-intervention. The fact that “State sovereignty and international norms and prin- ciples that flow from sovereignty” apply to cyberspace constitutes by now established consensus.113 As has been pointed out above, Western states have re-embraced the principle of non-interven- tion114 and the protective dimension of sovereignty more generally.115 Yet, this renewed focus on sovereignty is balanced with an emphasis on the free flow of information. Austria has explicitly highlighted that “State sovereignty must not serve as a pretext for tightening control over a State’s citizens, which undermines their basic human rights such as the right to privacy and the freedom of expression,”116 the latter consisting of the freedom to seek and receive information regardless of frontiers.117 Similarly, the “EU has always advocated that the internet should be treated as one single unfragmented space, where all resources should be accessible in the same manner, irrespective of the location of the user or provider.”118 In contrast, other states have advanced a much more robust understanding of cyber sovereignty. For China, “Safeguarding Sovereignty and Security” is its primary strategic goal in cyberspace.119 One manifestation of Chinese cyber sovereignty is the ability of states to “prohibit overseas organizations from fabricating and distorting facts and disseminating The proposals by Ohlin and Sloss do not directly concern the principle of non-intervention. Sloss does not even rely on international law, but his ideas also rest on the premise that a political community may govern itself without participation from foreigners.111 Ohlin rejects non- intervention as a useful framework for addressing election interferences.112 The potential effect on the principle of non-intervention is rather an indirect one. 105Id. at 159. 106Id. at 168. 107Id. At 169. 108Id. at 165. 109Id. at 156–157. 110See Franck, supra note 84. 111Sloss, supra note 104, at 11. 112Ohlin, supra note 81, at 88. 113GGE Report 2013, supra note 16, para. 19; G.A. Res 75/240 (Dec. 31, 2020). 114See, e.g., New Zealand, supra note 22, para. 10; Germany supra note 7, at 5; see also France, supra note 22, at 7. 115Lahmann, supra note 46, at 90. 116Pre-Draft Report of the Open Ended Working Group Comments by Austria 3 (Mar. 31, 2020) https://front.un-arm.org/ wp-content/uploads/2020/04/comments-by-austria.pdf. 117International Covenant on Civil and Political Rights, art. 19, Dec. 16, 1966, 999 U.N.T.S. 171. 118Patryk Pawlak, Operational Guidance for the EU’s International Cooperation on Cyber Capacity Building, EUROPEAN COMMISSION 39 (2018). 119International Strategy of Cooperation on Cyberspace, Chapter III.1 (2017) http://www.xinhuanet.com//english/china/ 2017-03/01/c_136094371.htm; see also Zhang Xinbao, China’s Strategy for International Cooperation on Cyberspace, 16 CHINESE J. INT’L L. 377, 380 (2017). p 111Sloss, supra note 104, at 11. https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 120China’s Views on the Application of the Principle of Sovereignty in Cyberspace 5 (2021) https://documents.unoda.org/ wp-content/uploads/2021/12/Chinese-Position-Paper-on-the-Application-of-the-Principle-of-Sovereignty-ENG.pdf. 121Zhixiong Huang & Kubo Mačák, Towards the International Rule of Law in Cyberspace: Contrasting Chinese and Western Approaches, 16 CHINESE J. INT’L L. 271, 293 (2017). 122Justin Sherman, Russia and Iran Plan to Fundamentally Isolate the Internet, WIRED (Jun. 6, 2019) https://www.wired. com/story/russia-and-iran-plan-to-fundamentally-isolate-the-internet/. 123Krieger, supra note 12, at 991. 124Id. at 990. 125Lahmann, supra note 46, at 68. 126See, e.g., Sergei Lavrov, О праве, правах и правилах (The Law, the Rights and the Rules) KOMMERSANT (Jun. 28, 2021) https://www.kommersant.ru/doc/4877702 (“The multipolar world is becoming reality”). 127Krieger, supra note 12, at 990. II. Shielding Domestic Discourses from Foreign Interference 112Ohlin, supra note 81, at 88. p p p 114See, e.g., New Zealand, supra note 22, para. 10; Germany supra note 7, at 5; see also France, supra note 22, at 7. 115Lahmann, supra note 46, at 90. p p p 114See, e.g., New Zealand, supra note 22, para. 10; Germany supra note 7, at 5; see also France, supra note 22, at 7. 115Lahmann, supra note 46, at 90. 116Pre-Draft Report of the Open Ended Working Group Comments by Austria 3 (Mar. 31, 2020) https://front.un-arm.org/ wp-content/uploads/2020/04/comments-by-austria.pdf. 116Pre-Draft Report of the Open Ended Working Group Comments by Austria 3 (Mar. 31, 2020) https://front.un-arm.org/ wp-content/uploads/2020/04/comments-by-austria.pdf. 117International Covenant on Civil and Political Rights, art. 19, Dec. 16, 1966, 999 U.N.T.S. 171. 118Patryk Pawlak, Operational Guidance for the EU’s International Cooperation on Cyber Capacity Building, EUROPEAN COMMISSION 39 (2018). 118Patryk Pawlak, Operational Guidance for the EU’s International Cooperation on Cyber Capacity Building, EUROPEAN COMMISSION 39 (2018). 118Patryk Pawlak, Operational Guidance for the EU’s International Cooperation on Cyber Capacity Building, EUROPEAN COMMISSION 39 (2018). 119International Strategy of Cooperation on Cyberspace, Chapter III.1 (2017) http://www.xinhuanet.com//english/china/ 2017-03/01/c_136094371.htm; see also Zhang Xinbao, China’s Strategy for International Cooperation on Cyberspace, 16 CHINESE J. INT’L L. 377, 380 (2017). 119International Strategy of Cooperation on Cyberspace, Chapter III.1 (2017) http://www.xinhuanet.com//english/china/ 2017-03/01/c_136094371.htm; see also Zhang Xinbao, China’s Strategy for International Cooperation on Cyberspace, 16 CHINESE J. INT’L L. 377, 380 (2017). https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press Lukas Willmer 520 online information content in their territories that seriously damages their national security and public interests.”120 China’s “great firewall” or “Golden Shield Project” allows it to control what information enters the country.121 While the Chinese approach is limited to—extremely effective —content control, Russia and Iran have taken steps to completely disconnect their domestic networks from the global internet.122 These understandings of cyber sovereignty do thus not give any regard to the free flow of information. Arguably, limiting this flow is one of its main purposes. II. Shielding Domestic Discourses from Foreign Interference online information content in their territories that seriously damages their national security and public interests.”120 China’s “great firewall” or “Golden Shield Project” allows it to control what information enters the country.121 While the Chinese approach is limited to—extremely effective online information content in their territories that seriously damages their national security and public interests.”120 China’s “great firewall” or “Golden Shield Project” allows it to control what information enters the country.121 While the Chinese approach is limited to—extremely effective —content control, Russia and Iran have taken steps to completely disconnect their domestic networks from the global internet.122 These understandings of cyber sovereignty do thus not give any regard to the free flow of information. Arguably, limiting this flow is one of its main purposes. This corresponds with other attempts at limiting foreign influence on domestic discourses. Various states such as China, Russia, Hungary, and Venezuela have passed legislation to restrict foreign funding for NGOs, which has also been interpreted as an expression of a more robust understanding of the principle of non-intervention.123 —content control, Russia and Iran have taken steps to completely disconnect their domestic networks from the global internet.122 These understandings of cyber sovereignty do thus not give any regard to the free flow of information. Arguably, limiting this flow is one of its main purposes. This corresponds with other attempts at limiting foreign influence on domestic discourses. Various states such as China, Russia, Hungary, and Venezuela have passed legislation to restrict foreign funding for NGOs, which has also been interpreted as an expression of a more robust understanding of the principle of non-intervention.123 p p The proposals by Sloss and Ohlin are clearly less restrictive, but they still go in a similar direc- tion by limiting the free flow of information. Focusing solely on the identity of the speaker, and not the content of speech, leads to blanket limitations that appear to be too restrictive. In trying to protect free and open discourses domestically, these limitations curtail the ability to lead free and open discourses transnationally and inadvertently advance a more robust understanding of sover- eignty and the principle of non-intervention. https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press III. The Same Challenge – Diametrically Opposed Answers Taking all the proposals discussed above together, a curious picture emerges. Since the end of the cold war, the promotion of democracy relied on de-emphasizing the principle of non-interven- tion. Democratic values were promoted by NGOs and other private actors to whom the principle did not apply.124 The internet became part of that very promise.125 The contemporary challenge for democratic states is a radically different one. Rather than exporting their ideals and values, they struggle to safeguard their own democratic structures that are under pressure, vulnerable precisely because of their openness. This leads to proposals that have direct or indirect repercussions on the values underlying the principle of non-intervention. Interestingly, even though the ideas discussed here all draw from the principle of self-determination, they lead to starkly different results. One, despite being conceived in a fairly defensive way, potentially justifies pro-democratic interventions reshaping the principle of non-intervention into a norm entrenched with democratic values. The principle of non-intervention would no longer be an obstacle to, but a vehicle for democracy promotion. The other proposals mark an inward turn. They would preserve the right of states to freely choose their political system, but in doing so risk giving up some of the foundational promises of the internet and lead from an interconnected world to a world of more closed, coex- isting societies. The world thus envisioned is one that is paradoxically also advocated for by some of the authoritarian actors against which the proponents of the latter proposal wish to protect themselves.126 It would be a world with sharp ideological differences in which a strong principle of non-intervention plays an important role in minimizing tensions—as it already has during the Cold War.127 , p , 126See, e.g., Sergei Lavrov, О праве, правах и правилах (The Law, the Rights and the Rules) KOMMERSANT (Jun. 28, 2021) https://www.kommersant.ru/doc/4877702 (“The multipolar world is becoming reality”). 127K i t 12 t 990 https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press 521 German Law Journal 128Ginsburg, supra note 1, 187. 129Krieger, supra note 12, 996. 128Ginsburg, supra note 1, 187. 129Krieger, supra note 12, 996. 130See, e.g., France, supra note 22, at 7. 130See, e.g., France, supra note 22, at 7. E. Conclusion Does digitalization reshape the principle of non-intervention? Does it create a new type of international law? Digitalization has sparked debates that have the potential to reshape the prin- ciple. Adopting a cyber treaty would change the nature of non-intervention in cyberspace, as would the expansion of informal rules. Incorporating democratic values would alter the central purpose of non-intervention as an embodiment of the regime neutrality of international law. Compared to that, advancing a more robust understanding of non-intervention seems much more conventional, even though the origin of certain proposals that at least have an indirect effect in that regard may be surprising. What has not changed, is the fact that geopolitical tensions surround the principle and the debate over its interpretation evidenced by the rivalries regarding the GGE/OEWG processes. The principle of non-intervention has always been politically charged and continues to be so. As a result, the principle’s regulatory vagueness persists. But above all, digitalization has reinvigorated an old idea, namely that a certain sphere of a state’s domestic affairs is protected from outside interference. The exact contours remain unclear, but the fact that there are limits somewhere is firmly accepted. Increasing interconnectedness does not make the principle of non-intervention obsolete, rather, it leads even Western states to rediscover the protective dimension of their sovereignty. While this rediscovery is currently limited to the cyber sphere, it coincides with more general perceptions that a rising “authoritarian international law” may lead to a reassertion of norms of noninterference,128 or that “populist governments” re- advance a concept of international law as a law of coordination among independent nations.129 Overly broad invocations of non-intervention in cyberspace130 may therefore inadvertently reinforce trends towards a stronger non-intervention principle even outside the cyber context. Digitalization has, at least so far, not so much reshaped the principle of non-intervention, as it has given it a renewed emphasis. This has implications for the type of international law created by digitalization in general, where sovereignty and non-intervention may play a more prominent role again. The Westphalian castle is not so out of fashion after all. Acknowledgement. The author would like to thank Janina Barkholdt and Sophie Schuberth as well as the convenors of this special issue for their valuable comments on earlier drafts of this paper. Cite this article: Willmer L (2023). Does Digitalization Reshape the Principle of Non-Intervention?. German Law Journal 24, 508–521. Acknowledgement. The author would like to thank Janina Barkholdt and Sophie Schuberth as well as the convenors of this special issue for their valuable comments on earlier drafts of this paper. 128Ginsburg, supra note 1, 187. 129Krieger, supra note 12, 996. 130See, e.g., France, supra note 22, at 7. Cit thi ti l Will L (2023) D Di i li i R h h P i Cite this article: Willmer L (2023). Does Digitalization Reshape the Principle of Non-Intervention?. German Law Journal 24, 508–521. https://doi.org/10.1017/glj.2023.27 Note. This paper has been finalized in August 2021 with state practice cursorily being updated in June 2022. Acknowledgement. The author would like to thank Janina Barkholdt and Sophie Schuberth as well as the convenors of this special issue for their valuable comments on earlier drafts of this paper. Competing Interests. The authors declare none. Funding Statement. No specific funding has been declared in relation to this article. Note. This paper has been finalized in August 2021 with state practice cursorily being updated in June 2022. E. Conclusion https://doi.org/10.1017/glj.2023.27 https://doi.org/10.1017/glj.2023.27 Published online by Cambridge University Press
https://openalex.org/W2273873073	https://journals.eco-vector.com/RCF/article/download/742/347	Russian	null	Perspectives of D1 dopamine antagonists using in treatment of nervous and psychic disorders with (+)-SCH-23390 as an example	Obzory po kliničeskoj farmakologii i lekarstvennoj terapii	2,014	cc-by	3,635	оригинальные статьи оригинальные статьи Возможности использования антагонистов D1‑рецепторов дофамина для лечения нервнопси- хических заболеваний на примере (+)-SCH‑23390 УДК 615.21 Ключевые слова Ключевые слова дофамин; SCH‑23390; квинпирол; ГАМК-ергические ней- роны; личинки миноги Lampetra planeri; пэтч-кламп. © А. А. Букинич © А. А. Букинич © А. А. Букинич ФГБУ «Научно-исследовательский институт экспериментальной медицины» СЗО РАМН, Санкт-Петербург ФГБУ «Научно-исследовательский институт экспериментальной медицины» СЗО РАМН, Санкт-Петербург ФГБУ «Научно-исследовательский институт экспериментальной медицины» СЗО РАМН, Са в модификации «целая клетка» было исследовано модулирующее действие дофамина на амплитуду ГАМК-активируемых токов. Перед каждым тестом проверялось наличие потенциалактивируемых Na+-K+-токов, которые указывали на жизнеспособ- ность клеток, и выбирался командный потенциал, при котором амплитуда ГАМК-активируемых токов была максимальной или приближена к максималь- ной. Командный потенциал в большинстве случа- ев составлял –100 мВ. Концентрация ГАМК во всех сериях экспериментов составляла 2 мМ. Соглас- но проведенным исследованиям,. на пескоройке и на миноге концентрация насыщения для ГАМК была обусловлена этими цифрами [7–8], поэтому эта концентрация была использована в наших ис- следованиях как рабочая. Дофамин, (+)-SKF‑38393 (агонист D1‑рецепторов), (–)-quinpirole (квинпи- рол) — агонист D2‑рецепторов, (+)-SCH‑23390 (ан- тагонист D1‑рецепторов), (–)-sulpiride (сульпирид) — антагонист D2‑рецепторов (все препараты Sigma, США), в нужной дозировке разводили в дистилли- рованной воде и помещали в морозильную камеру (-20 0 С). Непосредственно перед тестированием раствор размораживали и доводили физиологи- ческим раствором до концентрации необходимой для тестирования. Все указанные реактивы разла- гаются при воздействии света, поэтому хранение и все операции с ними проводили в темноте. Исходя из тех же соображений, экспериментальная часть работы также была выполнена в условиях затемне- ния. Концентрации дофамина, его агонистов и анта- гонистов во всех сериях экспериментов составляли 5 мкМ. Протокол записи был следующий: вначале апплицировалась ГАМК, затем после 2 минут от- мыва — апплицировалась ГАМК с агонистом дофа- миновых рецепторов, после чего следовал отмыв в течение 2 мин и снова апплицировалась ГАМК. При изучении эффектов антагонистов перед тестиро- ванием проводилась преинкубация антагонистом в течение 2 минут. Для регистрации токов исполь- зовали усилитель AXOPATH‑1D с головкой CV‑4 (AxonInstrument, США) и аналого-цифровой преоб- разователь DigiData‑1200 (AxonInstrument, США). Данные получали с использованием программ CLAMPEX пакета pCLAMP 6.2 и анализировали с ис- пользованием программ CLAMРFIT 6.2, CLAMРFIT 8.1, MicrosoftExcel, SigmaPlot. Ключевые слова дофамин; SCH‑23390; квинпирол; ГАМК-ергические ней- роны; личинки миноги Lampetra planeri; пэтч-кламп. оригинальные статьи n Рисунок 1. Действие дофамина 5 мкМ и агони D1‑рецепторов (+)-SKF‑38393 5 мкМ на амплит ГАМК-активируемых токов 2 мМ. а — гистограмма, показывающая нормированную ам туду ГАМК-активируемых токов (2 мМ); (n=8, p<0.0 1 — при перфузии нейрона физиологическим раство Рисунок 1. Действие дофамина 5 мкМ и агон ‑рецепторов (+)-SKF‑38393 5 мкМ на ампли МК-активируемых токов 2 мМ. — гистограмма, показывающая нормированную ам уду ГАМК-активируемых токов (2 мМ); (n=8, p<0. — при перфузии нейрона физиологическим раств — при действии 5 мкМ дофамина; 3 — при отмыве — гистограмма, показывающая нормированную итуду ГАМК-активируемых токов (2 мМ); ( 0 007) 1 ф й ф В результате экспериментов было показано, что аппликация дофамина вызывает разнонаправлен- ное действие на амплитуду ГАМК-активируемого тока на разных исследуемых нейронах. А имен- но, на 8 исследованных нейронах отмечалось уменьшение амплитуды ГАМК-активируемого тока в среднем на 33,3 ± 8,7 % (p < 0,004) (рис. 1, а). На 5 исследованных нейронах происходило уве- личение амплитуды, в среднем, на 37,3 ± 11,8 % (p < 0,007) (рис. 1, б). Восстановление амплитуды ГАМК-активируемого тока после отмыва дофами- на составило 98,7 ± 14,4 % в случае уменьшения амплитуды ГАМК-активируемого тока, и 100,0 ± 0,9 % — в случае увеличения амплитуды. В кон- трольных тестах, когда вместо дофамина подава- ли физиологический раствор, подобные эффек- ты не наблюдали: уменьшение амплитуды ГАМК- активируемого тока составило в среднем 4,44 ± 1,91 % (n = 9, р = 0,04), а увеличение — 4,24 ± 1,39 % (n = 8, р = 0,02). Простое взвешенное отклонение или размах вариации при действии дофамина на ам- плитуду ГАМК-активируемого тока составил 10,0 ± 4,2 %, а в контроле (при действии физиологиче- ского раствора на амплитуду ГАМК-активируемого тока) — 0,1 ± 0,06 %. Таким образом, доказано, что дофамин оказывает действие на амплитуду ГАМК- активируемого тока. При изучении действия агонистов D1 и D2‑ре цепторов на амплитуду хемоуправляемых токов было показано, что агонист D1‑рецепторов (+)-SKF‑3839 уменьшает амплитуду ГАМК-активируемого тока, в среднем, на 63,1 ± 11,7 % (n = 8, p < 0,01) (рис. 1, в). После отмыва амплитуда ГАМК-активируемого тока составила 78,7 ± 15,0 % от амплитуды токов, регистрируемых при перфузии нейронов физио- логическим раствором. Агонист D2‑рецепторов (–)-квинпирол вызывает в разных клетках эффекты подобные дофамину: увеличение амплитуды ГАМК- активируемого тока, в среднем, на 61,0 ± 13,8 % (n = 8, p < 0,002) (рис. 2, б), и уменьшение амплитуды, в среднем, на 55,7 ± 2,0 % (n = 6, p < 0,001) (рис. 2, а). Резюме Резюме На изолированных мультиполярных нейронах спинно- го мозга пескоройки (личинки миноги Lampetra planeri) методом пэтч-кламп в модификации «целая клетка» было исследовано модулирующее действие дофамина и его агонистов и антагонистов на амплитуду ГАМК- активируемых токов. Показано, что антагонист D1‑рецепторов (+)-SCH‑23390 блокирует эффекты дофамина на амплитуду ГАМК-активируемых токов на 63,0 ± 4,7 % и на 77,1 ± 2,0 %. Эффекты, вызванные агонистом D2‑рецепторов (–)-квинпиролом на ам- плитуду ГАМК-активируемых токов, были заблокиро- ваны на 78,8 ± 0,4 % и на 85,0 ± 5,7 % антагонистом D1‑рецепторов (+)-SCH‑23390. Так как хемоуправляе- мые токи подчиняются градуальному закону, резуль- таты по блокированию антагонистом D1‑рецепторов (+)-SCH‑23390 эффектов дофамина является идеальны- ми, что может быть основанием для рассмотрения во- проса о клинической апробации и возможности примене- ния (+)-SCH‑23390 и подобных соединений для лечения эпилепсии, невротических расстройств, депрессии. Целью настоящего исследования является вы- явление новых подходов к лечению эпилепсии, невротических расстройств, депрессии, в частно- сти возможность применения являющегося на на- стоящее время сугубо тестовым препаратом анта- гониста D1‑рецепторов (+)-SCH‑23390 в качестве лекарственного препарата. Методом для подтверж- дения возможности внедрения данного препарата в лечебный процесс был выбран прогрессивный электрофизиологический метод пэтч-кламп в мо- дификации «целая клетка», с помощью которого можно проследить блокирование ионотропных хе- моуправляемых токов, вызываемых ГАМК, на мем- бране единичного нейрона центральной нервной системы позвоночных животных. Так как в основе вышеописанных заболеваний лежит сопряжен- ный механизм, вовлекающий системы дофамина и ГАМК, находящиеся в реципрокных функциональ- ных отношениях [1–6], на изолированных мультипо- лярных нейронах спинного мозга пескоройки (ли- чинки миноги Lampetra planeri) методом пэтч-кламп 54 ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ ТОМ 12/2014/2 оригинальные статьи б — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 8, p < 0,002): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (–) квинпирола; 3 —после 2 минут отмыва n Рисунок 2. Действие агониста D2‑рецепторов (–)-квинпирола 5 мкМ на амплитуду ГАМК-активируемых 2 М n Рисунок 2. Действие агониста D2‑рецепторов (–)-квинпирола 5 мкМ на амплитуду ГАМК-активируемых токов 2 мМ. д ГАМК (2 М) ( 6 0 001) 1 токов 2 мМ. а — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 6, p < 0,001): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (–)-квинпирола; 3 — после 2 минут отмыва. б — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 8, p < 0,002): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (–)-квинпирола; 3 —после 2 минут отмыва Эффекты дофамина были частично заблокиро- ваны антагонистом D1‑рецепторов (+)-SCH‑23390: уменьшение амплитуды ГАМК-активируемых токов составило в среднем, 11,7 ± 1,8 % (n = 7, p < 0,001) (рис. 3, а), увеличение амплитуды — 8,3 ± 2,0 % (n = 5, твор, уменьшение амплитуды ГАМК-активируемого тока составило, в среднем 4,44 ± 1,91 %, а увеличе- ние — 4,24 ± 1,39 %). Таким образом, было показано, что (+)-SCH‑23390 не оказывает действия на ампли- туду ГАМК-активируемого тока. твор, уменьшение амплитуды ГАМК-активируемого тока составило, в среднем 4,44 ± 1,91 %, а увеличе- ние — 4,24 ± 1,39 %). Таким образом, было показано, что (+)-SCH‑23390 не оказывает действия на ампли- туду ГАМК-активируемого тока. n Рисунок 3. Амплитуды ГАМК-активируемых токов (2 мМ) в условиях инкубации с антагонистом D1‑рецепторов (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ) и/или с (–)-квинпиролом (5 мкМ) а — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 7, p < 0,001): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — в условиях инкубации в растворе с (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ); 4 — после 2 минут отмыва. б — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 5, p < 0,01): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — в условиях инкубации в рас- творе с (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ); 4 — после 2 минут отмыва. оригинальные статьи Восстановление ГАМК-активируемого тока после отмыва (–)-квинпирола в случае уменьшения ампли- туды составило 90,6 ± 6,1 %, в случае увеличения ам- плитуды — 91,3 ± 15,1 %. n Рисунок 1. Действие дофамина 5 мкМ и агониста D1‑рецепторов (+)-SKF‑38393 5 мкМ на амплитуду ГАМК-активируемых токов 2 мМ. а — гистограмма, показывающая нормированную ампли- туду ГАМК-активируемых токов (2 мМ); (n=8, p<0.004): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — при отмыве. б — гистограмма, показывающая нормированную ам- плитуду ГАМК-активируемых токов (2 мМ); (n=5, p<0.007): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — после 2 минут отмыва. в — гистограмма, показывающая нормированную ампли- туду ГАМК-активируемых токов (2 мМ); (n=8, p<0.01): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (+)-SKF‑38393; 3 — после 2 ми- нут отмыва. n Рисунок 1. Действие дофамина 5 мкМ и агониста D1‑рецепторов (+)-SKF‑38393 5 мкМ на амплитуду ГАМК-активируемых токов 2 мМ. Дальнейшее исследование было направлено на изучение действия антагониста D1‑рецепторов (+)-SCH‑23390 наэффекты вызванные агониста- ми дофаминовых рецепторов на амплитуду ГАМК- активируемого тока. Так как исследования дей- ствия самого (+)-SCH‑23390 на амплитуду ГАМК- активируемого тока не проводилось, нами вначале была проведена данная серия опытов. В результате было показано, что амплитуда ГАМК-активируемого тока уменьшалась, в среднем, на 4,9 ± 1,4 % (n = 7, p = 0,04) и увеличивалась, в среднем, на 4,5 ± 1,1 % (n = 6, p = 0,02). (Для сравнения, когда вместо (–)-сульпирида подавался физиологический рас- б — гистограмма, показывающая нормированную ам- плитуду ГАМК-активируемых токов (2 мМ); (n=5, p<0.007): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — после 2 минут отмыва. в — гистограмма, показывающая нормированную ампли- туду ГАМК-активируемых токов (2 мМ); (n=8, p<0.01): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (+)-SKF‑38393; 3 — после 2 ми- нут отмыва. 55 ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ ТОМ 12/2014/2 ТОМ 12/2014/2 оригинальные статьи n Рисунок 2. Действие агониста D2‑рецепторов (–)-квинпирола 5 мкМ на амплитуду ГАМК-активируемых токов 2 мМ. а — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 6, p < 0,001): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (–)-квинпирола; 3 — после 2 минут отмыва. оригинальные статьи в — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 6, p < 0,01): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (-)-квинпирола; 3 — в условиях инкубации в растворе с (+)-SCH‑23390 (5 мкМ) и с (-)-квинпиролом (5 мкМ); 4 — после 2 минут отмыва. г — гистограмма, показывающая амплитуду ГАМК-активируемых токов (2 мМ); (n = 10, p < 0,005): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (–)-квинпирола; 3 — в условиях инкубации в растворе с (+)-SCH‑23390 (5 мкМ) и с (–)-квинпиролом (5 мкМ); 4 — после 2 минут отмыва n Рисунок 3. Амплитуды ГАМК-активируемых токов (2 мМ) в условиях инкубации с антагонистом D1‑рецепторов (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ) и/или с (–)-квинпиролом (5 мкМ) а — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 7, p < 0,001): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — в условиях инкубации в растворе с (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ); 4 — после 2 минут отмыва. б гистограмма показывающая нормированную амплитуду ГАМК активируемых токов (2 мМ); (n 5 p<0 01): 1 n Рисунок 3. Амплитуды ГАМК-активируемых токов (2 мМ) в условиях инкубации с антагонистом D1‑рецепторов (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ) и/или с (–)-квинпиролом (5 мкМ) а — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 7, p < 0,001): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — в условиях инкубации в растворе с (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ); 4 — после 2 минут отмыва. б — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 5, p < 0,01): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ дофамина; 3 — в условиях инкубации в рас- творе с (+)-SCH‑23390 (5 мкМ) и с дофамином (5 мкМ); 4 — после 2 минут отмыва. в — гистограмма, показывающая нормированную амплитуду ГАМК-активируемых токов (2 мМ); (n = 6, p < 0,01): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (-)-квинпирола; 3 — в условиях инкубации в растворе с (+)-SCH‑23390 (5 мкМ) и с (-)-квинпиролом (5 мкМ); 4 — после 2 минут отмыва. ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ Литература Шабанов П. Д., Лебедев А. A. 1. Участие ГАМК- и дофами- нергических механизмов ядра ложа конечной полоски в подкрепляющих эффектах психотропных средств, реализуемых через латеральный гипоталамус // Рос. физиол. журн. им. И. М. Сеченова. — 2011. — Т. 97, № 8. — С. 804–813. 2. Shabanov P. D., Lebedev A. A., Sheveleva M. V. 2. Modula- tion of rewarding effects of psychotropic drugs by GABA and dopamine mechanisms from the bed nucleus of the stria terminalis // Eur. Neuropsychopharmacol. — 2011. — Vol. 21,Suppl. 2. — P. S161–S162. В качестве контрольного теста нами были проведены эксперименты, где действие агони- ста D2‑рецепторов (–)-квинпирола на амплиту- ду ГАМК-активируемого тока блокировали анта- гонистом D1‑рецепторов (+)-SCH‑23390. Нами было протестировано 16 мультиполярных ней- ронов. На 6 нейронах было показано уменьше- ние амплитуды ГАМК-активируемых токов в усло- виях инкубации в растворе с (+)-SCH‑23390 и с (–)-квинпиролом, в среднем, на 9,2 ± 3,4 % (p < 0,01) (рис. 3, в), на 10 нейронах — увеличение амплитуды ГАМК-активируемых токов на 6,3 ± 1,8 % (p < 0,005) (рис. 3, г). (Для сравнения, (–)-квинпи- рол уменьшал амплитуду ГАМК-активируемых то- ков на 55,7 ± 2,0 % и увеличивал на 61,0 ± 13,8 %). После отмыва амплитуда исследуемого тока со- ставила 100,0 ± 19,4 % от амплитуды токов, реги- стрируемых при перфузии нейронов физиологиче- ским раствором в случае уменьшения амплитуды ГАМК-активируемого тока, и 97,1 ± 2,3 % — в слу- чае увеличения. В данных сериях экспериментов эффекты агониста D2‑рецепторов (–)-квинпиро- ла 5 мкМ на амплитуду ГАМК-активируемых токов были заблокированы антагонистом D1‑рецепторов (+)-SCH‑23390 5 мкМ (при учете, что эффекты (–)-квинпирола на ГАМК-активируемые токи были приняты за сто процентов) на 78,8 ± 0,4 % в случае уменьшения и на 85,0 ± 5,7 % в случае увеличения амплитуды ГАМК-активируемых токов. Шабанов П. Д., Лебедев А. A. 3. Модуляция подкрепляю- щих эффектов психотропных средств ГАМК- и дофа- минергическими механизмами ядра ложа конечной полоски // Мед. акад. журн. — 2011. — Т. 11, №. 4. — С. 71–77. Шабанов П. Д., Лебедев А. A. 4. Сопряженность работы ГАМК- и дофаминергических механизмов ядра ложа конечной полоски в обеспечении подкрепляющих эф- фектов психотропных средств // Вестник Смоленск. госуд. акад. наук. — 2012. — Т. 11, №. 4. — С. 3–12. 5. у у Shabanov P. D., Lebedev A. A. 5. Литература Involvement of GABA and dopaminergic mechanisms of the bed nuckeus of the stria terminalis in the reinforcing effects of psychotropic sub- stances mediated via the lateral hypothalamus // Neurosci. Behav. Physiol. — 2013. — Vol. 43, N. 4. — P. 485–491. y Лебедев А. A., Роик Р. О., Шевелева М. В., Шуми- 6. лов Е. Г., Смирнов А. А. Значение системы кортиколи- берина, дофамина и ГА МК в структурах расширенной миндалины для подкрепляющих эффектов нарко- генов // Обз. по клин. фармакол. и лек. терапии. — 2013. — Т. 11. Спецвып. — С. 92–94. ц Дудко Н. Б., Цветков Е. А., Судеревская Е. И., Малки- 7. ель А. И., Веселкин Н. П. Потенциал-активируемые и хемочувствительные токи мембран изолированных спинальных нейронов пескоройки // Рос. физиол. журн. им. И. М. Сеченова. — 2004. — Т. 90, № 8. — С. 370. 8. Батуева И. В., Судеревская Е. И., Цветков Е. А., 8. Веселкин Н. П. Исследование влияния гамма- аминомасляной кислоты на дорсальные чувстви- тельные клетки изолированного спинного мозга миноги // Журн. эволюц. биохим. и физиол. — 1995. — Т. 31. — С. 286–291. Таким образом, было показано, что антагонист D1‑рецепторов (+)-SCH‑23390 блокирует эффекты дофамина на амплитуду ГАМК-активируемых токов на 63,0 ± 4,7 % и на 77,1 ± 2,0 %. Эффекты, вызван- ные агонистом D2‑рецепторов (–)-квинпиролом на амплитуду ГАМК-активируемых токов, были за- блокированы на 78,8 ± 0,4 % и на 85,0 ± 5,7 % анта- гонистом D1‑рецепторов (+)-SCH‑23390. оригинальные статьи оригинальные статьи p < 0,01) (рис. 3, б). (Для сравнения, дофамин вызы- вает уменьшение амплитуды ГАМК-активируемого тока в среднем на 33,3 ± 8,7 %, или увеличение ам- плитуды в среднем на 37,3 ± 11,8 %). После отмыва амплитуда ГАМК-активируемого тока восстанови- лась до 100,0 ± 10,7 % от амплитуды токов, реги- стрируемых при перфузии нейронов физиологиче- ским раствором, в случае уменьшения амплитуды ГАМК-активируемого тока, и до 93,2 ± 4,9 % — в слу- чае увеличения. Таким образом, эффекты дофами- на были частично заблокированы антагонистом D1‑рецепторов (+)-SCH‑23390 (при учете, что эф- фекты дофамина на ГАМК-активируемые токи были приняты за сто процентов) на 63,0 ± 4,7 % в случае уменьшения, и на 77,1 ± 2,0 % — в случае увеличе- ния амплитуды ГАМК-активируемых токов. са о клинической апробации и возможности приме- нения (+)-SCH‑23390 и подобных соединений для лечения эпилепсии, невротических расстройств, депрессии. p < 0,01) (рис. 3, б). (Для сравнения, дофамин вызы- вает уменьшение амплитуды ГАМК-активируемого тока в среднем на 33,3 ± 8,7 %, или увеличение ам- плитуды в среднем на 37,3 ± 11,8 %). После отмыва амплитуда ГАМК-активируемого тока восстанови- лась до 100,0 ± 10,7 % от амплитуды токов, реги- стрируемых при перфузии нейронов физиологиче- ским раствором, в случае уменьшения амплитуды ГАМК-активируемого тока, и до 93,2 ± 4,9 % — в слу- чае увеличения. Таким образом, эффекты дофами- на были частично заблокированы антагонистом D1‑рецепторов (+)-SCH‑23390 (при учете, что эф- фекты дофамина на ГАМК-активируемые токи были приняты за сто процентов) на 63,0 ± 4,7 % в случае уменьшения, и на 77,1 ± 2,0 % — в случае увеличе- ния амплитуды ГАМК-активируемых токов. са о клинической апробации и возможности приме- нения (+)-SCH‑23390 и подобных соединений для лечения эпилепсии, невротических расстройств, депрессии. p < 0,01) (рис. 3, б). (Для сравнения, дофамин вызы- вает уменьшение амплитуды ГАМК-активируемого тока в среднем на 33,3 ± 8,7 %, или увеличение ам- плитуды в среднем на 37,3 ± 11,8 %). После отмыва амплитуда ГАМК-активируемого тока восстанови- лась до 100,0 ± 10,7 % от амплитуды токов, реги- стрируемых при перфузии нейронов физиологиче- ским раствором, в случае уменьшения амплитуды ГАМК-активируемого тока, и до 93,2 ± 4,9 % — в слу- чае увеличения. Таким образом, эффекты дофами- на были частично заблокированы антагонистом D1‑рецепторов (+)-SCH‑23390 (при учете, что эф- фекты дофамина на ГАМК-активируемые токи были приняты за сто процентов) на 63,0 ± 4,7 % в случае уменьшения, и на 77,1 ± 2,0 % — в случае увеличе- ния амплитуды ГАМК-активируемых токов. ТОМ 12/2014/2 оригинальные статьи г — гистограмма, показывающая амплитуду ГАМК-активируемых токов (2 мМ); (n = 10, p < 0,005): 1 — при перфузии нейрона физиологическим раствором; 2 — при действии 5 мкМ (–)-квинпирола; 3 — в условиях инкубации в растворе с (+)-SCH‑23390 (5 мкМ) и с (–)-квинпиролом (5 мкМ); 4 — после 2 минут отмыва мплитуды ГАМК-активируемых токов (2 мМ) в условиях инкубации с антагонистом D1‑рецептор (5 мкМ) и с дофамином (5 мкМ) и/или с (–)-квинпиролом (5 мкМ) 56 ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ ТОМ 12/2014/2 Bukinich Anna Alexandrovna — PhD, Researcher, Dept. of Neu- ropharmacology, Institute of Experimental Medicine NWB RAMS, St.Petersburg 197376, Acad. Pavlov street, 12. E-mail: bukinich@ mail.ru A. A. Bukinich The effect of dopamine (DA), its agonists Summary: and antagonists on the amplitude of GABA-activated currents of isolated multipolar spinal cord neurons (both motoneurons and interneurons) of larva of the lamprey Lampetra planeri by means of patch-clamp method in the whole cell configuration was studied. (+)-SCH‑23390, a D1‑DA receptors antagonist was shown to block dopamine effects on GABA-activated currents by 63.0 ± 4.7 % and Так как хемоуправляемые токи подчиняются градуальному закону, результаты по блокирова- нию антагонистом D1‑рецепторов (+)-SCH‑23390 эффектов дофамина являются идеальными, что может быть основанием для рассмотрения вопро- 57 ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ ТОМ 12/2014/2 оригинальные статьи by 77.1 ± 2.0 %. Effects of (-)quinpirol, a D2‑DA receptors agonist, on GABA-activated currents were blocked by means of (+)-SCH‑23390 by 78.8 ± 0.4 % and by 85.0 ± 5.7 %. Because of chemoactivated currents are in full accordance with a gradual scale, the results on blocking D1‑DA receptors by (+)-SCH‑23390 are ideal ones and that is the possible basis to further clinical aprobation of (+)-SCH‑23390 for treatment of epilepsy, neurotic reactions and depression. by 77.1 ± 2.0 %. Effects of (-)quinpirol, a D2‑DA receptors agonist, on GABA-activated currents were blocked by means of (+)-SCH‑23390 by 78.8 ± 0.4 % and by 85.0 ± 5.7 %. Because of chemoactivated currents are in full accordance with a gradual scale, the results on blocking D1‑DA receptors by (+)-SCH‑23390 are ideal ones and that is the possible basis to further clinical aprobation of (+)-SCH‑23390 for treatment of epilepsy, neurotic reactions and depression. Key words: dopamine; SCH‑23390; quinpirol; GABA- ergic neurons; larva of the lamprey Lampetra planeri; patch- clamp. ◆ Информация об авторах Букинич Анна Александровна — кандидат медицинских наук, на- учный сотрудник отдела нейрофармакологии им. С. В. Аничкова. ФГБУ «Научно-исследовательский институт экспериментальной медицины» СЗО РАМН. Санкт-Петербург 197376, ул. Акад. Пав- лова, 12. E-mail: bukinich@mail.ru 58 ТОМ 12/2014/2 ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ ОБЗОРЫ ПО КЛИНИЧЕСКОЙ ФАРМАКОЛОГИИ И ЛЕКАРСТВЕННОЙ ТЕРАПИИ
https://openalex.org/W3020693183	https://www.preprints.org/manuscript/202004.0488/v1/download	English	null	A Note on the Degenerate Poly-Cauchy Polynomials and Numbers of the Second Kind	Symmetry	2,020	cc-by	4,748	Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 doi:10.20944/preprints202004.0488.v Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 doi:10.20944/preprints202004.0488.v doi:10.20944/preprints202004.0488.v1 A NOTE ON THE DEGENERATE POLY-CAUCHY POLYNOMIALS AND NUMBERS OF THE SECOND KIND HYE KYUNG KIM1 AND LEE-CHAE JANG2,∗ Abstract. Kim(2015) introduced the degenerate Cauchy numbers of the second kind and gave some identities of them. In this paper, by using the modiﬁed polyexponential functions which are introduced by Kim-Kim(2019), we deﬁne the degenerate poly-Cauchy polynomials of the second kind and investigate some properties of them. Furthermore, we consider the degenerate unipoly-Cauchy polynomials of the second kind and discuss some properties of them. Key words and phrases. polylogarithm functions, unipoly functions, Cauchy polynomials, poly- Cauchy polynomials, unipoly-Cauchy polynomials. 2010 Mathematics Subject Classiﬁcation. 11B83, 11S80. 1. Introduction As is well known, the Cauchy polynomials Cn(x) (or the Bernoulli polynomials of the second kind) are derived from the integral as follows: Z 1 0 (1+t)x+ydy = t log(1 + t)(1+t)x = ∞ X n=0 Cn(x)tn n!, (see [1, 14, 15, 19] ). (1.1) Z 1 0 (1+t)x+ydy = t log(1 + t)(1+t)x = ∞ X n=0 Cn(x)tn n!, (see [1, 14, 15, 19] ). (1.1) When x = 0, Cn = Cn(0) are called the Cauchy numbers. In [22], the Daehee polynomials Dn(x) are deﬁned by the generating function to be When x = 0, Cn = Cn(0) are called the Cauchy numbers. In [22], the Daehee polynomials Dn(x) are deﬁned by the generating function to be log(1 + t) t (1 + t)x = ∞ X n=0 Dn(x)tn n!, (see [4, 22] ). (1.2) (1.2) When x = 0, Dn = Dn(0) are called the Cauchy numbers. Kim [14] deﬁned the degenerate Cauchy polynomials Cn,λ(x) as follows: Z 1 0 1 + log(1 + λt) 1 λ x+y dy = 1 λ log(1 + λt) log 1 + 1 λ log(1 + λt) 1 + log(1 + λt) 1 λ x = ∞ X n=0 Cn,λ(x)tn n!, (see [2, 7, 18, 21] ). (1.3) (1.3) When x = 0, Cn,λ = Cn,λ(0) are the degenerate Cauchy numbers. The degenerate Cauchy polynomials Cn,λ,2(x) of the second kind are introduced in [14] as follows: When x = 0, Cn,λ = Cn,λ(0) are the degenerate Cauchy numbers. The degenerate Cauchy polynomials Cn,λ,2(x) of the second kind are introduced in [14] as follows: t log 1 + 1 λ log(1 + λt) 1 + 1 λ log(1 + λt) x = ∞ X n=0 Cn,λ,2(x)tn n!, (see [9, 10, 24] ). (1.4) (1.4) Key words and phrases. polylogarithm functions, unipoly functions, Cauchy polynomials, poly- Cauchy polynomials, unipoly-Cauchy polynomials. © doi:10.20944/preprints202004.0488.v1 L.C. Jang 2 When x = 0, Cn,λ,2 = Cn,λ,2(0) are called the degenerate Cauchy polynomials of the second kind. 1. Introduction Pyo-Kim-Kim [24] introduced the degenerate Cauchy polynomials Cn,λ,3(x) of the third kind as follows: λ (1 + λ log(1 + t)) 1 λ −1 log(1 + λ log(1 + t)) (1 + λ log(1 + t)) x λ = ∞ X n=0 Cn,λ,3(x)tn n!, (see [9, 10, 24] ) (1.5) (1.5) and also introduced the degenerate Cauchy polynomials Cn,λ,4(x) of the fourth kind as follows: λt log(1 + λ log(1 + t)) (1 + λ log(1 + t)) x λ = ∞ X n=0 Cn,λ,4(x)tn n!, (see [11, 12, 23] ). (1.6) (1.6) It is well known that the Stirling numbers of the ﬁrst kind are deﬁned by (x)n = n X l=0 S1(n, l)xl, (see [3, 15] ), (1.7) (1.7) where (x)0 = 1, (x)n = x(x −1) . . . (x −n + 1), (n ≥1). From (1.7), it is easily to see that where (x)0 = 1, (x)n = x(x −1) . . . (x −n + 1), (n ≥1). From (1.7), it is easily to see that 1 k!(log(1 + t))k = ∞ X n=k S1(n, k)tn n!, (see [3, 15] ). (1.8) (1.8) In the inverse expression to (1.7), for n ≥0, the Stirling numbers of the second kind are deﬁned by xn = n X l=0 S2(n, l)(x)l, (see [4, 5, 19] ). (1.9) (1.9) From (1.10), it is easily to see that From (1.10), it is easily to see that 1 k!(et −1)k = ∞ X n=k S2(n, k)tn n!, (see [3, 25] ). (1.10) (1.10) In this paper, with a motive similar to the degenerate poly-Bernoulli polynomails, we deﬁne the degenerate poly-Cauchy polynomials of the second kind by using the polyexponential functions, and investigate their properties. Furthermore, we con- sider the degenerate unipoly Cauchy polynomials of the second kind and discuss some identities of them. A note on the degenerate poly-Cauchy polynomials 3 Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 doi:10.20944/preprints202004.0488.v1 Peer-reviewed version available at Symmetry 2020, 12, 1066; doi:10.3390/sym12071066 doi:10.20944/preprints202004.0488.v1 A note on the degenerate poly-Cauchy polynomials 3 A note on the degenerate poly-Cauchy polynomials 2. The degenerate poly-Cauchy polynomials of the second kind Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 doi:10.20944/preprints202004.0488.v1 Peer-reviewed version available at Symmetry 2020, 12, 1066; doi:10.3390/sym12071066 Peer-reviewed version available at Symmetry 2020, 12, 1066; doi:10.3390/sym12071066 4 4 L.C. Jang (1) 4 L.C. Jang By (2.3) and (2.4), we see that C(1) n,λ,2 = Cn,λ,2. From (2.3) with x = 0, we observe that By (2.3) and (2.4), we see that C(1) n,λ,2 = Cn,λ,2. From (2.3) with x = 0, we observe that By (2.3) and (2.4), we see that C(1) n,λ,2 = Cn,λ,2. From (2.3) with x = 0, we observe th t ∞ X n=0 C(k) n,λ,2 tn n! = Eik(log(1 + t)) log 1 + 1 λ log(1 + λt) = t log 1 + 1 λ log(1 + λt) 1 t ∞ X m=1 (log(1 + t))m (m −1)!mk = t log 1 + 1 λ log(1 + λt) 1 t ∞ X m=0 (log(1 + t))m+1 (m + 1)!(m + 1)k−1 = t log 1 + 1 λ log(1 + λt) 1 t ∞ X m=0 1 (m + 1)k−1 ∞ X l=m+1 S1(l, m + 1)tl l! = t log 1 + 1 λ log(1 + λt) ∞ X m=0 1 (m + 1)k−1 ∞ X l=m S1(l + 1, m + 1) tl (l + 1)! = ∞ X s=0 Cs,λ,2 ts s! ! ∞ X l=0 l X m=0 1 (m + 1)k−1 S1(l + 1, m + 1) l + 1 tl l! ! = ∞ X n=0 n X l=0 l X m=0 n l Cn−l,λ,2 S1(l + 1, m + 1) (l + 1)(m + 1)k−1 ! tn n!. (2 = ∞ X n=0 n X l=0 l X m=0 n l Cn−l,λ,2 S1(l + 1, m + 1) (l + 1)(m + 1)k−1 ! tn n!. (2.5) (2.5) Therefore, by (2.5), we obtain the following theorem. Theorem 2.1. For n ≥0, k ∈Z, we have Therefore, by (2.5), we obtain the following theorem. Therefore, by (2.5), we obtain the following theorem. Theorem 2.1. For n ≥0, k ∈Z, we have Theorem 2.1. For n ≥0, k ∈Z, we have C(k) n,λ,2 = n X l=0 l X m=0 n l Cn−l,λ,2 S1(l + 1, m + 1) (l + 1)((m + 1)k−1 . (2.6) (2.6) Let us take k = 1. From (2.6), we get Let us take k = 1. 2. The degenerate poly-Cauchy polynomials of the second kind For k ∈Z, it is well known that the polylogarithm function Lik(x) is deﬁned by a power series in x to be Lik(x) = ∞ X n=1 xn nk = x + x2 2k + x3 3k + · · · , (see [5, 6, 16, 18] ). (2.1) (2.1) Kim-Kim [?] deﬁned the polyexponential function, as an inverse to the polylogarithm function Eik(x) = ∞ X n=1 xn (n −1)!nk , (see [8, 13, 17, 20] ). (2.2) (2.2) When k = 1, by (2.2), we get Ei1(x) = ex −1. In the viewpoint of (1.4) and (2.2), we deﬁne the degenerate poly-Cauchy polynomials of the second kind as follows: Eik(log(1 + t)) log 1 + 1 λ log(1 + λt) 1 + 1 λ log(1 + λt) x = ∞ X n=0 C(k) n,λ,2(x)tn n!. (2.3) (2.3) When x = 0, C(k) n,λ,2 = C(k) n,λ,2(0) are called the degenerate poly-Cauchy numbers of the second kind. For k = 1, by (2.2), we note that Ei1(log(1 + t)) = ∞ X n=1 (log(1 + t))n (n −1)!n = elog(1+t) −1 = t. (2.4) (2.4) Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 From (2.6), we get n X l=1 l X m=0 n l Cn−l,λ,2 S1(l + 1, m + 1) (l + 1) = 0. (2.7) (2.7) In [5], it is well known that d dxEik(log(1 + x)) = 1 (1 + x) log(1 + x)Eik−1(log(1 + x)). (2.8) From (2.8), we note that Eik(log(1 + x)) = Z x 0 1 (1 + t) log(1 + t)Eik−1(log(1 + t))dt = Z x 0 1 (1 + t) log(1 + t) Z t 0 1 (1 + t) log(1 + t) Z t 0 · · · Z t 0 \| {z } (k−2)times t (1 + t) log(1 + t)dtdt · · · dt. d dxEik(log(1 + x)) = 1 (1 + x) log(1 + x)Eik−1(log(1 + x)). (2.8) (2.8) From (2.8), we note that Eik(log(1 + x)) = Z 0 1 (1 + t) log(1 + t)Eik−1(log(1 + t))dt = Z x 0 1 (1 + t) log(1 + t) Z t 0 1 (1 + t) log(1 + t) Z t 0 · · · Z t 0 \| {z } (k−2)times t (1 + t) log(1 + t)dtdt · · · dt. = Z x 0 1 (1 + t) log(1 + t) Z t 0 1 (1 + t) log(1 + t) Z t 0 · · · Z t 0 \| {z } (k−2)times t (1 + t) log(1 + t)dtdt · · · dt. (2.9) (2.9) (2.9) doi:10.20944/preprints202004.0488.v1 A note on the degenerate poly-Cauchy polynomials 5 A note on the degenerate poly-Cauchy polynomials It is well known that It is well known that t (1 + t) log(1 + t) = ∞ X l=0 B(l) l tl l!. (2.10) (2.10) From (2.4), (2.9), and (2.10), we get ∞ X n=0 C(k) n,λ,2 xn n! = Eik(log(1 + x)) log 1 + 1 λ log(1 + λx) = 1 log 1 + 1 λ log(1 + λx) Z x 0 1 (1 + t) log(1 + t)Eik−1(log(1 + t))dt = 1 log 1 + 1 λ log(1 + λx) Z x 0 1 (1 + t) log(1 + t) × Z t 0 1 (1 + t) log(1 + t) Z t 0 · · · Z t 0 \| {z } (k−2)times t (1 + t) log(1 + t)dt · · · dtdt. Peer-reviewed version available at Symmetry 2020, 12, 1066; doi:10.3390/sym12071066 From (2.3), we observe that ∞ X n=0 C(k) n,λ,2(x)tn n! = Eik(log(1 + t)) log 1 + 1 λ log(1 + λt) 1 + 1 λ log(1 + λt) x = ∞ X l=0 C(k) l,λ,2 tl l! ! ∞ X m=0 x m 1 λ log(1 + λt) m! = ∞ X l=0 C(k) l,λ,2 tl l! ! ∞ X m=0 (x)mλ−m ∞ X s=m S1(s, m)λs ts s! ! = ∞ X l=0 C(k) l,λ,2 tl l! ! ∞ X s=0 s X m=0 (x)mλ−mS1(s, m)λs ts s! ! = ∞ X n=0 n X l=0 n−l X m=0 n l C(k) l,λ,2(x)mλn−l−mS1(n −l, m) ! = ∞ X n=0 n X l=0 n−l X m=0 n l C(k) l,λ,2(x)mλn−l−mS1(n −l, m) ! tn n!. (2.14) (2.14) By comparing the coeﬃcients on both sides of (2.14), we obtain the following theorem. By comparing the coeﬃcients on both sides of (2.14), we obtain the following theorem. Theorem 2.3. Let n ≥0 and k ∈Z. Then we have C(k) n,λ,2(x) = n X l=0 n−l X m=0 n l C(k) l,λ,2(x)mλn−l−mS1(n −l, m). (2.15) C(k) n,λ,2(x) = n X l=0 n−l X m=0 n l C(k) l,λ,2(x)mλn−l−mS1(n −l, m). (2.15) (2.15) Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 × (2.11) Let us take k = 2. Then we have Let us take k = 2. Then we have ∞ X n=0 C(2) n,λ,2 tn n! = 1 log 1 + 1 λ log(1 + λx) Z x 0 t (1 + t) log(1 + t)dt = 1 log 1 + 1 λ log(1 + λx) ∞ X l=0 B(l) l l! Z x 0 tldt = x log 1 + 1 λ log(1 + λx) ∞ X l=0 B(l) l l + 1 xl l! = ∞ X m=0 Cm,λ,2 xm m! ! ∞ X l=0 B(l) l l + 1 xl l! ! = ∞ X n=0 n X l=0 n l Cn−l,λ,2B(l) l l + 1 ! xn n! . (2.12) Therefore, by (2.12), we obtain the following theorem. Theorem 2.2. Let n ≥0. Then we have C(2) n,λ,2 = n X l=0 n l Cn−l,λ,2B(l) l l + 1 . (2.13) (2.13) 3. The degenerate unipoly-Cauchy polynomials of the second kind Let p be any arithmetic function which is a real or complex valued function deﬁned on the set of positive integers N. Then Kim-Kim [5] deﬁned the unipoly function attached to p by uk(x\|p) = ∞ X n=1 p(n)xn nk , (k ∈Z). (3.1) (3.1) It is well known that uk(x\|1) = ∞ X n=1 xn nk = Lik(x) (3.2) (3.2) is ordinary polylogarithm function, and for k ≥2, is ordinary polylogarithm function, and for k ≥2, d dxuk(x\|p) = 1 xuk−1(x\|p), (3.3) (3.3) and uk(x\|p) = Z x 0 1 t Z t 0 · · · Z t 0 \| {z } (k−2)times 1 t u1(t\|p)dt · · · dtdt (see [?] ). (3.4) (3.4) doi:10.20944/preprints202004.0488.v1 A note on the degenerate poly-Cauchy polynomials 7 A note on the degenerate poly-Cauchy polynomials By using (3.1), we deﬁne the degenerate unipoly-Cauchy polynomials of the second kind as follows: By using (3.1), we deﬁne the degenerate unipoly-Cauchy polynomials of the second kind as follows: uk(log(1 + t)\|p) log 1 + 1 λ log(1 + λt) 1 + 1 λ log(1 + λt) x λ = ∞ X n=0 C(k,p) n,λ,2(x)tn n!. (3.5) (3.5) When x = 0, C(k,p) n,λ,2 = C(k,p) n,λ,2(0) are called the degenerate unipoly-Cauchy numbers of the second kind. Let us take p(n) = 1 Γ(n). Then we have When x = 0, C(k,p) n,λ,2 = C(k,p) n,λ,2(0) are called the degenerate unipoly-Cauchy numbers of the second kind. Let us take p(n) = 1 Γ(n). Then we have ∞ X n=0 C(k,p) n,λ,2(x)tn n! = uk log(1 + t)\| 1 Γp log 1 + 1 λ log(1 + λt) 1 + 1 λ log(1 + λt) x λ = 1 log 1 + 1 λ log(1 + λt) ∞ X m=1 (log(1 + t))m mk(m −1)! 1 + 1 λ log(1 + λt) x λ = Eik(log(1 + t)) log 1 + 1 λ log(1 + λt) 1 + 1 λ log(1 + λt) x λ = ∞ X n=0 C(k) n,λ,2(x)tn n!. = ∞ X n=0 C(k) n,λ,2(x)tn n!. (3.6) = ∞ X n=0 C(k) n,λ,2(x)tn n!. (3.6) (3.6) Thus, by (3.6), we have the following theorem. Theorem 3.1. Let n ≥0 and k ∈Z, and Γ(n) be a Gamma function. Then, we have Theorem 3.1. 3. The degenerate unipoly-Cauchy polynomials of the second kind Let n ≥0 and k ∈Z, and Γ(n) be a Gamma function. Then, we have Theorem 3.1. Let n ≥0 and k ∈Z, and Γ(n) be a Gamma function. Then, we have C(k, 1 Γ) n,λ,2 (x) = C(k) n,λ,2(x). (3.7) (3.7) From (3.5), we get ∞ X n=0 C(k,p) n,λ,2 tn n! = uk(log(1 + t)\|p) log 1 + 1 λ log(1 + λt) = 1 log 1 + 1 λ log(1 + λt) ∞ X m=1 p(m) mk (log(1 + t))m = 1 log 1 + 1 λ log(1 + λt) ∞ X m=0 p(m + 1)(m + 1)! (m + 1)k ∞ X l=m+1 S1(m + 1, l)tl l! =   ∞ X j=0 Cj,λ,2 tj j!   ∞ X l=0 l X m=0 p(m + 1)(m + 1)! (m + 1)k S1(m + 1, l)tl l! ! = ∞ X n=0 ∞ X l=0 l X m=0 n l p(m + 1)(m + 1)! (m + 1)k S1(m + 1, l)Cn−l,λ,2 l + 1 ! tn n!. (3.8) ∞ X n=0 C(k,p) n,λ,2 tn n! = uk(log(1 + t)\|p) log 1 + 1 λ log(1 + λt) ∞ X n=0 C(k,p) n,λ,2 tn n! = uk(log(1 + t)\|p) log 1 + 1 λ log(1 + λt) = 1 log 1 + 1 λ log(1 + λt) ∞ X m=0 p(m + 1)(m + 1)! (m + 1)k ∞ X l=m+1 S1(m + 1, l)tl l! =   ∞ X j=0 Cj,λ,2 tj j!   ∞ X l=0 l X m=0 p(m + 1)(m + 1)! (m + 1)k S1(m + 1, l)tl l! ! = ∞ X n=0 ∞ X l=0 l X m=0 n l p(m + 1)(m + 1)! (m + 1)k S1(m + 1, l)Cn−l,λ,2 l + 1 ! tn n!. (3.8) Therefore, by comparing the coeﬃcients on both sides of (3.8), we obtain the following theorem. Therefore, by comparing the coeﬃcients on both sides of (3.8), we obtain the following theorem. Peer-reviewed version available at Symmetry 2020, 12, 1066; doi:10.3390/sym12071066 L.C. Jang Theorem 3.2. Let n ∈N and k ∈Z. Then we have Theorem 3.2. Let n ∈N and k ∈Z. Then we have C(k,p) n,λ,2 = ∞ X l=0 l X m=0 n l p(m + 1)(m + 1)! (m + 1)k S1(m + 1, l)Cn−l,λ,2 l + 1 . Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 doi A note on the degenerate poly-Cauchy polynomials A note on the degenerate poly-Cauchy polynomials From (3.5), we observe that From (3.5), we observe that From (3.5), we observe that From (3.5), we observe that ∞ X n=0 C(k,p) n,λ,2 tn n! = uk(log(1 + t)\|p) log 1 + 1 λ log(1 + λt) = 1 log 1 + 1 λ log(1 + λt) ∞ X m=0 p(m + 1) (m + 1)k m! m!(log(1 + t))m+1 = log(1 + t) log 1 + 1 λ log(1 + λt) ∞ X m=0 p(m + 1) (m + 1)k m! m!(log(1 + t))m = log(1 + t) log 1 + 1 λ log(1 + λt) ∞ X m=0 p(m + 1) (m + 1)k m! m!(log(1 + t))m = log(1 + t) t t log 1 + 1 λ log(1 + λt) ∞ X m=0 p(m + 1)m! (m + 1)k ∞ X l=m S1(l, m)tl l! = ∞ X s=0 Ds ts s! ! ∞ X a=0 Ca,λ,2 ta a! ! ∞ X l=0 l X m=0 p(m + 1)m! (m + 1)k S1(l, m)tl l! ! = ∞ X b=0 b X a=0 b a Db−aCa,λ,2 tb b! ! ∞ X l=0 l X m=0 p(m + 1)m! (m + 1)k S1(l, m)tl l! ! = ∞ X n=0 n X l=0 n−l X a=0 l X m=0 n l Dn−l−aCa,λ,2 p(m + 1)m! (m + 1)k S1(l, m) ! tn n!. (3.13 = ∞ X s=0 Ds ts s! ! ∞ X a=0 Ca,λ,2 ta a! ! ∞ X l=0 l X m=0 p(m + 1)m! (m + 1)k S1(l, m)tl l! ! = ∞ X b=0 b X a=0 b a Db−aCa,λ,2 tb b! ! ∞ X l=0 l X m=0 p(m + 1)m! (m + 1)k S1(l, m)tl l! ! = ∞ X n=0 n X l=0 n−l X a=0 l X m=0 n l Dn−l−aCa,λ,2 p(m + 1)m! (m + 1)k S1(l, m) ! tn n!. (3.13) (3.13) By comparing coeﬃcients on both sides of (3.13), we obtain the following theorem. Theorem 3.4. Let n ≥0 and k ∈Z. Then we have Theorem 3.4. Let n ≥0 and k ∈Z. Then we have Theorem 3.4. Let n ≥0 and k ∈Z. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 doi Then we have C(k,p) n,λ,2 = n X l=0 n−l X a=0 l X m=0 n l Dn−l−aCa,λ,2 p(m + 1)m! (m + 1)k S1(l.m). (3.14) (3.14) 3. The degenerate unipoly-Cauchy polynomials of the second kind (3.9) (3.9) In particular, C (k, 1 Γ ) n,λ,2 = C(k) n,λ,2 = ∞ X l=0 l X m=0 n l S1(m + 1, l)Cn−l,λ,2 (m + 1)k−1(l + 1) . (3.10) (3.10) From (3.5), we observe that From (3.5), we observe that From (3.5), we observe that From (3.5), we observe that ∞ X n=0 C(k,p) n,λ,2(x)tn n! = uk(log(1 + t)\|p) log 1 + 1 λ log(1 + λt) 1 + 1 λ log(1 + λt) x λ = uk(log(1 + t)\|p) log 1 + 1 λ log(1 + λt) ∞ X m=0 x λ m 1 λ log(1 + λt) m = ∞ X l=0 C(k,p) l,λ,2 tl l! ! ∞ X m=0 (x)m,λλ−2m ∞ X s=m S1(s, m)ts s! ! = ∞ X l=0 C(k,p) l,λ,2 tl l! ! ∞ X s=0 s X m=0 (x)m,λλ−2mS1(s, m)ts s! ! = ∞ X n=0 n X l=0 n−l X m=0 C(k,p) l,λ,2 (x)m,λλ−2mS1(n −l, m) ! tn n!. (3 (3.11) From (3.11) , we obtain the following theorem. From (3.11) , we obtain the following theorem. Theorem 3.3. Let n ≥0 and k ∈Z. Then we have Theorem 3.3. Let n ≥0 and k ∈Z. Then we have Theorem 3.3. Let n ≥0 and k ∈Z. Then we have C(k,p) n,λ,2(x) = n X l=0 n−l X m=0 C(k,p) l,λ,2 (x)m,λλ−2mS1(n −l, m). (3.12) (3.12) References 1. B. S. Borisov, The p -binomial transform Cauchy numbers and ﬁgurate numbers, Proc. Jang- jeon Math. Soc. 19(4),(2016), 631-644. 2. S.-K. Chung, G.-W. Jang, D. S. Kim and J. Kwon, Some identities of the type 2 degenerate Bernoulli and Euler numbers, Adv. Stud. Contemp. Math. (Kyungshang) 29(4),(2019), 613- 632. 3. U. Duran, M. Acikgoz, S. Araci, Hermite based poly-Bernoulli polynomials with a q-parameter, Adv. Stud. Contemp. Math. (Kyungshang) 28(2),(2018), 285-296. 4. L.C. Jang, W. Kim, H.-I. Kwon, T. Kim, On degenerate Daehee polynomials and numbers of the third kind, J. Comput. Appl. Math. 364, (2020), 112343, 9 pp. 5. D.S. Kim, T. Kim, A note on polyexponential and unipoly functions, Russ. J. Math. Phys. 26(1),(2019), 40-49. 6. D. S. Kim, T. Kim, On sums of ﬁnite products of balancing polynomials, Comput. Appl. Math. 377, (2020), 112913. 7. T. Kim, D. S. Kim, H. Y. Kim, L.C.Jang, Degenerate poly-Bernoulli numbers and polynomials, Informatica , 31(3),(2020), 2-8. 8. T. Kim, D. S. Kim, D. V. Dolgy, J. Kwon, Some identities on generalized degenerate Genocchi and Euler numbers, Informatica, 31(4),(2020), 42-51. 9. T. Kim, D. S. Kim, J. Kwon, H. Lee, A note on degenerate gamma random variables, Revista de Educacion 388(4),(2020), 39-44. 10. T. Kim, D.S. Kim, Some identities of extended degenerate r-central Bell polynomials aris- ing from umbral calculus, Rev. R. Acad. Cienc. Exactas Fs. Nat. Ser. A Mat. RACSAM 114(1),(2020), 19pp. 11. T. Kim, D.S. Kim, H. Lee, J. Kwon, Degenerate binomial coeﬃcients and degenerate hyperge- ometric functions, Adv. Diﬀerence Equ. 2020:115,(2020), 17pp. 12. T. Kim, D. S. Kim, Degenerate polyexponential functions and degenerate Bell polynomials, J. Math. Anal. Appl. 487(2),(2020),124017. ( ) ( ) 13. T. Kim, D. S. Kim, L.C. Jang, H. Y. Kim, A note on discrete degenerate random variables,, Proc. Jangjeon Math. Soc. 23(1),(2020), 125-135. ( ) ( ) 14. T. Kim, Degenerate Cauchy numbers and polynomials of the second kind, Adv. Stud. Contemp. Math. (Kyungshang) 27,(2017), 441-449. ( ) ( ) 15. T. Kim , On degenerate Cauchy numbers and polynomials, Proc. Jangjeon Math. Soc. 18(3),(2015), 307-312. ( ) ( ) 16. M. Kaneko, Poly-Bernoulli numbers, J. Theor. Nombres Bordeanes 9(1),(1997), 221-228. 17. W. A. Khan and M. Ahmad , Partially degenerate poly-Bernoulli polynomials associated with Hermite polynomials, Adv. Stud. Contemp. Math. (Kyungshang) 28(3),(2018), 487-496. 18. W. A. Khan, A new class of degenerate Frobenius-Euler-Hermite polynomials, Adv. Stud. Contemp. Math. (Kyungshang) 28(4),(2018), 567-576. 19. 4. Conclusions In 2019 Kim-Kim considered the polyexponential functions and poly-Bernoulli polynomials and Kim [14] introduced the degenerate Cauchy numbers of the second kind. In the same view as these functions and polynomials, we deﬁned the degenerate poly-Cauchy polynomials of the second kind (Eq.(2.3)) and obtained some identities of the degenerate poly-Cauchy numbers of the second kind (Theorems 2.1, and 2.2). In particular, we obtained an identity of the degenerate poly-Cauchy polynomials of the second kind in Theorem 2.3. Furthermore, by using the unipoly functions, we deﬁned the degenerate unipoly-Cauchy polynomials of the second kind(Eq. (3.5)) and obtained some properties of the degenerate unipoly-Cauchy numbers of the sec- ond kind(Theorems 3.1, and 3.3). Finally, we obtained an identity of the degenerate unipoly-Cauchy polynomials of the second kind in Theorem 3.3 and gave the identity indicating the relationship of the degenerate unipoly-Cauchy numbers of the second doi:10.20944/preprints202004.0488.v1 L.C. Jang 10 kind and the Daehee numbers and degenerate Cauchy numbers of the second kind in Theorem 3.4. kind and the Daehee numbers and degenerate Cauchy numbers of the second kind in Theorem 3.4. kind and the Daehee numbers and degenerate Cauchy numbers of the second kind in Theorem 3.4. LCJ conceived of the framework and structured the whole paper; HKK and LCJ checked the results of the paper and completed the revision of the article. All authors have read and agreed to the published version of the manuscript. Supported by the Basic Science Research Program, the National Research Foun- dation of Korea, the Ministry of Education, (NRF-2018R1D1A1B07049584).. The authors declare that they have no competing interests. Preprints (www.preprints.org) \| NOT PEER-REVIEWED \| Posted: 28 April 2020 doi:10.20944/preprints202004.0488.v1 References T. Komatsu, Higher-order convolution identities for Cauchy numbers of the second kind,Proc. Jangjeon Math. Soc. 18(3),(2015), 369-383. ( ) ( ) 20. D. V. Kruchinin, V. V. Kruchinin, Explicit formula for reciprocal generating function and its application, Adv. Stud. Contemp. Math. (Kyungshang) 29(3),(2019), 365-372 Peer-reviewed version available at Symmetry 2020, 12, 1066; doi:10.3390/sym12071066 A note on the degenerate poly-Cauchy polynomials A note on the degenerate poly-Cauchy polynomials 11 11 21. W. A. Khan, Some identities for degenerate complete and incomplete r-Bell polynomials, J. Inequal. Appl. 2020:23 ,(2020), 9pp. 21. W. A. Khan, Some identities for degenerate complete and incomplete r-Bell polynomials, J. Inequal. Appl. 2020:23 ,(2020), 9pp. 22. D. Lim, Modiﬁed degenerate Daehee numbers and polynomials arising from diﬀerential equa- tions, Adv. Stud. Contemp. Math. (Kyungshang) 28(3) ,(2018), 497-506 22. D. Lim, Modiﬁed degenerate Daehee numbers and polynomials arising from diﬀerential equa- tions, Adv. Stud. Contemp. Math. (Kyungshang) 28(3) ,(2018), 497-506 ( g g) ( ) ( ) 23. S.-S. Pyo, Degenerate Cauchy numbers and polynomials of the fourth kind, Adv. Stud. Con- temp. Math. (Kyungshang) 28(1),(2018), 127-138 ( ) ( ) ( ) 23. S.-S. Pyo, Degenerate Cauchy numbers and polynomials of the fourth kind, Adv. Stud. Con- temp. Math. (Kyungshang) 28(1),(2018), 127-138 ( ) ( ) ( ) 24. S. -S. Pyo, T. Kim, S. -H. Rim, Degenerate Cauchy numbers of the third kind, J. Ineq. Appl. 2018:32,(2018), 12pp. 24. S. -S. Pyo, T. Kim, S. -H. Rim, Degenerate Cauchy numbers of the third kind, J. Ineq. Appl. 2018:32,(2018), 12pp. 25. S. Roman, The umbral calculus, Pure and Applied Mathematics, 111. Academic Press, Inc. [Harcourt Brace Jovanovich, Publishers], New York, 1984. x+193 pp. ISBN: 0-12-594380-6 25. S. Roman, The umbral calculus, Pure and Applied Mathematics, 111. Academic Press, Inc. [Harcourt Brace Jovanovich, Publishers], New York, 1984. x+193 pp. ISBN: 0-12-594380-6 1 Department of Mathematics Education, Daegu Catholic University, Gyeongsan 38430, Republic of Korea 1 Department of Mathematics Education, Daegu Catholic University, Gyeongsan 38430, Republic of Korea E-mail address: hkkim@cu.ac.kr 2 Graduate School of Education, Konkuk University, Seoul, 05029, Republic of Korea, corresponding author 2 Graduate School of Education, Konkuk University, Seoul, 05029, Republic of Korea, corresponding author E-mail address: Lcjang@konkuk.ac.kr E-mail address: Lcjang@konkuk.ac.kr
https://openalex.org/W103580373	https://informesdelaconstruccion.revistas.csic.es/index.php/informesdelaconstruccion/article/download/4893/5698/8738	Spanish; Castilian	null	Elclor, Brasil	Informes de la construcción	1,962	cc-by	884	Informes de la Construcción Vol. 14, nº 139 Abril de 1962 Informes de la Construcción Vol. 14, nº 139 Abril de 1962 Informes de la Construcción Vol. 14, nº 139 Abril de 1962 Informes de la Construcción Vol. 14, nº 139 Abril de 1962 Informes de la Construcción Vol. 14, nº 139 Abril de 1962 A i\ l i i l A i\ planta principal 131 - 19 elclor B r a s i l RINO LEVI, arquifecto ROBERTO CERQUEIRA CESAR y L R. CARV^^LHO FRANCO, arquitectos asociados * i © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es A i\ planta principal 131 - elclor B r a s i RINO LEVI, arquifec ROBERTO CERQUEIRA CESA y L R. CARV^^LHO FRANC arquitectos asociad * i © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es planta principal e ROB y L * i © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconst planta principal planta principal 131 - 19 elclor B r a s i l RINO LEVI, arquifecto ROBERTO CERQUEIRA CESAR y L R. CARV^^LHO FRANCO, arquitectos asociados © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es http://informesdelaconstruccion.revistas.csic.es El edificio ha sido construido para sede social de las Industrias Químicas Elec- tro Cloro, S. A., y residencia de los ingenieros solteros de la empresa, en Río Grande, en el Estado de San Pablo. La construcción presentada está enclavada en los terrenos de la propia indus- tria, en Sierra del Mar, a unos 900 m de altura sobre el nivel del mar, con clima húmedo y frío, contando con el elevado índice pluviometrico de la zona y el cielo frecuentemente cubierto por espesa neblina. El edificio se levanta en la cúspide de un promontorio que domina todo el complejo de la industria y se ha procurado una adaptación perfecta a la topo- grafía del terreno, sin modificarlo en absoluto. La zona de representación y de servicio se ha organizado en un nivel interme- dio, en relación con las estancias de habitación nocturna. f a c h a d a s © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es p l a n t a b a j a Los dormitorios han sido distribuidos en el lugar más idóneo, procurando la mejor orientación y el soleamiento óptimo. p l a n t a b a j a '/T- Hay un elemento vertical que alberga el depósito de agua y sirve de contraste al ritmo horizontal del conjunto. En él se halla también la chimenea de ventilación de los baños del cuerpo de habitación nocturno. Esta torre es de hormigón visto, con lo que adquiere una recia textura y un color adecuado al clima y de feliz envejecimiento. Hay un garaje en el sector de servicio y una te- rraza cubierta destinada al tendido y secado de ropa. Los materiales empleados y el tipo de construcción no presentan peculiaridades notables: estructura de hormigón armado; muros de fábrica de ladrillo y de piedra granítica del país (con aparejo irregular y labra basta) ; revestimiento exterior del bloque de dormitorios en placas onduladas de fibrocemento; te- chos de hormigón armado con aislamiento; cubierta de placas onduladas de fibrocemento; calefac- ción de tipo radiante; carpintería de aluminio natural;... todo ello se ha elegido de tal ma- nera que pueda soportar airosamente el clima riguroso de la región y con coloraciones ade- cuadas, con objeto de que acuse escasamente el paso del tiempo. s e c c i ó n © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es de placas onduladas de fibrocemento; calefac- ción de tipo radiante; carpintería de aluminio natural;... todo ello se ha elegido de tal ma- nera que pueda soportar airosamente el clima riguroso de la región y con coloraciones ade- cuadas, con objeto de que acuse escasamente el paso del tiempo. s e c c i ó n © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es http://informesdelaconstruccion.revistas.csic.es ' sección planta baia +--_30- --rjQ_-¡ 7 1 - + sección 2.50 pasamanos +772.00 alzado planta alta 2.50 barandilla barandilla barandilla 72.00 esoalers El estudio se ha llevado a cabo en su totalidad, incluyendo mobiliario y jardinería Una airosa rotonda blanca, con pérgola atrevida, recibe al visitante, y en su fachada curva acristalada se acusan fuertemente las sombras arrojadas de dicha pérgola radial. f a c h a d a s Es, realmente, el cuerpo constructivo más alegre del conjunto que, por necesidades ambientales y de entreteni- miento, ha de presentar una fisonomía gris y severa. La jardinería es sencilla, paisajista, y prestan movimiento al complejo los desniveles del te- rreno cuidadosamente conservados y aprovechados. © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) El estudio se ha llevado a cabo en su totalidad, incluyendo mobiliario y jardinería. © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es http://informesdelaconstruccion.revistas.csic.es © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es © Consejo Superior de Investigaciones Científicas Licencia Creative Commons 3.0 España (by-nc) http://informesdelaconstruccion.revistas.csic.es http://informesdelaconstruccion.revistas.csic.es
https://openalex.org/W3015175342	https://pureadmin.qub.ac.uk/ws/files/203449491/476_E2026.pdf	English	null	Are Subsidies Required for Marine Hydrokinetic Energy Devices in Ireland? A Technological and Site Assessment	International journal of materials, mechanics and manufacturing	2,020	cc-by	4,573	Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Porta Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal y Link to publication record in Queen's University g Copyright 2020 the authors. py g This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Are Subsidies Required for Marine Hydrokinetic Energy Devices in Ireland? A Technological and Site Assessment Kumar, N., McCallum, C., Britton, G., & Doran, W. J. (2020). Are Subsidies Required for Marine Hydrokinetic Energy Devices in Ireland? A Technological and Site Assessment. International Journal of Materials, Mechanics and Manufacturing, 8(1), 12-16. https://doi.org/10.18178/ijmmm.2020.8.1.476 Kumar, N., McCallum, C., Britton, G., & Doran, W. J. (2020). Are Subsidies Required for Marine Hydrokinetic Energy Devices in Ireland? A Technological and Site Assessment. International Journal of Materials, Mechanics and Manufacturing, 8(1), 12-16. https://doi.org/10.18178/ijmmm.2020.8.1.476 Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Published in: Published in: International Journal of Materials, Mechanics and Manufacturing Published in: International Journal of Materials, Mechanics and Manufacturing Document Version: Publisher's PDF, also known as Version of record Document Version: Publisher's PDF, also known as Version of record General rights C f General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Manuscript received November 1, 2019; revised January 22, 2020. N. Kumar is with Bryden Centre, Letterkenny Institute of Technology, Letterkenny, Ireland. (e-mail address: Narendran.kumar@lyit.ie) C. McCallum, G. J. Britton, and J. Doran are with Bryden Centre, Letterkenny Institute of Technology, Letterkenny, Ireland and School of Chemistry and Chemical Engineering, Queen’s University Belfast, Belfast. I. INTRODUCTION The main forms of ocean energy are tides, marine currents, waves, salinity and temperature gradient [1] . Harnessing energy from tides encompasses tidal range and tidal stream technologies. The tidal range technologies harness energy form the potential energy created due to the ocean surface levels. The natural barriers or the fabricated structures (barrages) are used to create this potential energy and employs simple hydropower principles to generate energy. The tidal stream technologies harness energy from the kinetic energy of the flowing water. The potential locations where the tidal streams are strong are estuaries, river mouths, tidal inlets, bays, straits, etc. In these locations, the incoming (flood) and outgoing (ebb) flow of water generates stronger currents and the hydrokinetic energy devices are used to generate energy. The tidal energy potential of 1200 TWh/year [2] or 1 TW [3] could become a large part of the energy mix to meet global electricity demand, 26700 TWh/year [4] . However, considering the constraints and limitations such as fishing sites, harbours, ports, shipping lanes, technological viability, Natura sites, human activities, siting and environmental issues, the estimated theoretical figures could be reduced substantially. Although experts have identified a huge potential in offshore renewable energy sector with regards to economic growth, job creation, energy security, reducing carbon emissions and providing sustainable energy [5], [6] . In this article potential deployment of marine hydrokinetic (MHK) energy devices in Ireland are analysed. y ) In ROI the Department of Communications, Energy and Natural Resources have estimated the tidal energy development potential in ROI ranging from 1.5 to 3.0 GW [12] . There are a series of challenges and issues that will have to be overcome to achieve this energy potential such as Technological Readiness Level (TRL), financing, market establishments, and grid connections in remote areas. The delay in the tidal energy development is caused by significant challenges based on technology development, reduction of financial risks in deployment, operation and maintenance (O&M), and retrieval costs, engagement with manufacturers and supply chain [13]. Open Access Thi h Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love t this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 International Journal of Materials, Mechanics and Manufacturing, Vol. 8, No. 1, February 2020 Are Subsidies Required for Marine Hydrokinetic Energy Devices in Ireland?ATechnological and SiteAssessment Narendran Kumar, Christopher McCallum, Gary James Britton, and John Doran Abstract—To achieve the renewable energy target, it is necessary to carry out technological assessment of marine hydrokinetic energy devices. The objective of this study is to present the suitability of existing devices in the established resource sites in Ireland. The analysis is based on the technical data of the turbines and tidal current velocity profiles from the established numerical model. From this study, we conclude that there are certain locations where all kinds of marine hydrokinetic devices could be deployed, while in a few locations’ technology feasibility is limited. A. Tidal and marine Currents Energy Potential in Europe A. Tidal and marine Currents Energy Potential in Europe The tidal and wave energy resource potential for EU is 100 GW [7] . The renewable energy directive (2009/28/EC) sets rules for the EU to achieve 20% renewable target by 2020 [8] . All member states have set their own targets of energy from renewable sources by 2020. The Republic of Ireland (ROI) target is 16% [8] and Northern Ireland (NI) had set its domestic target of 40% renewable electricity and 10% renewable heat from overall 15% share of United Kingdom (UK) [9] . So far, in ROI, 10.6% of gross final energy consumption in 2017 is from renewable sources [10]. The wind energy is the main contributing technology (84% in 2017) to achieve so far [10]. Every member state in EU has implemented respective polices, plans and consenting processes that would set the mechanism in motion to develop ocean energy installations. In the year 2016, France ranked number one in electricity generation of 501 GWh/year from marine renewable sources followed by the Republic of Korea (496 GWh/year) and Canada (18 GWh/year) [11]. Index Terms—Hydrokinetic energy devices, LCOE, annual energy output, EU ETS. III. OVERVIEW OF THE RESOURCE IN IRELAND The Sustainable Energy Authority of Ireland (SEAI) has calculated the theoretical tidal energy resource to be around 230 TWh/year in the island of Ireland. However, considering the practical constraints such as flow velocities, shipping lanes, military zones, etc. the estimate is reduced to 0.92 TWh/year [16] . Table I presents the MHK energy devices based on its Technological Readiness Level (TRL) [17] . The manufacturer of MHK energy devices, its rated power, rated speed, LCOE (Levelised Cost of Energy), AEP (Annual Energy Production) and TRL parameters of the devices are presented. Fig. 1. MHK energy projects across Europe. From Fig. 1, it is evident that only a few projects are From Fig. 1, it is evident that only a few projects are TABLE I: STATE-OF-THE-ART TECHNOLOGIES OF MHK ENERGY DEVICES Device and type Manufacturer Rated Power (MW) Rated speed (m/s) LCOE (€/MWh) AEP (MWh) TRL [17] AR1500 Atlantis 1.5 3-5 [18] - 5256 9 [19] E35 Evopod Ocean Flow Energy 0.035 2.3 - 91.98 5 [20] Oceade 18 Alstom 1.4 - 2452.8* 7 [19] T200 Tocardo 0.2 - 201.48 7 [19] SR2000 Orbital 2 3.0 [21] - 5956.8 7 [19] HS1000 Hammerfest 1 2.8 [22] - 4029.60 7 [19, 20] SeaGen S-2 Marine Current Turbines (MCT) Ltd 2 2.5 146 [23] 10336.8 7 [20] Sabella D10 Sabella 1 4.0 [24] - 1752* 6-7 [19, 20] SIT250 Sustainable Marine Energy Ltd. 0.062 - 108.62* 6 [19] M100 Nova Innovation 0.1 [25, 26] 2.0 477-786 175.2* 5-7 [20] Deep Green Tidal kite 500 Minesto 0.5 100 [27] 1686.30 5 [20] CoRMAT 500 Nautricity Ltd 0.5 2.5 [28] - 876* 5 [20] TidGen ORPC 0.7 3.0 [29] 540† 3985.8 5 [20] P154 Guinard energies 0.02 2.5 [30] 130-260 100 Unknown *capacity factor is assumed as 20% [31], †private communication with ORPC TABLE I: STATE-OF-THE-ART TECHNOLOGIES OF MHK ENERGY DEVICES Manufacturer Rated Power (MW) Rated speed (m/s) LCOE (€/MWh) AEP (MWh) TRL [17] TABLE II: ANNUAL ELECTRICAL CONSUMPTION IN PRACTICAL RESOURCE SITES [16] S. No. Site Capacity (GWh/year) 1. Inishtrahull Sound 514 2. Bulls Mouth 6 3. Shannon Estuary 367 4. Dursey Island 4 5. Gascanane Sound 1 6. Tuskar Rock 420 7. Arklow 791 TABLE II: ANNUAL ELECTRICAL CONSUMPTION IN PRACTICAL RESOURCE SITES [16] water depth and tidal current velocity greater than 1.5 m/s. II. DEPLOYMENTS OF MHK DEVICES IN EUROPE Ocean Energy Europe reported that since 2010 there has been 26.8 MW of installed tidal capacity, although only 11.9 MW is still operating while the remainder has been decommissioned [14] . The recent developments has provided knowledge and experiences in the aspects of technological, economic and social aspects. Tidal devices are versatile devices as they can be deployed both inland and at water outlets at sea. The MHK energy devices are classified as axial-flow, cross-flow turbines, tidal kite, Archimedes screw device, oscillating hydrofoil [15] . Among the tidal stream technologies, large number of axial flow turbines are employed with small number of cross-flow turbines under development. Moreover, the axial-flow type is the most common and economical device in the present scenario. The additional advantage over other renewable doi: 10.18178/ijmmm.2020.8.1.476 doi: 10.18178/ijmmm.2020.8.1.476 12 International Journal of Materials, Mechanics and Manufacturing, Vol. 8, No. 1, February 2020 International Journal of Materials, Mechanics and Manufacturing, Vol. 8, No. 1, February 2020 energy technologies is that the resources of tidal energy is more predictable and the energy devices are effective at lower fluid flow speeds in comparison with wind energy devices. currently deployed. Magagna et al. (2014) proposes twin- track approach such as improving the reliability and robustness of the devices through technology development and implementing appropriate mechanisms to push the sector from pilot array deployments to large-scale farms [13]. Fig. 1. MHK energy projects across Europe. From Fig. 1, it is evident that only a few projects are V. CASE STUDIES This section investigates the projected cost of electricity from natural gas, on/offshore wind, nuclear and MHK energy. The price of energy from gas from 2018 to 2035 is based on the price reported by by the UK Department for Business, Energy and Industrial Strategy (BEIS) [32]. The price of wind energy (onshore and offshore) is based on the latest report on energy prices in Ireland [33] . The price of nuclear energy was calculated from £2012 price from Hinkley Point C [34] using a 17.11 % increase [35] and exchange rate of £1 = €1.16. The carbon tax scenarios, reported in Table IV, are applied to the 0.37 tonne CO2/MWh generated from gas, according the IPCC [36]. TABLE III: ANNUAL ENERGY OUTPUT (AEO) OF THE HYDROKINETIC ENERGY DEVICES FOR THE RESPECTIVE RESOURCE SITES TABLE III: ANNUAL ENERGY OUTPUT (AEO) OF THE HYDROKINETIC ENERGY DEVICES FOR THE RESPECTIVE RESOURCE SITES TABLE IV: SCENARIOS FOR PRICE DETERMINATION Scenario Number Scenario Conditions 1 Business as usual  Energy continues to be generated from gas with electricity prices tracking the increase in gas price [32]  No significant form of carbon taxation is placed on energy generated from natural gas 2 EU Recommended Price (Low Price)  Energy continues to be generated from gas with electricity prices tracking the increase in gas price [32]  From 2020 a USD$ 40/tCO2 (€ 36.40) is added with USD$ 50/tCO2 (€ 45.50) from 2030 [37] 3 EU Recommended Price (High Price)  Energy continues to be generated from gas with electricity prices tracking the increase in gas price [32]  From 2020 a USD$ 80/tCO2 (€ 72.80) is added with USD $ 100/tCO2 (€ 91.00) from 2030 [37] 4 Real Price of Carbon  Energy continues to be generated from gas with electricity prices tracking the increase in gas price [32]  An additional cost of USD$ 200/tCO2 (€ 182.00) is added from 2020 [38] The prices of tidal devices were initially set at €340/MWh. [39] the projected prices were determined via the potential growth of the tidal capacity as projected by the European Commission [40] applied via (1). prices of electricity prices from a variety of energy generation sources, if the gas price follows the central expectation of the BEIS. Fig. 3 reports the prices for the central BEIS price changes for gas, and tidal prices. III. OVERVIEW OF THE RESOURCE IN IRELAND In this study the velocity of all the sites are taken from the numerical model, discussed in the next section. Fig. 2. Histogram of tidal current velocity and power curves of hydrokinetic energy devices. ig. 2. Histogram of tidal current velocity and power curves of hydrokinet energy devices. Table I shows the MHK energy devices starting from AR1500 has the highest TRL, 9, followed by other devices with relatively lower TRL. The suitability of these devices in the established practical resource sites of ROI is discussed. The LCOE and capacity factor (CF) of certain devices are available and the corresponding AEP is estimated. For some devices, the CF is assumed to be 20% as suggested by [31] . The assessment of technology resources is conducted on the sites suggested by SEAI report [16], tabulated in Table II. The objective of this study is to present the suitability of existing devices in the established resource sites. The capacity values provided in the Table II represents the theoretical estimation of annual energy generation in GWh based on the tidal array spacing, Fig. 2. Histogram of tidal current velocity and power curves of hydrokinetic energy devices. Fig. 2 consists of a histogram of tidal current velocity and power curves of MHK energy devices considered with corresponding values presented in right and left y-axis respectively. For Inishtrahull Sound the velocity distribution ranges from 0.1-1.6 m/s, with 1 m/s as the maximum 13 International Journal of Materials, Mechanics and Manufacturing, Vol. 8, No. 1, February 2020 (a) (b) (c) (d) (e) (f) (a) Inishtrahull Sound (b) Bulls Mouth (c) Shannon Estuary (d) Dursey Island (e) Gascanane Sound (f) Tuskar Rock (g) Arklow. (g) (a) number of occurrences in a given time. Where the power curves are above the histogram this the area of “useful” energy which the device can generate energy from, this is multiplied to make an Annual Energy Output (AEO). The suitability of the MHK energy devices are proposed for potential sites from the estimated AEO value ranges as shown in Table III. The ordinate of Table III is in log-scale in order to show the AEO values for all devices regardless of plate capacity. (b) (b) (a) (a) (c) ( ) (d) (d) (c) (c) (e) (g) IV. ASSESSMENT OF TECHNOLOGY RESOURCES IN IRELAND (c) (e) ( ) (f) For Inishtrahull Sound, we have observed that all the hydrokinetic devices considered in this study could be deployed to generate useful power as shown in Table IIIa. The power curves of all the MHK energy devices are presented in Fig. 2 except for CoRMAT, M100, Deep Green 500 and Sabella in due to the unavailability of its power curve data. Therefore, the assessment of these devices could not be made in this study. In the Bulls Mouth site as shown in Table IIIb, the appropriate devices which could be deployed are AR1500, TidGen 8, SR2000 and SIT250. While the power curves of other devices such as HS1000, SeaGen, etc., does not fall within the projected tidal current velocity regime, therefore these devices could produce no energy in Bulls Mouth site. Similarly, we observe that the appropriate devices which could be deployed in Shannon Estuary, Gascanane Sound and Arlow are AR1500, TidGen, SR2000, SIT250 and E35 as shown in Table IIIc, e and g. In Dursey Sound Table IIId, AR1500, TidGen, SR2000 and SIT250 could be deployed. Table IIIf presents the technology suitability in Tuskar Rock, which has velocity distribution similar to Inishtrauhal Sound, therefore all the MHK energy devices is feasible to be deployed. (f) (e) (g) REFERENCES [1] L. Mofor, J. Goldsmith, and F. Jones, Ocean Energy: Technology Readiness, Patents, Deployment Status and Outlook, Abu Dhabi, 2014. Fig. 3. Central BEIS price of gas model. [2] Ocean Energy Systems, An International Vision for Ocean energy, 2017. [3] R. Kempener and F. Neumann, Tidal Energy: Technology Brief, Abu Dhabi, 2014. [4] World electricity demand, Data and Statistics, The International Energy Agency. gy g y [5] Marine renewable energy: Commonwealth blue economy series, The Commonwealth, no. 4, London, 2016. [6] Powering the Blue Economy, The U.S. Department of Energy (DOE), 2019. Fig. 3. Central BEIS price of gas model. [7] Industry Vision Paper, Ocean Energy Europe., 2013. [8] Directive 2009/28/EC of the European parliament and of the council. [Online]. Available: https://eur- lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:140:0016:0 062:EN:PDF VI. CONCLUSIONS lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:140:0016:0 062:EN:PDF From this assessment, we propose the most common and appropriate MHK energy devices considered for the sites in Ireland are AR1500, TidGen8, SR2000, SIT250 and E35. At present, the LCOE of MHK energy devices are higher but eventually will decrease as the TRL increases along with mass production, innovations, improved CF and lower operation and maintenance (O&M) costs. The issue with carbon taxation is it will cause people who cannot afford costly capital for renewable generation sources to pay higher energy prices. Instead, subsidies usual placed on fossil fuels, estimated at €112 billion per year (2014-2016) across the EU [41] , and should be diverted to subsidize renewable energy installation, operation and generation. In doing so need for carbon taxation isn’t required while also securing energy for the future of countries in a sustainable manner which reduces climate change. From the existing MHK deployments, it is clear that these devices present an economically viable LCOE where site specific constraints on other energy generation devices exist. Research and Development supports, alongside renewable electricity feed in tariffs or similar, are key enablers in lowering the LCOE of these devices, which will play a major role in a wider renewable energy mix. [9] UK Renewable Energy Roadmap, Department of Energy and Climate Change, 2011. [10] Energy in Ireland, SEAI, 2018. [11] IRENA. Country rankings of marine renewable energy projects installed. [Online]. Available: https://www.irena.org/Statistics/View- Data-by-Topic/Capacity-and-Generation/Country-Rankings [12] Offshore Renewable Energy Development Plan, Department of Communications, Energy and Natural Resources, 2014. [13] D. Magagna, A. MacGillivray, H. Jeffrey, C. Hanmer, A. Raventos, A. Badcock-Broe, and E. Tzimas, "Wave and tidal energy strategic technology agenda," SI Ocean, 2014. [14] Ocean energy Key trends and statistics, Ocean Energy Europe Report, 2018. [15] EMEC tidal devices. [Online]. Available: http://www.emec.org.uk/marine-energy/tidal-devices [16] Tidal and Current Energy Resources in Ireland, SEAI. [17] Technology readiness levels (TRL). [Online]. Available: https://ec.europa.eu/research/participants/data/ref/h2020/wp/2014_20 15/annexes/h2020-wp1415-annex-g-trl_en.pdf [18] Specifications of AR1500. [Online]. Available: https://www.atlantisresourcesltd.com/wp/wp- content/uploads/2016/08/AR1500-Brochure-Final-1.pdf [19] M. Soede, Study on lessons for ocean energy development: Final Report, European Commission, 2017. [20] P. Mascarenhas , J. Bald, I. Menchaca , A. M. O’Hagan, and T. Simas, "Report on RiCORE project novel technology selection,” Deliverable 3.2, RICORE Project, 2015. [21] Specifications of SR2000. [Online]. Available: https://orbitalmarine.com/technology-development/sr2000 [21] Specifications of SR2000. [Online]. Available: https://orbitalmarine.com/technology-development/sr2000 CONFLICT OF INTEREST The authors declare no conflict of interest. [24] Sabella D10 tidal turbine. [Online]. Available: https://www.sabella.bzh/en/projects/d10. V. CASE STUDIES Although there is an initial decrease in electricity generation from natural gas in this model without carbon taxation, as shown in Scenario 1, until 2021 the ability to tax the carbon can make the price of generation from natural gas competitive with that of tidal as early as late 2024. Without carbon taxation tidal energy would not be competitive with gas generation until 2026. If Difference in Price = €340 × 1 Expected Capacity in year 20XX/Capacity of tidal in 2017 (1) Difference in Price = €340 × 1 Expected Capacity in year 20XX/Capacity of tidal in 2017 (1) (1) The prices for alternative technologies are (€70/MWh) onshore wind [33], offshore wind (€80/MWh) [33], nuclear power (€125.65/MWh) [34] . Figure 3 reports the various 14 International Journal of Materials, Mechanics and Manufacturing, Vol. 8, No. 1, February 2020 the results, checking the facts and proofreading. All authors had approved the final version. tidal turbine capacity followed the pessimistic model then tidal energy would not be competitive with gas until at least 2030 and that would require a carbon tax of €182/tCO2 which hasn’t been put forward by any country to date. With respect to other renewable or carbon neutral technologies tidal energy can also remain competitive. Unless the pessimistic scenario is followed the energy generated will be equal to nuclear by 2027 or 2024. A competitive price with the more established renewable energy of wind will be the larger challenge as even in an optimistic scenario the price wouldn’t be equal until 2029 at the earliest. ACKNOWLEDGMENT The Bryden Centre project is supported by the European Union's INTERREG VA Programme, managed by the Special EU Programmes Body (SEUPB). The Bryden Centre project is supported by the European Union's INTERREG VA Programme, managed by the Special EU Programmes Body (SEUPB). This research was supported by the European Union under the H2020, TAOIDE project grant no: 727465. This research was supported by the European Union under the H2020, TAOIDE project grant no: 727465. DISCLAIMER The views and opinions expressed in this paper do not necessarily reflect those of the European Commission or the Special EU Programmes Body (SEUPB). [22] Specifications of HS1000. [Online]. Available: https://tethys.pnnl.gov/sites/default/files/publications/ScottishPower- Renewables-2010.pdf [22] Specifications of HS1000. [Online]. Available: https://tethys.pnnl.gov/sites/default/files/publications/ScottishPower- Renewables-2010.pdf [23] G. L. Zupone, M. Amelio, S. Barbarelli, G. Florio, N. M. Scornaienchi, and A. Cutrupi, "Levelized cost of energy: A first evaluation for a self balancing kinetic turbine," Energy Procedia, vol. 75, pp. 283-293, 2015. International Journal of Materials, Mechanics and Manufacturing, Vol. 8, No. 1, February 2020 [27] LCOE of Minesto tidal kite. [Online]. Available: https://renewablesnow.com/news/minesto-says-lcoe-below-eur- 50mwh-possible-for-kite-turbine-586850/ [52] State and trends of carbon pricing 2018. World Bank Group, [Online]. Available: https://openknowledge.worldbank.org/handle/10986/29687 [53] Conversion factors: Energy and carbon conversions 2011 update," Carbon Trust. [28] Specifications of CoRMAT 500. [Online]. Available: https://tethys.pnnl.gov/annex-iv-sites/argyll-tidal-demonstrator- project [54] Beyond the EU ETS: Strengthening Europe’s carbon market through national action. Carbon Market Watch. [Online]. Available: https://carbonmarketwatch.org/wp/wp- content/uploads/2017/12/CMW-BEYOND-THE-EU-ETS- STRENGTHENING-EUROPE%E2%80%99S-CARBON-MARKET- THROUGH-NATIONAL-ACTION.pdf [54] Beyond the EU ETS: Strengthening Europe’s carbon market through national action. Carbon Market Watch. [Online]. Available: https://carbonmarketwatch.org/wp/wp- [29] Specifications of TidGen. [Online]. Available: http://www.orpc.co/our-solutions/scalable-grid-integrated- systems/tidgen-power-system content/uploads/2017/12/CMW-BEYOND-THE-EU-ETS- STRENGTHENING-EUROPE%E2%80%99S-CARBON-MARKET- THROUGH-NATIONAL-ACTION.pdf [30] Guinard energies P154 tidal turbine. [Online]. Available: https://www.guinard-energies.bzh/en/our-products/p154- hydrokinetic-turbine/ [55] Estimated social cost. Stanford University, [Online]. Available: https://pangea.stanford.edu/news/estimated-social-cost-climate- change-too-low y [31] D. Magagna, R. Monfardini, and A. Uihlein, JRC Ocean Energy Status Report, European Union, 2016. Copyright © 2020 by the authors. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0). [32] UK Department for Business, Energy & Industrial Strategy, BEIS 2018 Fossil Fuel Price Assumptions, 2018. [33] Baringa, 70 by 30; A 70 % Renewable Electricity Vision for Ireland in 2030, 2018. [34] UK National Audit Office; Department for Business, Energy and Industrial Strategy, Hinkely Point C, 2017. Narendran Kumar received his Ph.D. in ocean engineering from Indian Institute of Technology Madras, India. He was working in Mechanical Engineering Department, Civil and Environmental Engineering Department, National University of Singapore as a research fellow. He joined Bryden Centre, Letterkenny Institute of Technology, Ireland since February 2019 as a postdoctoral fellow. His research interests includes marine renewable energy, engineering design and techno economic analysis. Narendran Kumar received his Ph.D. in ocean engineering from Indian Institute of Technology Madras, India. He was working in Mechanical Engineering Department, Civil and Environmental Engineering Department, National University of Singapore as a research fellow. He joined Bryden Centre, Letterkenny Institute of Technology, Ireland since February 2019 as a postdoctoral fellow. His research interests includes marine renewable energy, engineering design and techno economic analysis. [35] UK Office of National Statisitics. (August 2019). Inflation and Price Indices. [Online]. Available: https://www.ons.gov.uk/economy/inflationandpriceindices [36] R5 climate change 2014: Mitigation of climate change, Intergovernmental Panel on Climate Change, A Cambridge University Press , 2014. [37] Carbon Market Watch, Beyond the EU ETS: Strengthening Europe's Carbon Market Through National Action, 2017. [38] F. C. AUTHOR CONTRIBUTIONS [25] Enabling Future Arrays in Tidal (EnFAIT) LCOE and Financial Models, Nova Innovation Ltd., 2019. The first and second authors produced results and wrote the paper. The third and fourth authors helped in analysing [26] Nova Innovation Tidal Array Shetland (www.grebeproject.eu), Nova Innovation Ltd. 15 International Journal of Materials, Mechanics and Manufacturing, Vol. 8, No. 1, February 2020 Moore and D. B. Diaz, "Temperature impacts on economic growth warrant stringent mitigation policy," Nature Climate Change, vol. 5, pp. 127-131, 2015. mari techno economic analysis. [39] G. Smart and M. Noonan, "Tidal stream and wave energy cost reduction and industrial benefit," Catapult Offshore Renewable Energy, 2018. Christopher McCallum is a postdoctoral fellow at Letterkenny Institute of Technology working with the Bryden Centre, since February 2019. He obtained his PhD in chemical engineering from Queen’s University Belfast where he also obtained a BEng and MPhil. He is currently working in bioenergy, lifecycle assessment and techno economic analysis. 0] European Commission, Market Study on Ocean Energy, 2018. [41] Climate Action Network Europe, Phase-out 2020; Monitoring Europe's Fossil Fuel Subsidies, 2017. [42] International Energy Agency. World electricity demand. [Online]. Available: https://web.archive.org/web/20150522054948/http://www.iea.org/tec hinitiatives/renewableenergy/ocean/ [43] M. Holland and M. Howley, "Renewable electricity in Ireland 2015." SEAI Report, 2016. Gary James Britton is a research assistant and technician at Letterkenny Institute of Technology (LYIT), working within the Bryden Centre. He received his HNC and BEng civil engineering from LYIT, his BEng (Hons) from the University of Edinburgh, and MSc Industrial Management from Edinburgh Napier University. He is currently researching best practice in marine governance and policy development. p , [44] EvoPod E35. [Online]. Available: https://openei.org/wiki/MHK_Technologies/Evopod_E35 ] [ ] https://openei.org/wiki/MHK_Technologies/Evopod_E35 [45] Irish Marine Institute Northeast Atlantic Model. Marine Institute. [Online]. Available: https://erddap.marine.ie/erddap/griddap/IMI_NEATL.graph?sea_surf ace_x_velocity[(2019-09-25T00:00:00Z):(2019-10- 06T00:00:00Z)][(55.13125)][(- 7.518750000000001)]&.draw=linesAndMarkers&.vars=time%7Csea _surface_x_velocity%7C&.marker=5%7C5&.color=0x000000&.col [46] IEA World Energy Outlook. [Online]. Available: https://www.thepriceofcarbon.com/2018/11/international-energy- agency-2018-world-energy-outlook-sustainable-development-with- well-below-2-c-in-surface-warming/ John Doran is the regional manager of the Bryden Centre at Letterkenny of Institute of Technology and a principal researcher for TAOIDE a H2020 funded research project on Tidal Energy. He obtained his PhD in mechanical engineering from Queen’s University of Belfast where he is currently a Visiting Research Professor in the School of Chemistry and Chemical Engineering. His research interests are in the fields of renewable energy, techno-economic analysis and social acceptance of marine and bio- [47] Electricity market price (2). [Online]. Available: https://www.sseairtricity.com/ie/home/products/product-details?p=54 [48] Electricity market price (1). [Online]. Available: https://www.bonkers.ie/compare-gas-electricity-prices/electric- ireland/LEGJH2/standard-domestic-electricity/ [49] LCOE of Nuclear energy. [Online]. Available: https://atb.nrel.gov/electricity/2018/index.html?t=cn [50] IRENA, Renewable Power Generation Costs in 2018, 2019. [51] European Commission. EU Emissions Trading System (EU ETS). [Online]. Available: https://ec.europa.eu/clima/policies/ets_en 16
https://openalex.org/W4235527547	https://www.qeios.com/read/B0EAQC/pdf	English	null	Mucocutaneous Candidiasis	Definitions	2,020	cc-by	75	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Mucocutaneous Candidiasis National Cancer Institute National Cancer Institute Qeios ID: B0EAQC · https://doi.org/10.32388/B0EAQC Source National Cancer Institute. Mucocutaneous Candidiasis. NCI Thesaurus. Code C35576. A fungal infection of the skin, nails, oral and vaginal mucosal sites caused by species of the genus Candida. It manifests with white discoloration of the tongue and swelling, redness, and tenderness of the nails. Qeios ID: B0EAQC · https://doi.org/10.32388/B0EAQC 1/1
https://openalex.org/W3042529793	https://digitalcommons.chapman.edu/cgi/viewcontent.cgi?article=1097&context=art_articles	English	null	Museums as Realms of (dis)Enchantment	Latin American & latinx visual culture	2,020	cc-by-sa	3,246	Museums as Realms of (dis)Enchantment Museums as Realms of (dis)Enchantment Amy Buono Chapman University, buono@chapman.edu Follow this and additional works at: https://digitalcommons.chapman.edu/art_articles Part of the Latin American History Commons, Latin American Languages and Societies Commons, Museum Studies Commons, and the Other History of Art, Architecture, and Archaeology Commons Art Faculty Articles and Research Recommended Citation Recommended Citation Amy Buono, “Museums, and Other Realms of (dis)Enchantment,” Latin American and Latinx Visual Culture, Dialogues section, Special eds. Tatiana Flores and Harper Montgomery, Vol. 2, No. 2, April 2020, 79-82. https://doi.org/10.1525/lavc.2020.220007 This Article is brought to you for free and open access by the Art at Chapman University Digital Commons. It has been accepted for inclusion in Art Faculty Articles and Research by an authorized administrator of Chapman University Digital Commons. For more information, please contact laughtin@chapman.edu. Comments Comments This article was originally published in Latin American and Latinx Visual Culture, volume 2, issue 2, in 2020. https://doi.org/10.1525/lavc.2020.220007 This article is available at Chapman University Digital Commons: https://digitalcommons.chapman.edu/art_articles/ 97 Copyright The Regents of the University of California Museums as Realms of (dis)Enchantment On the night of September , , a blaze swept through Brazil’s national museum, Museu Nacional, in Rio de Janeiro, destroying not only the colonial building, portions of which dated to the sixteenth century, but roughly percent of the million objects in its holdings. This was Brazil’s greatest encyclopedic museum, incorporating (among many others) collections of natural history, anthro- pology, archaeology, and art, thus forming the most com- prehensive museum collection in the nation. Along with its many unique and irreplaceable collections, the Museu Nacional was also home to the country’s oldest Indigenous Brazilian and Afro-Brazilian materials (fig. ). the Getúlio Vargas regime, the collection and their manage- ment became part of the university system. Budding and seasoned anthropologists, archaeologists, paleontologists, zoologists, botanists, linguists, and other specialists work with objects and specimens, teaching, studying, researching, documenting, curating, and conserving. Integrated into the public university system, it has long served as a space where students, staff and faculty worked on and in the presence of mummies, meteorites, funerary urns, insects, paintings, sculpture, and featherwork, to name just a few of the muse- um’s myriad objects. The exhibition spaces of the museum were physically and conceptually attached to offices, class- rooms, research labs, archives, and to Brazil’s oldest research library. Through its long history, the museum has been a living point of connection between the historic and con- temporary spaces of Brazil, binding communities within and well beyond Rio, from Amazonian and quilombola (Afro-descendant) communities to scholars, artists, and in- tellectuals from throughout the country and world. Downloaded from http://online.ucpress.edu/lalvc/article-pdf/2/2/79/405309/lavc_2_2_079.pdf by guest on 16 July 2020 With origins in the nineteenth century, the museum de- veloped from the Portuguese Royal collections and libraries brought to Rio in , after the court fled the Napoleonic invasion of Portugal at the end of . Dom João VI do- nated the natural history specimens that constituted the core collections of the museum when it was formally estab- lished in Rio as the Royal Museum in . The Museu Na- cional, as it was renamed, expanded over the next two centuries through expeditions, donations, excavations, and strategic acquisitions and exchanges. Latin American and Latinx Visual Culture, Vol. , Number , pp. –. Electronic ISSN: -© by The Regents of the University of California. All rights reserved. Please direct all requests for permission to photocopy or reproduce article content through the University of California Press’s Reprints and Permissions web page, https://www.ucpress.edu/journals/reprints-permissions. DOI: https://doi.org/./lavc.. Copyright Thus, from the time of its foundation, the Museu Nacional was not only one of Brazil’s oldest and most venerable institutions but also among the most central public establishments devoted to knowledge production in the biological and social sciences. The museum sits at some distance from the city’s more ex- clusive beach neighborhoods of Ipanema, Copacabana, and Leblon. Located in the northern São Cristóvão neighbor- hood, within the majestic (and now partially abandoned) Quinta da Boa Vista Park, the Museu Nacional—along with the park’s Zoological Gardens—has been a destination point and memory marker since the late nineteenth century. Until the fire, schoolchildren and families strolling through the park and zoo, together with throngs of tourists from across Brazil and the world, visited the museum. Downloaded from http://online.ucpress.edu/lalvc/article-pdf/2/2/79/405309/lavc_2_2_079.pdf by guest on 16 July 2020 The world watched in horror as the fire’s progress was streamed live on television. The overall list of materials lost is breathtaking. Among those that perished—and this is only a token list—were innumerable holotype biological specimens, fossils, dinosaur bones, Egyptian and Greco- Roman artifacts, pre-Columbian ceramics and textiles, fres- coes from Pompei, and the oldest human skeleton in the Americas. The library included one of the largest collections of anthropological literature in Latin America. These losses are appalling not only due to the vast numbers of things destroyed and the uniqueness of so many individual items, which could be the case if any other museum suffered a sim- ilar catastrophe, but also because the destruction included entire corpuses of material that existed nowhere else in the world and therefore have vanished entirely. Although only a small fraction of the millions of arti- facts destroyed in a single night, the loss of Brazil’s Indig- enous material culture is immeasurable, including unique collections of Brazilian featherwork, ceramics, and bas- ketry. Current estimates suggest that the fire destroyed over forty thousand artifacts related to Brazil’s Indigenous Perhaps less known to those outside Brazil, the Museu Nacional is also a part of the Federal University of Rio de Janeiro (UFRJ), a “museum-campus” that serves as a preem- inent institution for teaching and research. In , during 79 Dialogues FIGURE 1. Fire, Museu Nacional/ Universidade Federal do Rio de Janeiro, Rio de Janeiro, September , . Photo by Felipe Milanez. Wikimedia Commons, licensed under the Creative Commons Attribution-Share Alike .International license, commons.wikimedia.org/wiki/File:Fire_-_Museu_Nacional_.jpg (accessed March , ). FIGURE 1. . For losses, see Ana Lucia Araujo, “The Death of Brazil’s National Museum,” American Historical Review , no. (April ): ; For a historic look at the catalogs of anthropological and ethnographic holdings of the National Museum, see Crenivaldo Regis Veloso Júnior, “Índice de objetos, índice de histórias: o catálogo geral das coleções de antropologia e etnografia do Museu Nacional,” Ventilando Acervos, special issue, no. (September ): –. Special thanks Tatiana Flores and Harper Montgomery for creating a forum for this discussion, and to Flávia Nogueira de Sá, Roberto Conduru, and Wendy Salmond for valuable conversations on museums in peril. Special thanks Tatiana Flores and Harper Montgomery for creating a forum for this discussion, and to Flávia Nogueira de Sá, Roberto Conduru, and Wendy Salmond for valuable conversations on museums in peril. . For losses, see Ana Lucia Araujo, “The Death of Brazil’s National Museum,” American Historical Review , no. (April ): ; For a historic look at the catalogs of anthropological and ethnographic holdings of the National Museum, see Crenivaldo Regis Veloso Júnior, “Índice de objetos, índice de histórias: o catálogo geral das coleções de antropologia e etnografia do Museu Nacional,” Ventilando Acervos, special issue, no. (September ): –. Copyright Fire, Museu Nacional/ Universidade Federal do Rio de Janeiro, Rio de Janeiro, September , . Photo by Felipe Milanez. Wikimedia Commons, licensed under the Creative Commons Attribution-Share Alike .International license, commons.wikimedia.org/wiki/File:Fire_-_Museu_Nacional_.jpg (accessed March , ). populations alone.1 In quantity, historical depth, scope and range, this was a corpus of Brazilian Indigenous ma- terial and intangible heritage unequalled by any collections still extant in the world. significant: these were the oldest and best documented col- lections in Brazil, dating from the time of slavery and giving testimony to everyday life, religious life, and histories of en- slavement and repression on a systemic level. While an un- known amount of related material survives elsewhere, it lies piecemeal in a variety of institutions across the country, some in hard to access police and legal medicine collections, others in more accessible state museums and university col- lections. The fragmentation of the collections makes their public contemplation and scholarly study logistically and practically problematic, if not impossible, to study. It is also very important to recognize that the losses were not only of the material objects but also field notes and con- textual materials, such as audio recordings of languages ei- ther no longer spoken or in decline. All of these materials were of crucial importance for contemporary Indigenous groups across Brazil, who used them to trace relationships to the past and to constitute memory. In a similar manner, the Museu Nacional’s Africana collections, as well as objects produced by Afro-descendants in Brazil, were particularly As the largest and oldest collection of historical and cultural artifacts in Latin America, this extraordinary loss has global consequences for Indigenous rights, the histories and collective memories of entire populations. It also ren- ders future scholarship that might have been based upon these collections, including my own, now impossible. Like the Africana materials, what does survive of the historical and material record of Indigenous cultures of Brazil from the sixteenth through eighteenth centuries now exists only in fragmented and scattered form throughout archives, museums, and other institutional collections across the LATIN AMERICAN AND LATINX VISUAL CULTURE 80 The same financial and political tinder-by-neglect is re- peated in too many cities across Latin America and the Global South to name. Copyright In fact, although the Museu Nacional fire was certainly the most catastrophic in Brazil, it was by no means the first or only such disaster. Brazil has witnessed multiple museum fires that signal consistent problems with both infrastructure and staff oversight, resulting in devastating effects for the cultural and scientific sector and its valorization in Brazil. Most notable for readers in the art world, no doubt, is the devastating fire in Rio de Janeiro’s Museum of Modern Art (MAM), which destroyed percent of the famed modernist collections. In , a fire broke out in one of Brazil’s (and Latin America’s) most important biological and research centers, the Butantan Institute in São Paulo, destroying laboratories and the institute’s entire collection of ,snake specimens. In , a fire ravaged the much-beloved Museum of the Portuguese Language, also in São Paulo. These tragedies point to systemic negligence in the public sector for the sustaining of scientific and cultural heritage. Private safeguarding of artistic works has proved just as insecure, as was seen in the fire in a private home in Rio de Janeiro, where two thousand works by contemporary Brazilian artist Hélio Oiticica (–) were destroyed. Downloaded from http://online.ucpress.edu/lalvc/article-pdf/2/2/79/405309/lavc_2_2_079.pdf by guest on 16 July 2020 ownloaded from http://online.ucpress.edu/lalvc/article-pdf/2/2/79/405309/lavc_2_2_079.pdf by guest on 16 July 2020 Within the United States, art museums are facing de- mands for reckoning in the wake of the opioid epidemic, the #MeToo movement, and the Trump presidency. Increas- ingly, the ethical stakes of museums and cultural heritage sites are being raised; their powers to display, educate, and define history are being challenged; and, in a closely related area, the power of monuments and memorials to endorse or stage violence, as well as to assuage, heal, and embody grief, are being questioned. Museum publics are increasingly ask- ing institutions to face up to their fraught financial donor base and colonialist legacies, hiring policies, and exhibition and acquisition practices, the mechanics of which were for so long discreetly and deliberately hidden from view. The context of these dilemmas is both long-standing and intrac- table: the Eurocentric, patrician roots of art history and its founding subjects, methods, and practitioners; the gutting of public funding for the arts and humanities across the Amer- icas; and the power of the global contemporary art market to construct canons. . Fabio Serapião, “Curto em ar-condicionado causou fogo que destruiu Museu Nacional, diz perícia” [Short-circuit in the air conditioner caused the fire that destroyed the National Museum, says criminal investigation], Estadão (São Paulo), March , , https://brasil.estadao.com.br/noticias/ rio-de-janeiro,curto-em-ar-condicionado-causou-fogo-que-destruiu-museu- nacional-diz-pericia,. Copyright The scale and dramatic nature of the losses at Museu Nacional highlight the importance of safeguarding ethnographic collections, which are not only of importance to historians, but also vital for writing about modern and contemporary visual and material cul- ture in Latin America. As Latin American and Latinx Visual Culture readers, and as writers, art makers, curators, and cultural historians, we might do well to consider that ethnographic, historical, and archaeological collections help give voice to diverse histories and give testimony to diverse artistic practices. As art historian Ruth Philips has so poignantly stated: “Historical objects are witnesses, things that were there, then. They bear their makers’ marks in their weaves, textures and shapes, and have a compelling agency to cause people living in the present to enunciate their relationships to the past.”3 world, mostly in Europe. Long before modern-day museum tragedies, Brazil (like so many other nations in the Global South) had already been historically dispossessed of its material and intangible heritage through colonialism. As we have now seen, what remains in domestic collections, whether in Museu Nacional or in countless other places in the Global South, exists in perilous states of neglect or dis- repair from severe budget shortages. world, mostly in Europe. Long before modern-day museum tragedies, Brazil (like so many other nations in the Global South) had already been historically dispossessed of its material and intangible heritage through colonialism. As we have now seen, what remains in domestic collections, whether in Museu Nacional or in countless other places in the Global South, exists in perilous states of neglect or dis- repair from severe budget shortages. The Museu Nacional had experienced drastic budget cuts in recent years that made its compliance with proper safety standards impossible. As detailed in an April report by the Federal Police in Brazil, an overheated air conditioning system was believed to be the cause of the fire.2 Because of an inactive smoke detector system, malfunctioning sprin- klers, missing water hoses, open fire doors, and faulty secu- rity cameras, the inferno could not be contained. Thus, we know that the disaster was also completely preventable. The financial precariousness of the public sector in Rio de Janeiro, with its budget cuts, delayed infrastructural im- provements, poor oversight, and general neglect, was ulti- mately the kindling upon which the blaze was ignited. . Ruth B. Phillips, “Re-placing Objects: Historical Practices for the Second Museum Age,” Canadian Historical Review , no. (March ): . Copyright How do museums today begin to rec- oncile these legacies with their new realities—with intersec- tional identities, with social media, with the visual, material, and artistic worlds of local, global, and transnational com- munities? However important these ethical issues related to museums and the behavior of their donors and staff are— and I in no way dispute that importance—they are of a fun- damentally different order than the truly existential nature 81 Dialogues ethnological, historical, biological, and artistic works, analo- gous to losing the Smithsonian Institution or the Metropol- itan Museum of Art. In contrast to Notre Dame, however, the Museu Nacional fire elicited a briefer and less impas- sioned international response, and vastly smaller philan- thropic impulses. These losses push the conversation from one dominated by markets, values, canons, and identity politics—the issues swirling around US institutions—into the terrain of cultural memory, the relevance of the past to the present, and the construction of identities today. Fur- thermore, these collections help us both understand and question the very objects that we study and what we con- sider of cultural relevance. Rather than comparing what happened in Rio to either Notre Dame or to the current travails of the Met, perhaps the proper analogy is to the looming threat of species extinction writ large, also attribut- able to societal neglect. To push the analogy still further, the extinction of the entirety of the extant material and intangi- ble culture of many Indigenous populations of Brazil in a single night changes, diminishes, and imperils the larger so- cial and cultural ecosystem. Officials at the Museu Nacional are predicting that parts of the palace building will be recon- structed and reopened to the public in , in honor of the bicentenary of Brazilian Independence. Until then, portions of the surviving archives and collections will be exhibited in Rio and Brasilia, in part to raise private and public monies and awareness for Museu Nacional’s future. The museum is also collecting donations from around the world in its ef- forts to rebuild. Even those of us working and teaching in the Global North should not be complacent about the dura- bility of our own institutions, or of our government’s role in supporting them. The financial, cultural, and political eco- systems that sustain museums are fragile and need our im- mediate attention. of what has transpired in Brazil. . Vincent Noce, “Vote on Icom’s New Museum Definition Postponed,” Art Newspaper, September , , www.theartnewspaper.com/news/icom- kyoto. Copyright Do not governments, econ- omies, and societies have an obligation to take the necessary steps to ensure that the objects in these collections continue to exist? Indeed, the very definition of what a museum is has now become a source of confusion. Prior to the International Council of Museums (ICOM) Twenty-fifth General As- sembly in Kyoto this past September , ICOM held that “A museum is a non-profit, permanent institution, . . . which acquires, conserves, researches, communicates and ex- hibits the tangible and intangible heritage of humanity and its environment for the purposes of education, study and en- joyment.” ICOM’s recently drafted new definition began with a declaration that museums are “democratizing, inclu- sive and polyphonic spaces for critical dialogue about the past and future.”4 France was quick to critique the proposal, stating that the definition was too ideological, leading to a prolonged and unresolved debate which itself deserves rich study. After hours of deliberation, a consensus of non- consensus led to a postponement in formally adopting the revised definition, with France winning the support of out of national and regional delegations, and no date in sight for a revised definition or new vote. What is significant, in the context of this essay, is that ICOM’s new museum definition, despite the jargon, was trying to em- phasize the significance and role of objects and collections that lie in limbo outside dominant cultural patrimonies. This is the disenchanted realm—poorly funded and politi- cally marginalized—in which so many museums and re- search institutions of ethnology and culture in the Global South find themselves. In a larger framework, cultural heritage disasters bring even more questions to the surface in relation to museums and international spaces of cultural display, because they open up the stage for large-scale reckoning about the role of cultural sites for the modern nation-state. The fire that ripped through Notre Dame Cathedral in Paris in April received extraordinary international attention and led immediately to fundraising on a massive scale, no doubt spurred on by the iconic status of Notre-Dame de Paris not only as a marker of French national identity but also as an international tourist site. In a Latin American context, the Museu Nacional fire resulted in a near-total cultural loss on an almost unimaginable scale, including the archaeological, Amy Buono Chapman University ABOUT THE AUTHOR Amy Buono is assistant professor of art history at Chap- man University. Her interests include Indigenous and Afro- Brazilian artistic practices, material and intangible heritage studies, and colonialism and ethnopolitics. She is authoring Tupinambá Feathercraft in the Brazilian Atlantic (University of Pennsylvania Press), and coediting A Cultural History of Color in the Renaissance (Bloomsbury Press). LATIN AMERICAN AND LATINX VISUAL CULTURE 82
https://openalex.org/W4287377064	https://gmd.copernicus.org/articles/15/8439/2022/gmd-15-8439-2022.pdf	English	null	Development of an LSTM-Broadcasting deep-learning framework for regional air pollution forecast improvement	null	2,022	cc-by	9,784	Correspondence: Xingcheng Lu (xingchenglu2011@gmail.com) Correspondence: Xingcheng Lu (xingchenglu2011@gmail.com) Received: 24 June 2022 – Discussion started: 25 July 2022 evised: 10 October 2022 – Accepted: 21 October 2022 – Published: 21 November 2022 casting framework can be extended for air pollution forecast- ing in other regions of interest. casting framework can be extended for air pollution forecast- ing in other regions of interest. Abstract. Deep-learning frameworks can effectively fore- cast the air pollution data for individual stations by decod- ing time series data. However, most of the existing time- series-based deep-learning models use ofﬂine spatial interpo- lation strategies and thus cannot reliably project the station- based forecast to the spatial region of interest. In this study, the station-based long short-term memory (LSTM) technique was extended for spatial air quality forecasting by com- bining a novel deep-learning layer, termed the broadcasting layer, which incorporates a learnable weight decay parame- ter designed for point-to-area extension. Unlike most exist- ing deep-learning-based methods that isolate the interpola- tion from the model training process, the proposed end-to- end LSTM broadcasting framework can consider the tempo- ral characteristics of the time series and spatial relationships among different stations. To validate the proposed deep- learning framework, PM2.5 and O3 forecasts for the next 48 h were obtained using 3D chemical transport model simulation results and ground observation data as the inputs. The root mean square error associated with the proposed framework was 40 % and 20 % lower than those of the Weather Research and Forecasting–Community Multiscale Air Quality model and an ofﬂine combination of the deep-learning and spatial interpolation methods, respectively. The novel LSTM broad- Model description paper Model description paper Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 © Author(s) 2022. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Aggravated by industrialization and economic development, air pollution has received increasing attention in recent years. Fine suspended particulate matter (PM2.5) and ozone (O3), as prominent secondary air pollutants, can adversely inﬂu- ence human health and society (e.g., poor visibility may lead to trafﬁc delays). Accurately forecasting the levels of these two pollutants at the regional scale can provide the infor- mation necessary for relevant parties and the general pub- lic to address the threats posed by air pollution and imple- ment appropriate counteractive measures (e.g., emission re- duction or curtailment of unnecessary outdoor activities). To this end, several forecasting models have been developed. Three-dimensional (3D) numerical models have been ap- plied worldwide to obtain regional forecasts of air pollution levels. Based on historical emission inventories and physi- cal or chemical parameterization schemes, these numerical models simulate the formation, transmission, and destruction of air pollutants and forecast the regional air quality over a long prediction horizon (e.g., 120 h). However, the fore- Haochen Sun1,2, Jimmy C. H. Fung1,3,4, Yiang Chen3, Zhenning Li3, Dehao Yuan5, Wanying Chen3, and Xingcheng Lu6 1Department of Mathematics, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China 2Department of Computer Science and Engineering, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China y g g 3Division of Environment and Sustainability, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China Atmospheric Research Center, HKUST Fok Ying Tung Research Institute, Guangzhou, China Department of Computer Science, University of Maryland, College Park, Maryland, USA D t t f G h d R M t Chi U i it f H K Sh Ti 4Atmospheric Research Center, HKUST Fok Ying Tung Research Institute, Guangzhou, China 5Department of Computer Science, University of Maryland, College Park, Maryland, USA 6Department of Geography and Resource Management, Chinese University of Hong Kong, Sha Tin, Hong Kong SAR, China Atmospheric Research Center, HKUST Fok Ying Tung Research Institute, Guangzhou, China 5Department of Computer Science, University of Maryland, College Park, Maryland, USA 6Department of Geography and Resource Management, Chinese University of Hong Kong, Sha T 5Department of Computer Science, University of Maryland, College Park, Maryland, USA 6Department of Geography and Resource Management, Chinese University of Hong Kong, Sha Tin, Hong Kong SAR, China p p y y g y 6Department of Geography and Resource Management, Chinese University of Hong Kong, Sha Tin, Hong Kong SAR, China H. Sun et al.: Development of an LSTM broadcasting deep-learning framework In contrast, if the output and input are in the same temporal domain, then bidirectional LSTMs (Schuster and Paliwal, 1997) can be used. However, because the air quality depends on many factors other than historical values, the cor- relation between the future air pollution conditions and past ground observations is weak, especially in the case of large time lags, and the effective prediction horizon is constrained, typically to no more than 24 h (Bui et al., 2018; Li et al., 2020; Qin et al., 2019). Moreover, most of the abovemen- tioned studies focused on obtaining accurate forecasts for speciﬁc ground monitoring stations, and thus, deep-learning models that can forecast the air quality on a regional scale are lacking. With advances in deep-learning techniques, sophisticated architectures have been developed to incorporate spatiotem- poral correlations for regional air pollution forecasting. Pak et al. (2020) developed a spatiotemporal convolutional neu- ral network (CNN) LSTM network to predict the next day’s daily average PM2.5 concentration in Beijing, China. Qi et al. (2019) applied a graph neural network (GNN) to take into account the spatial correlations of multiple ground mon- itoring stations in the Jing-Jin-Ji region, China, and enhance the forecast accuracy at these stations. Han et al. (2021) proposed a MasterGNN structure to explore the spatiotem- poral information and forecast the air quality and weather at a given set of ground monitoring stations. However, the forecasts obtained by these architectures are restricted to a city-wide average or ﬁxed set of ground monitoring stations. Therefore, these models cannot be applied for regional fore- casting and predicting the pollutant concentrations at speciﬁc locations. Several studies have attempted to develop deep-learning- based models to obtain regional air pollution forecasts by combining ground observation data and numerical model re- sults through spatial interpolation methods. For example, the LSTM 3D-variational assimilation (3D-VAR) model (X. Lu et al., 2021) combines ground observations and 3D numerical models with the LSTM and 3D-VAR data assimilation tech- niques. This model can achieve accurate regional forecasts with a prediction horizon of 24 h; however, substantial com- putation power is required (1 h of computing time is required to obtain a 24 h forecast using two AMD EPYC 32 core processors). H. Sun et al.: Development of an LSTM broadcasting deep-learning framework H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8440 casts provided by such numerical models are prone to sig- niﬁcant errors, owing to the uncertainty and hysteresis of the emission inventories and bias in the simpliﬁed parameteriza- tion schemes and meteorological simulations (Gilliam et al., 2015; Holnicki and Nahorski, 2015; Tang et al., 2009). training. Sayeed et al. (2021a, b) and H. Lu et al. (2021) im- proved the accuracy of CMAQ forecast by ground observa- tions using deep-learning techniques, but the improvements were still limited to the ground monitor stations rather than the whole region. On the other hand, Lyu et al. (2019) devel- oped an ensemble model that combines the chemical trans- port models and the ground observations, but the regional forecast still depends on the traditional kriging method. Sim- ilarly, Bi et al. (2022) used the random forest algorithm to calibrate the numerical simulation based on chemical trans- port models. However, this model also relies on interpolation methods (e.g., ordinary kriging). Moreover, the parameters needed for the spatial interpolation schemes are not included in the training process when constructing the deep-learning framework, and the spatial correlations between different sta- tions cannot be introduced as a constraint (Zhou et al., 2020; Hähnel et al., 2020). ; , ; g , ) In recent years, machine learning algorithms have been widely applied to predict air quality (Janarthanan et al., 2021; Mao et al., 2021; Samal et al., 2021; Wu and Lin, 2019; Kim et al., 2019). As the future air quality is correlated with his- torical values, ground observations can be input to machine learning models to obtain forecasts. The forecasting process can be formulated as a time series task, with the input and training targets being hourly ground observations. Most stud- ies (Ayturan et al., 2018; Huang and Kuo, 2018; Tsai et al., 2018; Zhao et al., 2019) have applied long short-term mem- ory (LSTM; Hochreiter and Schmidhuber, 1997) frameworks – a variant of recurrent neural networks (RNNs) and a state- of-the-art deep-learning technique – to accomplish the time series tasks. Different LSTM frameworks (or other variants of RNNs) can be applied for different time series tasks. For example, if the output temporally postdates the input, then LSTM encoder–decoders (Sutskever et al., 2014) can be ap- plied. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.1 Data Ground observation data and WRF-CMAQ results from 2015 to 2021 in the GBA and surrounding regions (21.6–24.5◦N, 111.2–115.6◦E; referred to as the target region hereinafter), with a spatial resolution of 3 km, were extracted. Details of the model domain coverage and conﬁguration of the pa- rameterization schemes can be found in the work of Lu et al. (2015, 2018). The proposed model was built using the data from 2015 to 2020 (training period) and tested using the data from 2021 (testing period) to ensure temporal general- izability. y The ground observation data of air pollutant concentra- tions from several ground monitoring stations distributed across the region were used to partially represent the spa- tial distribution of the pollutants. In the training period, the ground monitoring stations with at least 90 % valid records (2015 to 2020) for both the target species, PM2.5 and O3, were selected as the training target stations. The same crite- rion was applied to select the testing target stations (2021) from the testing period. The ground monitoring stations with at least 95 % valid records for both the target species in both periods were selected as the source stations (denoted S), and the corresponding data were used as the ground truth for model training. Given these criteria, each source station was automatically a target station in both periods. As shown in Fig. 1, the criteria yielded 32 source stations, 90 training target stations, and 61 testing target stations. A total of 21 testing target stations that were neither source stations nor training target stations were used as the primary benchmark for quantitatively evaluating the results (referred to as bench- mark stations hereinafter; see Sect. 3). As the model did not encounter the data of these stations during training, satisfac- tory performances for these stations were expected to be in- dicative of spatial and temporal generalizability. Figure 1. Locations of the source and target stations (including the benchmark stations). For each day, d, the proposed model took the following inputs: 1. The hourly ground observation data at the source sta- tions from 09:00 on day d −3 to 08:00 on day d (both ends inclusive; 72 time steps). 2. The hourly WRF-CMAQ data at the locations of in- terest from 09:00 on day d to 08:00 on day d + 2 (48 time steps). 2 Method Figure 1. Locations of the source and target stations (including the benchmark stations). 2.1 Data Note that the WRF-CMAQ model can also work as a forecasting model, and therefore, these data are available and can be used before the beginning of the forecast. The model then outputs the hourly forecast of PM2.5 and O3 concentrations at the locations of interest from 09:00 on day d to 08:00 on day d + 2 (48 time steps). H. Sun et al.: Development of an LSTM broadcasting deep-learning framework The LSTM Weather Research and Forecasting– Community Multiscale Air Quality (WRF-CMAQ) model (Sun et al., 2021) combines ground observations and WRF- CMAQ models to achieve highly accurate regional forecasts with a prediction horizon of 48 h. However, the system re- quires a customized spatial correction (SC) scheme (e.g., nu- merical interpolation methods), and the accuracy at general locations is lower than that at the ground monitoring sta- tions, the data of which are used for deep-learning model In this study, to obtain accurate forecasts for a longer pe- riod and consider the spatial characteristics, an end-to-end deep-learning model that can forecast the regional air pollu- tion values for the next 48 h (starting at 09:00 LT (local time hereinafter, unless otherwise indicated) on each day) was developed. A novel broadcasting layer was incorporated in the model to introduce a spatial interpolation parameter into the deep-learning model training, and various LSTM-based deep-learning structures were used to support the end-to-end computation. The proposed model, which combines ground observation data and WRF-CMAQ numerical models as the inputs, can forecast the air quality for any location within a region. In tests pertaining to China’s Guangdong–Hong Kong–Macau Greater Bay Area (GBA) and the surrounding regions, the proposed model outperformed the CMAQ model and an of- ﬂine combination of the LSTM and SC methods in terms of the forecasting accuracy. Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8441 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8442 Table 1. Overall performance values for the PM2.5 forecast. Note: mean bias error (MBE), mean absolute value (MAE), root mean square error (RMSE), symmetric mean absolute percentage error (SMAPE), and Pearson correlation coefﬁcient (R) are shown. NN is for neural network, and IDW is for inverse distance weighting. Table 1. Overall performance values for the PM2.5 forecast. Note: mean bias error (MBE), mean absolute value (MAE), root mean square error (RMSE), symmetric mean absolute percentage error (SMAPE), and Pearson correlation coefﬁcient (R) are shown. NN is for neural network, and IDW is for inverse distance weighting. Time lags Model MBE (µg m−3) MAE (µg m−3) RMSE (µg m−3) SMAPE (%) R 0–23 h CMAQ 0.37 13 22 59 0.56 SC (NN) 0.29 12 19 63 0.50 SC (IDW) 0.30 11 18 54 0.51 SC (kriging) 0.20 10 17 56 0.50 Broadcasting 0.026 6.5 9.2 41 0.74 24–47 h CMAQ 0.40 13 22 60 0.55 SC (NN) 0.35 13 20 65 0.47 SC (IDW) 0.37 12 19 57 0.49 SC (kriging) 0.27 11 17 59 0.48 Broadcasting 0.097 7.2 9.8 45 0.70 The boldface values represent the highest performance for each period and metric. The boldface values represent the highest performance for each period and metric. of forecasting regional air quality to address the asymmetry between the availability of information at a limited number of locations and the need to predict the air quality for a com- plete region. For example, Sun et al. (2021) used the inverse distance weight to calibrate the difference between the deep- learning forecast and CMAQ forecast. Ma et al. (2019) pro- posed a geolayer to ﬁlter the data used for interpolation and combined this layer with LSTM-based models. However, in such ofﬂine combinations, the hidden connection among dif- ferent stations cannot be included in the deep-learning model building procedure. In addition, ofﬂine numerical interpola- tion methods do not have degrees of freedom. Several meth- ods of this type are not differentiable (e.g., nearest interpola- tion) or may incur numerical problems (e.g., inverse distance interpolation). Therefore, in order to better reveal the spatial characteristics of the air pollutant concentration ﬁeld, we in- troduced a novel broadcasting layer to enable the end-to-end deep-learning model for a regional air quality forecast. H. Sun et al.: Development of an LSTM broadcasting deep-learning framework vations) are input to the encoder LSTM to generate the en- coding vector of the input time series, h. Subsequently, h is passed to the decoder LSTM with Tout time steps, where Tout = 48 h is the length of the prediction. The hidden states of each time step n h(t)oTout t=1 are subsequently passed to a dense layer, activated by the rectiﬁed linear unit function (ReLU), where ReLU(z) = max(0, z), and applied to the output of the dense layer in an element-wise manner. In this study, the LSTM encoder–decoder associated with each source station, regardless of the number of ground ob- servation features, had an encoding dimension of 64. The output dimension of the dense layer was set as 64 (for each time step). The mathematical and technical details of encoder LSTM and decoder LSTM can be found in Sects. S1 and S2 in the Supplement. 2.3 Bidirectional LSTM In this framework, each ground observation station s ∈S is associated with a learnable weight decay parameter θs ≥ 0 (which can be trained while building the deep-learning model). At any target location t, when the input {Ys}s∈S is received from the source stations, the output of the layer at location t is computed as a weighted sum as follows: Because several inputs and intermediate outputs (e.g., the WRF-CMAQ input at the locations of interest and outputs of the LSTM encoder–decoders) were in the same temporal space as that of the ﬁnal output, bidirectional LSTMs were applied to extract the information embedded in these time series. A bidirectional LSTM contains two ordinary LSTM structures. When a time series x(t) T t=1 is input to a bidirec- tional LSTM, then it is passed to the two LSTM layers in the ordinary and reversed temporal orders, and the two hidden states of each time step are concatenated as the output of the bidirectional LSTM. More details regarding the bidirectional LSTM (as a variant of bidirectional RNNs) can be found in the work of Schuster and Paliwal (1997). Yt ′ = X s∈S ws,tYs, (1) (1) with the weights calculated as ws,t = exp(−θsd (s, t)) P s′∈S exp(−θs′d (s′, t)), (2) (2) where d(·, ·) denotes the distance between two locations, measured in kilometers. The computation of the weights is similar to that implemented in the conventional softmax function. Therefore, the weights sum to 1 for each location t, and the numerical problems that may occur during the dif- ferentiation of other forms (e.g., the inverse of the distance) 2.2 LSTM encoder–decoders Note that the threshold values of the selection criterion are determined adaptively from the nature of the dataset. The val- ues were set relatively high, such that the quality of the data could be guaranteed. However, to ensure that an adequate number of stations are selected to represent different areas of the target region, the threshold values could not be set too close to 100 %. The ground observation data at the source stations were processed using LSTM encoder–decoders, with one LSTM encoder–decoder associated with each source station. The LSTM encoder–decoder fs : Xs →H of a source station s is assumed to map the source-station-speciﬁc space Xs of the past 72 h ground observation data (which may contain a dif- ferent number of features for different source stations) to a homogeneous space H, which represents the information re- lated to the PM2.5 and O3 concentrations for the future 48 h of any location in the target region as derived from the past ground observations. The WRF and CMAQ models can output the future weather situations and air pollutant concentrations, which represent valuable information for the deep-learning model. Therefore, the hourly WRF and CMAQ results for the fore- cast period at the locations of interest were input to the model. In other words, the WRF and CMAQ results for the training target stations were used for the model training, and those for the testing target stations were used for the model testing. The WRF and CMAQ features are given in List 1 in the Supplement. Figure 2 shows the structure of the LSTM encoder– decoders used in this study. The LSTM encoder–decoder contains an encoder LSTM, a decoder LSTM, and a dense layer. First, the ground observation data for the past air pol- lutant concentrations and meteorological factors (denoted x(t) Tin t=1, where Tin = 72 h is the length of the past obser- https://doi.org/10.5194/gmd-15-8439-2022 Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 2.4 Broadcasting layer The spatial correction (SC) method, which is based on nu- merical interpolation, has been introduced into the process Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework Figure 2. LSTM encoder–decoder. H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8443 Table 2. Overall performance values for the O3 forecast. Note: ppbv is parts per billion by volume. Time lags Model MBE (ppbv) MAE (ppbv) RMSE (ppbv) SMAPE (%) R 0–23 h CMAQ 0.33 16 21 60 0.61 SC (NN) −0.065 12 17 58 0.66 SC (IDW) −0.050 12 16 54 0.68 SC (kriging) −0.051 11 16 54 0.70 Broadcasting −0.018 11 14 46 0.74 24–47 h CMAQ 0.33 16 21 61 0.60 SC (NN) −0.069 13 17 59 0.64 SC (IDW) −0.047 12 16 55 0.66 SC (kriging) −0.052 12 16 55 0.68 Broadcasting 0.017 11 15 48 0.71 The boldface values represent the highest performance for each period and metric. The boldface values represent the highest performance for each period and metric. The boldface values represent the highest performance for each period and metric. 2. A batch-normalization layer (Ioffe and Szegedy, 2015) was applied after each bidirectional LSTM layer (in- cluding the layers enclosed in the broadcasting layer). The WRF-CMAQ results and ground observations of the PM2.5 and O3 concentrations of the multiple train- ing target stations were simultaneously fed to the model during each minibatch to attain a larger batch size for the batch normalization layers to take effect. 2. A batch-normalization layer (Ioffe and Szegedy, 2015) was applied after each bidirectional LSTM layer (in- cluding the layers enclosed in the broadcasting layer). The WRF-CMAQ results and ground observations of the PM2.5 and O3 concentrations of the multiple train- ing target stations were simultaneously fed to the model during each minibatch to attain a larger batch size for the batch normalization layers to take effect. are avoided. Because the weighted sum preserves the dimen- sions, the output of the broadcasting layer (at a target location t) is a time series of 48 time steps and 64 dimensions. 2.5 Model structure and training Figure 3 shows the architecture of the proposed model. First, the ground observations of the source stations are passed through the LSTM encoder–decoders, as described in Sect. 2.2. Broadcasting to any location in the target region that requires using the novel layer is introduced in Sect. 2.4 (such a location is referred to as a target location). Then, the WRF-CMAQ result for the target location and target hours (as a time series with a length of 48 and dimension of 10) is passed through two bidirectional LSTM layers, both of which have an output dimension of 64. Next, the outputs of the broadcasting layers and bidirectional LSTM layers are concatenated at each time step, forming a time series with 48 time steps and 128 dimensions. Finally, the combined time series is passed to another bidirectional LSTM layer with an output dimension of 64 and a time-distributed dense layer (i.e., a dense layer associated with each of the 48 time steps) with an output dimension of 2, corresponding to the 48 h forecasts of the two air pollutant species. This model is referred to as the broadcasting model here- inafter. 3.1 Overview However, unlike PM2.5, the SC mod- els outperformed the CMAQ model in terms of all metrics for O3 forecasting, including R. The CMAQ was severely biased for both 24 h periods, although the SC solved the bias issue more effectively than that in the case of PM2.5. No- tably, the broadcasting model calibrated the bias such that the model was generally unbiased for both 24 h periods. In terms of the other metrics, the SC (especially with NN and IDW interpolations) exhibited signiﬁcant improvements over the CMAQ model (approximately 25 % in terms of the MAE and RMSE and approximately 10 % in terms of the SMAPE and R). Nevertheless, the broadcasting model outperformed the SC, with improvements of nearly 10 % for all the metrics. The broadcasting model outperformed the baselines for all metrics for both PM2.5 and O3. Tables 1 and 2 summarize the performance values of the broadcasting model and baselines, temporally differentiated by two classes of time lag, i.e., 0– 23 and 24–47 h. In terms of PM2.5, the performance of all models in the ﬁrst 24 h was superior to that in the second 24 h. According to the MBE values, the CMAQ model was highly biased, and the SC only partially resolved this issue. The broadcasting model exhibited a signiﬁcantly decreased bias for both the 24 h periods, and the forecast for the ﬁrst 24 h was generally unbiased. Moreover, although the SC method outperformed the CMAQ model in terms of the MAE, RMSE, and SMAPE (especially with NN and IDW interpolations), it exhibited an inferior R value. In contrast, the broadcasting model exhib- ited an improved R value, indicating a decreased variance. Therefore, the overall error for the proposed model was con- siderably lower than those for the baselines. For example, the Figure 4 shows the hourly RMSE (representing the abso- lute error) and SMAPE (representing the relative error) of the forecasts for the two pollutants. Owing to the daily scale vari- ations in the pollution levels, the RMSE and SMAPE trends were not always consistent with one another, especially for O3. In the case of PM2.5, the performance of the baselines was unsatisfactory at certain time lags (e.g., 11, 22, 35, and 46 h, corresponding to 08:00 and 07:00 on each day). In com- parison, the broadcasting model achieved satisfactory perfor- mance values over all time lags. 3.1 Overview This subsection describes the performance evaluation of the broadcasting model against the baselines on the benchmark stations. Once the WRF-CMAQ forecast was available, the LSTM broadcasting model required only several seconds to obtain the forecast for the next 48 h. Notably, the deep- learning-based structures of the SC were directly optimized to maximize the performance over the source stations. There- fore, the performance of SC on the target stations that were also source stations could not be taken to represent its re- gional forecast performance. For O3, similar to the case of PM2.5, all models were more accurate in forecasting the O3 concentrations in the ﬁrst 24 h than in the latter 24 h. However, unlike PM2.5, the SC mod- els outperformed the CMAQ model in terms of all metrics for O3 forecasting, including R. The CMAQ was severely biased for both 24 h periods, although the SC solved the bias issue more effectively than that in the case of PM2.5. No- tably, the broadcasting model calibrated the bias such that the model was generally unbiased for both 24 h periods. In terms of the other metrics, the SC (especially with NN and IDW interpolations) exhibited signiﬁcant improvements over the CMAQ model (approximately 25 % in terms of the MAE and RMSE and approximately 10 % in terms of the SMAPE and R). Nevertheless, the broadcasting model outperformed the SC, with improvements of nearly 10 % for all the metrics. Figure 4 shows the hourly RMSE (representing the abso- lute error) and SMAPE (representing the relative error) of the forecasts for the two pollutants. Owing to the daily scale vari- ations in the pollution levels, the RMSE and SMAPE trends were not always consistent with one another, especially for O3. In the case of PM2.5, the performance of the baselines was unsatisfactory at certain time lags (e.g., 11, 22, 35, and 46 h, corresponding to 08:00 and 07:00 on each day). In com- parison, the broadcasting model achieved satisfactory perfor- mance values over all time lags. In the case of O3, the SC (es- pecially with IDW and kriging interpolations) outperformed For O3, similar to the case of PM2.5, all models were more accurate in forecasting the O3 concentrations in the ﬁrst 24 h than in the latter 24 h. 3 Results The effectiveness of the broadcasting model was evaluated by comparing its results with the following two baselines: 1. The CMAQ model simulation. 2. The SC method introduced by Sun et al. (2021). Dif- ferent interpolation methods (nearest neighbor, NN, in- verse distance weighting, IDW, and kriging) were used to enhance the performance of the SC method on the test set. In this study, the model was trained for 32 epochs using the ADAM optimizer (Kingma and Ba, 2014) by minimizing the mean absolute error (MAE) of the prediction for all valid records, with a learning rate of 10−3 and batch size of 64. The following measures were introduced to prevent overﬁtting: The performance was evaluated using ﬁve metrics, namely mean bias error (MBE), mean absolute value (MAE), root mean square error (RMSE), symmetric mean absolute per- centage error (SMAPE), and Pearson correlation coefﬁcient (R). The formulas to determine these metrics are listed in Table S1 in the Supplement. 1. A dropout layer (Srivastava et al., 2014) with a rate of 0.5 was applied before the dense layer in each LSTM encoder–decoder and before the ﬁnal time-distributed dense layer. Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 8444 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework H. Sun et al.: Development of an LSTM broadcasting deep-learning framework Figure 3. LSTM broadcasting model structure. Figure 3. LSTM broadcasting model structure. Figure 3. LSTM broadcasting model structure. RMSE was 60 % and 50 % lower than those for the CMAQ and SC models, respectively, and the improvement margins for the other metrics were signiﬁcantly broader than those for the SC. https://doi.org/10.5194/gmd-15-8439-2022 3.2 Performance for different pollution levels As described in Sect. 3.1, the proposed model achieved en- hanced predictions compared with the baselines. The effec- tiveness of the broadcasting model was further evaluated considering different levels of air pollution. The same 21 benchmark stations as those in the analysis described in Sect. 3.1 were used. The ground observations (dots) were typically inconsis- tent with the predictions (background) made by the CMAQ model. In other words, the CMAQ forecasts were generally inaccurate and biased (mainly positively) and could not ac- curately model the regional air pollution. The SC only par- tially resolved this issue, with occasional incompatibilities between the ground observations and forecasts. Moreover, owing to the mathematical characteristics of the different in- terpolation methods, the spatial distribution modeled using the SC framework was evidently unrealistic. For example, the SC forecast with NN interpolation exhibited apparent spatial discontinuities over several straight-line segments in the region, which is highly unrealistic. For each target pollutant, the daily averages of the ground observation values at the different stations were divided into four quartiles (Q1, Q2, Q3, and Q4, in increasing order), as indicated in Table 3. Figures 6 and 7 show the performance values (absolute and relative errors, indicated by the RMSE and SMAPE, respec- tively) of the broadcasting model and baselines for the four quartiles. For both the pollutants and all models, as the pol- lution levels increased, the absolute error increased, and the relative error decreased. Similar to the overall performance trends, different models were generally more accurate in the ﬁrst 24 h in each quartile. However, the broadcasting model achieved signiﬁcantly improved PM2.5 forecasts for all pol- lution levels. In particular, the RMSE of the broadcasting model was around 50 % lower than that of the strongest base- line (SC with kriging interpolation) and was especially low at higher levels of pollution. A clear margin of improvement in the SMAPE was also observed at each quartile. In comparison, the ground observations and forecasts of the broadcasting model were consistent, which indicated that the broadcasting model could resolve the inaccuracies, espe- cially the bias issue, encountered by the other models. An- other key observation of the broadcasting model’s predic- tion is that the spatial distribution simulated by the broad- casting model was smoother than those of the other mod- els. 3.1 Overview In the case of O3, the SC (es- pecially with IDW and kriging interpolations) outperformed https://doi.org/10.5194/gmd-15-8439-2022 Geosci. Model Dev., 15, 8439–8452, 2022 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework Table 3. Quartiles of PM2.5 and O3 concentrations. Note: ppbv is parts per billion by volume. Pollutant Q1 Q2 Q3 Q4 PM2.5 (µg m−3) [0, 9.833) [9.833, 15.68) [15.68, 24.10) [24.10, +∞) O3 (ppbv) [0, 17.44) [17.44, 25.51) [25.51, 36.89) [36.89, +∞) H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8445 the CMAQ model for all metrics. The performance of the LSTM broadcasting model at each time lag was comparable to, if not better than, those of the baselines, and for most time lags, a signiﬁcant margin of improvement was observed. casts. In certain cases (e.g., RMSE of the second 24 h for Q1), the broadcasting model did not outperform the SC (but still signiﬁcantly outperformed the CMAQ model). However, in most cases, the broadcasting model still outperformed the CMAQ and SC models, even given that SC (especially with IDW and kriging interpolations) already supersedes CMAQ by a large margin in many cases. g g g p Figure 5 compares the forecasts of the broadcasting model and baselines with the ground observation data in February, May, August, and November 2021 (rows 1–4, respectively), considering the daily average over the benchmark stations. Consistent with the previous analyses, the forecast for the ﬁrst 24 h was more accurate than that for the second 24 h. In the case of PM2.5, the broadcasting model could better cap- ture the trends of ground observations and was less vulner- able to systematic bias over long periods than the baselines. In the case of O3, the SC model considerably outperformed CMAQ, and the broadcasting model was not evidently more accurate than SC. Nevertheless, the results of the previous quantitative analysis demonstrated the excellent capability of the broadcasting model in O3 forecasting. In conclusion, in addition to the overall improvement in the forecast performance, as described in Sect. 3.1, the broad- casting model exhibits a satisfactory performance at different pollution levels. Therefore, the broadcasting model is robust against different scenarios and can be applied for high or low pollution levels. 3.3 Regional forecast Figure 8 shows the regional forecast of the broadcasting model and baselines considering the monthly average of February 2021. The monthly average of the ground observa- tions at the testing target stations is also shown for compar- ison. The regional forecasts for May, August, and Novem- ber 2021 are presented in the Supplement (Figs. S1–S3). Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 3.2 Performance for different pollution levels However, the model that achieved the most realistic spa- tial smoothness cannot be identiﬁed from the given informa- tion owing to the lack of data in other regions. In fact, the smoothing effect may not align with the fact that some cities (e.g., Guangzhou and Foshan) have higher emission levels The improvements in the broadcasting model for the O3 forecasts were not as signiﬁcant as those for the PM2.5 fore- https://doi.org/10.5194/gmd-15-8439-2022 Geosci. Model Dev., 15, 8439–8452, 2022 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8446 p g p g Figure 4. Forecasting performance at each time lag. Figure 4. Forecasting performance at each time lag. discrete source stations and projects it to any location in the target region as a weighted sum over all source stations. This layer can help overcome the geographical barrier and is a promising alternative to traditional customized SC methods that are typically based on inﬂexible assumptions and result in exacerbated inaccuracies relative to the data of the ground monitoring stations. In addition, owing to the small number of parameters, the proposed model is unlikely to overﬁt spa- tially to the ground observation stations. The described struc- ture can also be extended to regional air pollution forecasting or other deep-learning tasks for regions for which informa- tion for only a limited number of locations is available. How- ever, this study only assumed that the impact of a source sta- tion decreases exponentially with the increase in the distance. Future work can be aimed at considering different patterns and factors other than the distance (e.g., terrains). than other locations in the target region. However, this in- consistency may also be attributable to the limited number of source stations in these cities (see Fig. 1). Nevertheless, in Hong Kong, in which the source stations are densely dis- tributed, the broadcasting model successfully predicted a sig- niﬁcantly lower pollution level, especially for PM2.5. Moreover, the running time of the Broadcasting model is also reasonable. With the graphics processing unit (GPU; K80 in the Google Colaboratory (CoLab) environment) sup- port, it only takes several seconds to ﬁnish the computation for the regional forecast of 1 d after the ground observation results and WRF-CMAQ data are available. Therefore, the broadcasting model satisﬁes the efﬁciency requirements of real applications (Lee et al., 2020; Zhang et al., 2012). 3.2 Performance for different pollution levels On the other hand, SC may take several seconds (NN and IDW) to about 3–5 min (kriging), depending on whether interpo- lation methods can be fully parallelized. By contrast, the LSTM 3D-VAR-CAMx (Comprehensive Air Quality Model with Extensions) will cost about 90 min (tested on a cluster machine with 40 cores and 128 GB of memory), given the ground observation and WRF-CAMx results as input, which may render the approach infeasible when instant forecasts are needed. g Also, our study has extensively exploited the power of LSTM in time-series-related deep-learning tasks. LSTM is one of the most powerful deep-learning tools for time series forecasting (Greff et al., 2017; Karim et al., 2017; Siami- Namini et al., 2018). As a variation in RNNs, it resolves the inherent gradient explosion and vanishing problem, signif- icantly extending the forecast horizon. By carefully exam- ining the nature of different input and output components, we proposed combining two variations in LSTM, i.e., LSTM encoder–decoders and bi-directional LSTMs to construct the model, and achieved relatively good results. From this, we ﬁnd that, in complex time-series-related deep-learning tasks, careful and ad hoc analysis of the nature of the different in- H. Sun et al.: Development of an LSTM broadcasting deep-learning framework H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8447 Figure 5. Comparisons of ground observations and forecasts for February, May, August, and November 2021 (rows 1–4, respectively). round observations and forecasts for February, May, August, and November 2021 (rows 1–4, respectively). Figure 5. Comparisons of ground observations and forecasts for February, May, August, and November 2 SC cannot be fully parallelized, then the forecasting is asso- ciated with a prohibitive runtime, which decreases the appli- cability of such methods. put and output time series is needed to construct the most effective model and achieve higher accuracy. Moreover, the end-to-end deep-learning forecast does not incur a signiﬁcant overhead, given that the ground observa- tions and WRF-CMAQ results are available. As in Sect. 3.3, with GPU acceleration, the proposed model can obtain fore- casts for thousands or even tens of thousands of locations spread across the target region within several seconds. In contrast, if the interpolation methods (e.g., kriging) used by Instead of the conventional random splitting of the training and test sets, two disjoint periods were used for training and testing in this study. This design was motivated by the sys- tematic long-term changes in the probability distributions of the pollutant concentrations, which partially arise because of the implementation of emission reduction (Lu et al., 2020) 4 Discussion This paper proposes an end-to-end deep-learning model for a regional air pollution forecast. The key structure enabling this feature is the broadcasting layer, which inputs the in- formation extracted from the past ground observations at the Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 https://doi.org/10.5194/gmd-15-8439-2022 https://doi.org/10.5194/gmd-15-8439-2022 Geosci. Model Dev., 15, 8439–8452, 2022 8448 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework Figure 6. PM2.5 forecast performances at each quartile. Figure 7. O3 forecast performances at each quartile. and COVID-19 control measures (Fan et al., 2020), which must be considered when ﬁne-tuning the model. If random splitting were applied, then the trained and ﬁne-tuned mod- els would only be guaranteed to be valid on the data from 2015 to 2021 and may fail beyond this period. In this study, a ﬁxed set of source stations was considered, tion. Moreover, this setting may cause some selected source stations to be invalidated in the future (e.g., if they are de- molished in 2023). This problem could only be solved by considering an alternative setting in which the source stations are not selected statically but dynamically at each time step (i.e., hour). However, this alternative setting would require H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8448 Figure 6. PM2.5 forecast performances at each quartile. Figure 6. PM2.5 forecast performances at each quartile. Figure 7. O3 forecast performances at each quartile. Figure 7. O3 forecast performances at each quartile. Figure 7. O3 forecast performances at each quartile. tion. Moreover, this setting may cause some selected source stations to be invalidated in the future (e.g., if they are de- molished in 2023). This problem could only be solved by considering an alternative setting in which the source stations are not selected statically but dynamically at each time step (i.e., hour). However, this alternative setting would require the efﬁcient management of the varying source stations (and even the variations in the number of these stations). and COVID-19 control measures (Fan et al., 2020), which must be considered when ﬁne-tuning the model. If random splitting were applied, then the trained and ﬁne-tuned mod- els would only be guaranteed to be valid on the data from 2015 to 2021 and may fail beyond this period. In this study, a ﬁxed set of source stations was considered, assuming that these stations would continuously output valid results over the years. However, this design may result in loss of information. 5 Conclusions target stations increases. Therefore, the model’s performance has been uneven across different areas of the target region. For example, as shown in Sect. 3.3, the performance in Hong Kong is generally better than that in other regions. In future works, other selection criteria of source stations and training target stations, in place of those introduced in Sect. 2.1, may be developed to resolve this issue. Ground observations of recent hours can provide information regarding the most immediate meteorological and air pollu- tion conditions. However, this information is typically avail- able only for ground monitoring stations, and the absence of information regarding the forecast period limits the accuracy of forecasts in the spatial and temporal dimensions. In this study, the parameters of spatial interpolation were incorpo- rated into the training process by introducing a novel broad- casting layer. This conﬁguration could overcome the prob- lems related to the ofﬂine SC methods and the spatial bar- rier, allowing information to be broadcast to all locations in the target region. Combined with the broadcasting layer, the end-to-end deep-learning model incorporated the ground ob- servation and WRF-CMAQ results through different LSTM- based structures suitable for various formats of time series data. The proposed model outperformed the existing mod- els in terms of the PM2.5 and O3 forecasts. For the two pollutants, the absolute error (e.g., RMSE) of the proposed model was 55 % and 30 % lower than those of the CMAQ model and 45 % and 10 % lower than those of the SC model. SMAPE of the proposed model was 30 % and 20 % lower than those of the CMAQ and 25 % and 15 % lower than those of the SC model. The proposed model structure can serve as a novel framework for regional air pollution forecasting. Speciﬁcally, this model can be applied to forecast the con- WRF-CMAQ simulation shows severe overestimations for both the PM2.5 and O3 forecasts, especially during 24–47 h. The errors can be caused by several factors, such as the emis- sion inventory, boundary and initial conditions, chemical and physical parameterization schemes, and meteorological fac- tors simulation. The emission inventory cannot always be up-to-date, since substantial efforts are needed to compile a new set of regional emission inventories in high resolution. In addition, the scientiﬁc community has not yet fully un- derstood many of the chemical and physical mechanisms in the atmosphere. https://doi.org/10.5194/gmd-15-8439-2022 For example, if a ground observation station produced high-quality records between 2015 and 2018 but was later demolished, then it was not selected as a source sta- In our setting, the source and training target stations play an essential role in the model’s accuracy. The forecast qual- ity generally increases as the number of source and training Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8449 Figure 8. Regional forecast results for February 2021. Figure 8. Regional forecast results for February 2021. References Ayturan, Y. A., Ayturan, Z. C., and Altun, H. O.: Air pollution mod- elling with deep learning: a review, International Journal of En- vironmental Pollution and Environmental Modelling, 1, 58–62, 2018. Bi, J., Knowland, K. E., Keller, C. A., and Liu, Y.: Combining Ma- chine Learning and Numerical Simulation for High-Resolution PM2.5 Concentration Forecast, Environ. Sci. Technol., 56, 1544– 1556, 2022. Bui, T.-C., Le, V.-D., and Cha, S.-K.: A deep learning approach for forecasting air pollution in South Korea using LSTM, arXiv [preprint], https://doi.org/10.48550/arXiv.1804.07891, 2018. Fan, C., Li, Y., Guang, J., Li, Z., Elnashar, A., Allam, M., and de Leeuw, G.: The impact of the control measures during the COVID-19 outbreak on air pollution in China, Remote Sensing, 12, 1613, https://doi.org/10.3390/rs12101613, 2020. Gilliam, R. C., Hogrefe, C., Godowitch, J. M., Napelenok, S., Mathur, R., and Rao, S. T.: Impact of inherent meteorology un- certainty on air quality model predictions, J. Geophys. Res.- Atmos., 120, 12259–12280, 2015. Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/gmd-15-8439-2022-supplement. Greff, K., Srivastava, R., Koutník, J., Steunebrink, B., and Schmidhuber, J.: LSTM: A search space odyssey, IEEE Transactions on Neural Networks Learning Systems, https://doi.org/10.1109/TNNLS.2016.2582924, 2017. Author contributions. HS, XL, and JCHF conceived the research. HS developed the model and performed the simulations. HS, XL, JCHF, and YC conducted the analysis. ZL, DY, and WC provided useful comments on the paper. HS prepared the paper, with contri- butions from all co-authors. Hähnel, P., Mareˇcek, J., Monteil, J., and O’Donncha, F.: Using deep learning to extend the range of air pollution monitoring and fore- casting, J. Comput. Phys., 408, 109278, 2020. Han, J., Liu, H., Zhu, H., Xiong, H., and Dou, D.: Joint air quality and weather prediction based on multi-adversarial spatiotempo- ral networks, Proceedings of the AAAI Conference on Artiﬁcial Intelligence, 2–9 February 2021, virtual conference, 4081–4089, https://doi.org/10.48550/arXiv.2012.15037, 2021. Competing interests. The contact author has declared that none of the authors has any competing interests. Hochreiter, S. and Schmidhuber, J.: Long short-term memory, Neu- ral Comput., 9, 1735–1780, 1997. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Holnicki, P. and Nahorski, Z.: Emission data uncertainty in urban air quality modeling–case study, Environ. Model. Assess., 20, 583– 597, 2015. Huang, C.-J. and Kuo, P.-H.: A deep CNN-LSTM model for par- ticulate matter (PM2.5) forecasting in smart cities, Sensors, 18, 2220, https://doi.org/10.3390/s18072220, 2018. Acknowledgements. H. Sun et al.: Development of an LSTM broadcasting deep-learning framework centrations of PM2.5, O3, and other pollutants in different re- gions worldwide if adequate ground observations for the re- gion are available and the numerical models (not necessarily WRF-CMAQ) can cover the target hours. The broadcasting layer may also be further applied to a wide range of tasks that would otherwise require interpolation, thereby facilitat- ing the development of end-to-end deep-learning models for these tasks. Considering the diverse nature of different tasks, ad hoc variations in the broadcasting layer may be designed to adapt to task-speciﬁc requirements. Financial support. This work has been supported by the Guangzhou Scientiﬁc and Technological Planning Project (grant no. 202102021297), the National Natural Science Foundation of China (grant no. 42007203), the Research Grants Council of Hong Kong Government (grant nos. 16305921, 16302220, and R6011- 18), the Guangdong–Hong Kong–Macau Joint Laboratory Grant (grant no. GDST20IP05), and the Environment and Conservation Fund (grant no. ECF 2020123). Review statement. This paper was edited by David Topping and re- viewed by two anonymous referees. Review statement. This paper was edited by David Topping and re- viewed by two anonymous referees. Code and data availability. The ground air pollutant observa- tion data were released by the China National Environmen- tal Monitoring Center and the Hong Kong Environmental Pro- tection Department. The ground observation data used in this study can be found at https://doi.org/10.5281/zenodo.6598377 (Sun et al., 2022a). The PM2.5 and O3 forecast results from different models and the setting of the WRF-CMAQ model are available at https://doi.org/10.5281/zenodo.6833673 (Sun et al., 2022b). The WRF model v3.7 and CMAQ model v5.0.2 can be downloaded from https://www2.mmm.ucar.edu/ wrf/users/download/get_source.html (Skamarock et al., 2008) and https://doi.org/10.5281/zenodo.1079898 (United States Envi- ronmental Protection Agency, 2014). The ofﬁcial implementa- tion of this work is at https://doi.org/10.5281/zenodo.7019243 (Sun, 2022), and the deep-learning model parameters and the input/output data with compatible formats are available at https://doi.org/10.5281/zenodo.6827585 (Sun et al., 2022c) and https://doi.org/10.5281/zenodo.6601173 (Sun et al., 2022d), respec- tively. 5 Conclusions Therefore, current state-of-the-art parame- terization schemes still have a long way to go to be further improved. Besides devoting time and effort to improving the performance of the prognostic model from the abovemen- tioned perspectives, from this work, we can ﬁnd that com- bining the observation data-driven skills (e.g., deep-learning methods) can work as a feasible and efﬁcient option to make up the current deﬁciency inherent in 3D chemical transport model and thus improve the forecast performance. https://doi.org/10.5194/gmd-15-8439-2022 Geosci. Model Dev., 15, 8439–8452, 2022 8450 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework Li, T., Hua, M., and Wu, X.: A hybrid CNN-LSTM model for fore- casting particulate matter (PM2.5), Ieee Access, 8, 26933–26940, 2020. Sayeed, A., Lops, Y., Choi, Y., Jung, J., and Salman, A. K.: Bias correcting and extending the PM forecast by CMAQ up to 7 days using deep convolutional neural networks, Atmos. Environ., 253, 118376, https://doi.org/10.1016/j.atmosenv.2021.118376, 2021b. Lu, H., Xie, M., Liu, X., Liu, B., Jiang, M., Gao, Y., and Zhao, X.: Adjusting prediction of ozone concentration based on CMAQ model and machine learning methods in Sichuan- Chongqing region, China, Atmos. Pollut. Res., 12, 101066, https://doi.org/10.1016/j.apr.2021.101066, 2021. Schuster, M. and Paliwal Kuldip, K.: Bidirectional recurrent neural networks, IEEE T. Signal Proces., 45, 2673–2681, 1997. Lu, X., Fung, J. C. H., and Wu, D.: Modeling wet deposition of acid substances over the PRD region in China, Atmos. Environ., 122, 819–828, 2015. Siami-Namini, S., Tavakoli, N., and Namin, A. S.: A comparison of ARIMA and LSTM in forecasting time series, 2018 17th IEEE international conference on machine learning and applications (ICMLA), 17–20 December 2018, Orlando, Florida, USA, 1394– 1401, https://doi.org/10.1109/ICMLA.2018.00227, 2018. Lu, X., Wang, Y., Li, J., Shen, L., and Fung, J. C.: Evidence of heterogeneous HONO formation from aerosols and the regional photochemical impact of this HONO source, Environ. Res. Lett., 13, 114002, https://doi.org/10.1088/1748-9326/aae492, 2018. Skamarock, W. C., Klemp, J. B., Dudhia, J., Gill, D. O., Barker, D., Duda, M. G., Huang, X. Y., Wang W., and Powers, J. G.: A description of the Advanced Research WRF version 3, NCAR Technical note-475+ STR, https://doi.org/10.5065/D68S4MVH, 2008 (data available at https://www2.mmm.ucar.edu/wrf/users/ download/get_source.html, last access: 14 November 2022). Lu, X., Zhang, S., Xing, J., Wang, Y., Chen, W., Ding, D., Wu, Y., Wang, S., Duan, L., and Hao, J.: Progress of air pollution control in China and its challenges and opportunities in the ecological civilization era, Engineering, 6, 1423–1431, 2020. Lu, X., Sha, Y. H., Li, Z., Huang, Y., Chen, W., Chen, D., Shen, J., Chen, Y., and Fung, J. C.: Development and application of a hybrid long-short term memory– three dimensional variational technique for the improvement of PM2.5 forecasting, Sci. Total Environ., 770, 144221, https://doi.org/10.1016/j.scitotenv.2020.144221, 2021. Srivastava, N., Hinton, G., Krizhevsky, A., Sutskever, I., and Salakhutdinov, R.: Dropout: a simple way to prevent neural net- works from overﬁtting, J. Mach. Learn. Res., 15, 1929–1958, 2014. Sun, H., Fung, J. H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8451 France, 448–456, https://doi.org/10.48550/arXiv.1502.03167, 2015. France, 448–456, https://doi.org/10.48550/arXiv.1502.03167, 2015. France, 448–456, https://doi.org/10.48550/arXiv.1502.03167, 2015. Mao, W., Wang, W., Jiao, L., Zhao, S., and Liu, A.: Modeling air quality prediction using a deep learning approach: Method optimization and evaluation, Sustain. Cities Soc., 65, 102567, https://doi.org/10.1016/j.scs.2020.102567, 2021. Janarthanan, R., Partheeban, P., Somasundaram, K., and Elampar- ithi, P. N.: A deep learning approach for prediction of air quality index in a metropolitan city, Sustain. Cities Soc., 67, 102720, 2021. Pak, U., Ma, J., Ryu, U., Ryom, K., Juhyok, U., Pak, K., and Pak, C.: Deep learning-based PM2.5 prediction con- sidering the spatiotemporal correlations: A case study of Beijing, China, Sci. Total Environ., 699, 133561, https://doi.org/10.1016/j.scitotenv.2019.07.367, 2020. Karim, F., Majumdar, S., Darabi, H., and Chen, S.: LSTM fully con- volutional networks for time series classiﬁcation, IEEE access, 6, 1662–1669, 2017. Kim, H. S., Park, I., Song, C. H., Lee, K., Yun, J. W., Kim, H. K., Jeon, M., Lee, J., and Han, K. M.: Development of a daily PM10 and PM2.5 prediction system using a deep long short-term memory neural network model, Atmos. Chem. Phys., 19, 12935– 12951, https://doi.org/10.5194/acp-19-12935-2019, 2019. Qi, Y., Li, Q., Karimian, H., and Liu, D.: A hybrid model for spa- tiotemporal forecasting of PM2.5 based on graph convolutional neural network and long short-term memory, Sci. Total Environ., 664, 1–10, 2019. Qin, D., Yu, J., Zou, G., Yong, R., Zhao, Q., and Zhang, B.: A novel combined prediction scheme based on CNN and LSTM for urban PM2.5 concentration, IEEE Access, 7, 20050–20059, 2019. Kingma, D. P. and Ba, J.: Adam: A method for stochastic optimiza- tion, arXiv [preprint],https://doi.org/10.48550/arXiv.1412.6980, 2014. Samal, K. K. R., Panda, A. K., Babu, K. S., and Das, S. K.: An improved pollution forecasting model with meteorological impact using multiple imputation and ﬁne-tuning approach, Sustain. Cities Soc., 70, 102923, https://doi.org/10.1016/j.scs.2021.102923, 2021. Lee, K., Yu, J., Lee, S., Park, M., Hong, H., Park, S. Y., Choi, M., Kim, J., Kim, Y., Woo, J.-H., Kim, S.-W., and Song, C. H.: Development of Korean Air Quality Prediction System ver- sion 1 (KAQPS v1) with focuses on practical issues, Geosci. Model Dev., 13, 1055–1073, https://doi.org/10.5194/gmd-13- 1055-2020, 2020. Sayeed, A., Choi, Y., Eslami, E., Jung, J., Lops, Y., Salman, A. K., Lee, J.-B., Park, H.-J., and Choi, M.-H.: A novel CMAQ-CNN hybrid model to forecast hourly surface-ozone concentrations 14 days in advance, Sci. Rep.-UK, 11, 1–8, 2021a. References We appreciate the assistance of the Hong Kong Observatory (HKO), which provided the meteorological data. We thank the constructive comments from reviewers and editor. Ioffe, S. and Szegedy, C.: Batch normalization: Accelerating deep network training by reducing internal covariate shift, Interna- tional conference on machine learning, 6–11 July 2015, Lille, Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 Geosci. Model Dev., 15, 8439–8452, 2022 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework C., Chen, Y., Chen, W., Li, Z., Huang, Y., Lin, C., Hu, M., and Lu, X.: Improvement of PM2.5 and O3 forecasting by integration of 3D numerical simulation with deep learning techniques, Sustain. Cities Soc., 75, 103372, https://doi.org/10.1016/j.scs.2021.103372, 2021. Lyu, B., Hu, Y., Zhang, W., Du, Y., Luo, B., Sun, X., Sun, Z., Deng, Z., Wang, X., and Liu, J.: Fusion method combining ground-level observations with chemical transport model predictions using an ensemble deep learning framework: application in China to esti- mate spatiotemporally-resolved PM2.5 exposure ﬁelds in 2014– 2017, Environ. Sci. Technol., 53, 7306–7315, 2019. Sun, H., Fung, J. C. H., Chen, Y., Li, Z., Yuan, D., Chen, W., and Lu, X.: Ground obeservation data (meteorological factors and air pollution) in Greater Bay Area, 2015–2021, Zenodo [data set], https://doi.org/10.5281/zenodo.6598377, 2022a. Ma, J., Ding, Y., Cheng, J. C., Jiang, F., and Wan, Z.: A temporal-spatial interpolation and extrapolation method based on geographic Long Short-Term Memory neu- ral network for PM2.5, J. Clean. Prod., 237, 117729, https://doi.org/10.1016/j.jclepro.2019.117729, 2019. Sun, H., Fung, J. C. H., Chen, Y., Li, Z., Yuan, D., Chen, W., and Lu, X.: Prediction of the broadcasting model and various baselines, https://doi.org/10.5194/gmd-15-8439-2022 Geosci. Model Dev., 15, 8439–8452, 2022 Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022 H. Sun et al.: Development of an LSTM broadcasting deep-learning framework 8452 Zenodo [data set], https://doi.org/10.5281/zenodo.6833673, 2022b. United States Environmental Protection Agency: CMAQ (Version 5.0.2), Zenodo [software], https://doi.org/10.5281/zenodo.1079898, 2014. Sun, H., Fung, J. C. H., Chen, Y., Li, Z., Yuan, D., Chen, W., and Lu, X.: Deep learning models in the study “Develop- ment of an LSTM-Broadcasting deep-learning framework for re- gional air pollution forecast improvement”, Zenodo [data set], https://doi.org/10.5281/zenodo.6827585, 2022c. Wu, Q. and Lin, H.: Daily urban air quality index forecast- ing based on variational mode decomposition, sample entropy and LSTM neural network, Sustain. Cities Soc., 50, 101657, https://doi.org/10.1016/j.scs.2019.101657, 2019. Zhang, Y., Bocquet, M., Mallet, V., Seigneur, C., and Baklanov, A.: Real-time air quality forecasting, part II: State of the science, current research needs, and future prospects, Atmos. Environ., 60, 656–676, 2012. Sun, H., Fung, J. C. H., Chen, Y., Li, Z., Yuan, D., Chen, W., and Lu, X.: Processed ground observation and WRF-CAMQ data for Greater Bay Area, 2015–2021, Zenodo [data set], https://doi.org/10.5281/zenodo.6601173, 2022d. Zhao, J., Deng, F., Cai, Y., and Chen, J.: Long short-term memory- Fully connected (LSTM-FC) neural network for PM2.5 concen- tration prediction, Chemosphere, 220, 486–492, 2019. Sun, J. H.: jvhs0706/regional-forecast-new: GMD paper code, Zen- odo [code], https://doi.org/10.5281/zenodo.7019243, 2022. Sutskever, I., Vinyals, O., and Le, Q. V.: Sequence to sequence learning with neural networks, Adv. Nur. In., 27, 3104–3112, https://doi.org/10.48550/arXiv.1409.3215, 2014. Zhou, X., Tong, W., and Li, L.: Deep learning spatiotemporal air pollution data in China using data fusion, Earth Sci. Inform., 13, 859–868, 2020. Tang, Y., Lee, P., Tsidulko, M., Huang, H.-C., McQueen, J. T., DiMego, G. J., Emmons, L. K., Pierce, R. B., Thompson, A. M., and Lin, H.-M.: The impact of chemical lateral boundary conditions on CMAQ predictions of tropospheric ozone over the continental United States, Environ. Fluid Mech., 9, 43–58, 2009. Tsai, Y.-T., Zeng, Y.-R., and Chang, Y.-S.: Air pollution forecast- ing using RNN with LSTM, 2018 IEEE 16th Intl Conf on De- pendable, Autonomic and Secure Computing, 16th Intl Conf on Pervasive Intelligence and Computing, 4th Intl Conf on Big Data Intelligence and Computing and Cyber Science and Technology Congress (DASC/PiCom/DataCom/CyberSciTech), 12–15 Au- gust 2018, Athens, Greece, 1074–1079, https://doi.org/10.1109/ DASC/PiCom/DataCom/CyberSciTec.2018.00178, 2018. Geosci. Model Dev., 15, 8439–8452, 2022 https://doi.org/10.5194/gmd-15-8439-2022
https://openalex.org/W2001155157	http://journals.iucr.org/c/issues/2005/04/00/sk1817/sk1817.pdf	English	null	Three isomeric<i>N</i>-(nitrophenyl)succinimides: isolated molecules, hydrogen-bonded sheets and a hydrogen-bonded three-dimensional framework	Acta crystallographica. Section C, Crystal structure communications/Acta crystallographica. Section C, Crystal structure communications.	2,005	cc-by	4,713	Acta Crystallographica Section C Acta Crystallographica Section C Crystal Structure Communications mean planes of the two rings are 57.4 (2), 46.0 (2) and 39.1 (2), respectively, while the dihedral angles between the aryl rings and the nitro groups are 40.0 (2), 4.9 (2) and 23.2 (2), respectively. In isomers (I) and (II), the molecules have point group C1, and in isomer (III), the molecular point group is C2; hence, in each isomer, the molecules are chiral. Thus, for isomer (II) in space group P21, each crystal contains just one enantiomer provided that inversion twinning is absent, although the bulk material is racemic. The bond distances and interbond angles in (I)±(III) show no unusual values. Crystal Structure Communications ISSN 0108-2701 Christopher Glidewell,a* John N. Low,b Janet M. S. Skakleb and James L. Wardellc Christopher Glidewell,a* John N. Low,b Janet M. S. Skakleb and James L. Wardellc aSchool of Chemistry, University of St Andrews, Fife KY16 9ST, Scotland, bDepartment of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen AB24 3UE, Scotland, and cInstituto de QuõÂmica, Departamento de QuõÂmica InorgaÃnica, Universidade Federal do Rio de Janeiro, 21945-970 Rio de Janeiro, RJ, Brazil Correspondence e-mail: cg@st-andrews.ac.uk Received 9 February 2005 Accepted 10 February 2005 Online 11 March 2005 Molecules of N-(2-nitrophenyl)succinimide, C10H8N2O4, are linked into sheets by a combination of CÐH O and CÐH (arene) hydrogen bonds. Molecules of N-(3-nitro- phenyl)succinimide are linked into a three-dimensional framework by a combination of a two-centre CÐH O hydrogen bond and a three-centre CÐH (O)2 hydrogen bond. Molecules of N-(4-nitrophenyl)succinimide which lie across twofold rotation axes in space group C2/c, participate in no direction-speci®c intermolecular interactions. The molecules of isomer (I) (Fig. 1) are linked into centrosymmetric dimers by a single CÐH O hydrogen bond, and these dimers are linked into sheets by a single CÐ H (arene) hydrogen bond (Table 1). Aromatic ± stacking interactions, on the other hand, are absent. Atom C3 in the molecule at (x, y, z) acts as a hydrogen-bond donor to nitro atom O21 in the molecule at (1 ÿ x, 1 ÿ y, 1 ÿ z), so generating a centrosymmetric R2 2(16) (Bernstein et al., 1995) dimer, centred at ( 1 2, 1 2, 1 2) (Fig. 4). The atoms of type C2 in this dimer, at (x, y, z) and (1 ÿ x, 1 ÿ y, 1 ÿ z), act as hydrogen- Comment We report here the structures of three isomeric N-(nitro- phenyl)succinimides, (I)±(III). These compounds offer, within the compass of a small molecular skeleton, a wide range of potential intermolecular interactions, including CÐH O(carbonyl/nitro) (each with aromatic and aliphatic CÐH units as potential donors) and CÐH (arene) hydrogen bonds, aromatic ± stacking interactions, and dipolar carbonyl±carbonyl and nitro±nitro interactions. Figure 1 The molecule of (I), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. In the 2-nitro and 3-nitro isomers (I) and (II) (Figs. 1 and 2), the succinimide rings are effectively planar. However, in the 4-nitro isomer, (III), where the molecules lie across twofold rotation axes in space group C2/c, with the reference molecule selected as one lying across the axis along (1 2, y, 3 4) (Fig. 3), the succinimide rings are markedly puckered. The total puckering amplitude Q2 (Cremer & Pople, 1975) is 0.161 (3) AÊ , and the ring-puckering parameter '2 is 90.0 (9) for the atom sequence N1ÐC1ÐC2ÐC2iiiÐC1iii [symmetry code: (iii) 1 ÿ x, y, 3 2 ÿ z], indicating a half-chair conformation for this ring. In isomers (I)±(III), the dihedral angles between the o216 # 2005 International Union of Crystallography organic compounds bond donors, respectively, to aryl rings C11±C16 in the mol- ecules at (1 ÿ x, ÿ1 2 + y, 3 2 ÿ z) and (x, 3 2 ÿ y, ÿ1 2 + z), which themselves form parts of the dimers centred at ( 1 2, 0, 1) and ( 1 2, 1, 0), respectively. In a similar way, aryl rings C11±C16 in the molecules at (x, y, z) and (1 ÿ x, 1 ÿ y, 1 ÿ z) accept hydrogen bonds from atoms C2 in the molecules at (1 ÿ x, 1 2 + y, 3 2 ÿ z) and (x, 1 2 ÿ y, ÿ1 2 + z), respectively, which are themselves components of the dimers centred at ( 1 2, 1, 1) and ( 1 2, 0, 0). In this manner, a (100) sheet is generated (Fig. 5). ecular structure; there are, in fact, no direction-speci®c inter- actions between adjacent sheets. The molecules of (II) are linked into a three-dimensional framework by a combination of one two-centre CÐH O hydrogen bond and one three-centre CÐH (O)2 hydrogen bond (Table 2), and the formation of the framework is readily analysed and described by consideration of each of these interactions in turn. In the two-centre hydrogen bond, aromatic atom C16 in the molecule at (x, y, z) acts as a donor to carbonyl atom O4 in the molecule at (ÿ1 + x, y, z), so generating by translation a C(6) (Bernstein et al., 1995) chain running parallel to the [100] direction (Fig. 6). There is a fairly short dipolar contact between carbonyl atoms O1 at (x, y, z) and C4 at (1 ÿ x, 1 2 + y, 3 2 ÿ z) [O1 C4iv = 2.959 (2) AÊ , O1 O4iv = 3.193 (2) AÊ , C1ÐO1 C4iv = 148.7 (2) and O1 C4ivÐO4iv = 50.4 (2); symmetry code: (iv) 1 ÿ x, 1 2 + y, 3 2 ÿ z], corresponding to an interaction part- way between the type I and type III motifs (Allen et al., 1998). Figure 2 Figure 2 The molecule of (II), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. Figure 3 The molecule of (III), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level, and atoms marked with the suf®x A are at the symmetry position (1 ÿ x, y, 3 2 ÿ z). Figure 4 Part of the crystal structure of (I), showing the formation of an R2 2(16) dimer centred at ( 1 2, 1 2, 1 2). Atoms marked with an asterisk () are at the symmetry position (1 ÿ x, 1 ÿ y, 1 ÿ z). Figure 4 Part of the crystal structure of (I), showing the formation of an R2 2(16) dimer centred at ( 1 2, 1 2, 1 2). Atoms marked with an asterisk () are at the symmetry position (1 ÿ x, 1 ÿ y, 1 ÿ z). Figure 2 The molecule of (II), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. Figure 2 The molecule of (II), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. g The molecule of (II), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. Figure 3 The molecule of (III), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level, and atoms marked with the suf®x A are at the symmetry position (1 ÿ x, y, 3 2 ÿ z). Figure 1 Figure 1 The molecule of (I), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. Figure 1 The molecule of (I), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. Figure 1 The molecule of (I), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. Acta Cryst. (2005). C61, o216±o220 Acta Cryst. (2005). C61, o216±o220 o216 o216 # 2005 International Union of Crystallography DOI: 10.1107/S0108270105004798 organic compounds organic compounds However, this interaction occurs within a (100) sheet and hence does not affect the dimensionality of the supramol- There is a fairly short dipolar contact between carbonyl atoms O1 at (x, y, z) and C4 at (1 ÿ x, 1 2 + y, 3 2 ÿ z) [O1 C4iv = 2.959 (2) AÊ , O1 O4iv = 3.193 (2) AÊ , C1ÐO1 C4iv = 148.7 (2) and O1 C4ivÐO4iv = 50.4 (2); symmetry code: (iv) 1 ÿ x, 1 2 + y, 3 2 ÿ z], corresponding to an interaction part- way between the type I and type III motifs (Allen et al., 1998). However, this interaction occurs within a (100) sheet and hence does not affect the dimensionality of the supramol- In the three-centre hydrogen bond, which is planar, atom C3 in the molecule at (x, y, z) acts as a donor, via H3A, to nitro atom O31 in the molecule at (x, y, ÿ1 + z) and to carbonyl atom O1 in the molecule at (ÿx, 1 2 + y, ÿz). The individual components of this three-centre system thus produce, respectively, a C(9) chain running parallel to the [001] direc- tion and generated by translation, and a C(5) chain running parallel to the [010] direction and generated by the 21 screw axis along ( 1 4, y, 1 4). The action of the two components of this system, acting together, forms a (100) sheet in the form of a (4,4)-net (Batten & Robson, 1998) built from a single type of R4 3(23) ring (Fig. 7). The combination of the [100] chain (Fig. 6) and the (100) sheet (Fig. 7) suf®ces to generate the three- dimensional framework. Figure 2 The molecule of (II), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level. In the structure of isomer (III) (Fig. 3) there are neither hydrogen bonds of any type nor aromatic ± stacking interactions or dipolar interactions; hence, the structure of (III) consists of isolated molecules. It is of interest to compare the supramolecular structures of isomers (I)±(III) with that of the isomeric C-(3-nitrophen- organic compounds hydrogen bonds involving one nitro O atom and one carbonyl O atom as acceptors; however, CÐH (arene) hydrogen bonds and aromatic ± stacking interactions are absent from the structure of (IV). We also note that, at 293 K, N-(4- nitrophenyl)maleimide, (V) [CSD refcodes BEDWOX (Fruk & Graham, 2003) and BEDWOX01 (Moreno-Fuquen et al., 2003)], is isostructural with (III). The structure of (V), like that of (III), contains no direction-speci®c intermolecular inter- actions, despite the different orientations of the CÐH bonds in the heterocyclic ring of (V). hydrogen bonds involving one nitro O atom and one carbonyl O atom as acceptors; however, CÐH (arene) hydrogen bonds and aromatic ± stacking interactions are absent from the structure of (IV). We also note that, at 293 K, N-(4- nitrophenyl)maleimide, (V) [CSD refcodes BEDWOX (Fruk & Graham, 2003) and BEDWOX01 (Moreno-Fuquen et al., 2003)], is isostructural with (III). The structure of (V), like that of (III), contains no direction-speci®c intermolecular inter- actions, despite the different orientations of the CÐH bonds in the heterocyclic ring of (V). yl)succinimide, (IV) [Cambridge Structural Databse (CSD; Allen, 2002) refcode TANPUT (Kwiatkowski & Karolak- Wojciechowska, 1992)]. Compound (IV) crystallizes in the centrosymmetric space group P21/c, with Z0 = 2, so that equal numbers of the R and S enantiomers are present in each crystal. The supramolecular structure is dominated by two NÐ H O hydrogen bonds, which generate C 2 2(8) chains along [010]. Although no CÐH O hydrogen bonds were mentioned in the original report, analysis of the reported atomic coordinates using PLATON (Spek, 2003) shows that the chains are, in fact, weakly linked into sheets by two such The intermolecular interactions manifest in the structures of (I)±(III) are different in all three isomers. In (I), the structure is determined by one CÐH O hydrogen bond and one CÐH (arene) hydrogen bond; both interactions involve a CH2 donor rather than an aryl CÐH bond as donor, and the CÐH O hydrogen bond has a nitro O-atom acceptor rather than the usual carbonyl O-atom acceptor; however, the carbonyl groups do participate in dipolar inter- actions. In isomer (II), by contrast, where only CÐH O hydrogen bonds occur, both CH2 and aryl donors participate, and the three-centre hydrogen bond involves both nitro and carbonyl O atoms as the acceptors. Figure 3 Figure 4 Figure 4 Part of the crystal structure of (I), showing the formation of an R2 2(16) dimer centred at ( 1 2, 1 2, 1 2). Atoms marked with an asterisk () are at the symmetry position (1 ÿ x, 1 ÿ y, 1 ÿ z). Figure 4 Part of the crystal structure of (I), showing the formation of an R2 2(16) dimer centred at ( 1 2, 1 2, 1 2). Atoms marked with an asterisk () are at the symmetry position (1 ÿ x, 1 ÿ y, 1 ÿ z). Figure 3 The molecule of (III), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level, and atoms marked with the suf®x A are at the symmetry position (1 ÿ x, y, 3 2 ÿ z). gu e 3 The molecule of (III), showing the atom-labelling scheme. Displacement ellipsoids are drawn at the 30% probability level, and atoms marked with the suf®x A are at the symmetry position (1 ÿ x, y, 3 2 ÿ z). Acta Cryst. (2005). C61, o216±o220 o217 Glidewell et al. Three isomers of C10H8N2O4 organic compounds Figure 6 Figure 6 Part of the crystal structure of (II), showing the formation, via a two- centre CÐH O hydrogen bond, of a C(6) chain along [100]. For clarity, H atoms not involved in the motif shown have been omitted. Atoms marked with an asterisk () or a hash (#) are at the symmetry positions (ÿ1 + x, y, z) and (1 + x, y, z), respectively. Figure 6 Part of the crystal structure of (II), showing the formation, via a two- centre CÐH O hydrogen bond, of a C(6) chain along [100]. For clarity, H atoms not involved in the motif shown have been omitted. Atoms marked with an asterisk () or a hash (#) are at the symmetry positions (ÿ1 + x, y, z) and (1 + x, y, z), respectively. organic compounds Likewise in (IV), the CÐ H O hydrogen bonds involve both nitro and carbonyl acceptors. In none of isomers (I)Ð(III) is there any aromatic ± stacking interactions, and these interactions are possibly precluded by the overall molecular conformations. Perhaps the most surprising feature of the structures of isomers (I)± (III) is the lack of any direction-speci®c intermolecular interactions in isomer (III). Figure 5 A stereoview of part of the crystal structure of (I), showing the formation of a (100) sheet built from CÐH O and CÐH (arene) hydrogen bonds. For clarity, H atoms not involved in the motifs shown have been omitted Figure 5 Figure 5 Figure 5 g A stereoview of part of the crystal structure of (I), showing the formation of a (100) sheet built from CÐH O and CÐH (arene) hydrogen bonds. For clarity, H atoms not involved in the motifs shown have been omitted Thus, each of the isomers (I)±(III) exhibits a different range of intermolecular interactions, and their supramolecular structures are all of different dimensionality, viz. two- and three-dimensional in (I) and (II), respectively, contrasted with isolated molecules in (III). Such differences within a series of positional isomers are not yet readily predictable, either heuristically or computationally. Figure 6 Part of the crystal structure of (II), showing the formation, via a two- centre CÐH O hydrogen bond, of a C(6) chain along [100]. For clarity, H atoms not involved in the motif shown have been omitted. Atoms marked with an asterisk (*) or a hash (#) are at the symmetry positions (ÿ1 + x, y, z) and (1 + x, y, z), respectively. Figure 7 A stereoview of part of the crystal structure of (II), showing the formation, via a three-centre CÐH (O)2 hydrogen bond, of a (100) sheet. For clarity, H atoms not involved in the motifs shown have been omitted. Figure 7 Figure 7 A stereoview of part of the crystal structure of (II), showing the formation, via a three-centre CÐH (O)2 hydrogen bond, of a (100) sheet. For clarity, H atoms not involved in the motifs shown have been omitted. Figure 7 A stereoview of part of the crystal structure of (II), showing the formation, via a three-centre CÐH (O)2 hydrogen bond, of a (100) sheet. For clarity, H atoms not involved in the motifs shown have been omitted. Acta Cryst. (2005). C61, o216±o220 o218 Glidewell et al. Three isomers of C10H8N2O4 organic compounds Data collection Nonius KappaCCD diffractometer ' scans, and ! scans with offsets Absorption correction: multi-scan (SORTAV; Blessing, 1995, 1997) Tmin = 0.947, Tmax = 0.964 5403 measured re¯ections 1152 independent re¯ections Re®nement Re®nement on F 2 R[F 2 > 2(F 2)] = 0.031 wR(F 2) = 0.078 S = 1.08 1152 re¯ections 146 parameters H-atom parameters constrained Data collection Nonius KappaCCD diffractometer ' scans, and ! scans with offsets Absorption correction: multi-scan (SORTAV; Blessing, 1995, 1997) Tmin = 0.947, Tmax = 0.964 5403 measured re¯ections 1152 independent re¯ections Experimental 10 Re®nement Re®nement on F 2 R[F 2 > 2(F 2)] = 0.058 wR(F 2) = 0.167 S = 1.06 1054 re¯ections 75 parameters H-atom parameters constrained w = 1/[2(F2 o) + (0.0889P)2 + 0.338P] where P = (F2 o + 2F2 c)/3 (/)max < 0.001 max = 0.25 e AÊ ÿ3 min = ÿ0.30 e AÊ ÿ3 For isomer (I), the space group P21/c was uniquely assigned from the systematic absences. For isomer (II), the systematic absences permitted P21 and P21/m as possible space groups; since the unit-cell volume suggested Z = 2, space group P21 was selected and subse- quently con®rmed by the successful structure analysis. For isomer (III), the systematic absences permitted C2/c and Cc as possible space groups; C2/c was selected and con®rmed by the successful structure 219 Table 1 Hydrogen-bond geometry (AÊ , ) for (I). Cg is the centroid of the C11±C16 ring. DÐH A DÐH H A D A DÐH A C3ÐH3B O21i 0.99 2.42 3.253 (2) 141 C2ÐH2B Cgii 0.99 2.75 3.638 (2) 149 Symmetry codes: (i) ÿx 1; ÿy 1; ÿz 1; (ii) ÿx 1; y ÿ 1 2; ÿz 3 2. Table 2 Hydrogen-bond geometry (AÊ , ) for (II). DÐH A DÐH H A D A DÐH A C3ÐH3A O31v 0.99 2.48 3.193 (2) 129 C3ÐH3A O1vi 0.99 2.47 3.158 (3) 127 C16ÐH16 O4vii 0.95 2.37 3.162 (2) 141 Symmetry codes: (v) x; y; z ÿ 1; (vi) ÿx; y 1 2; ÿz; (vii) x ÿ 1; y; z. Table 2 Hydrogen-bond geometry (AÊ , ) for (II). DÐH A DÐH H A D A DÐH A C3ÐH3A O31v 0.99 2.48 3.193 (2) 129 C3ÐH3A O1vi 0.99 2.47 3.158 (3) 127 C16ÐH16 O4vii 0.95 2.37 3.162 (2) 141 Symmetry codes: (v) x; y; z ÿ 1; (vi) ÿx; y 1 2; ÿz; (vii) x ÿ 1; y; z. Experimental Data collection 1067 re¯ections with I > 2(I) Rint = 0.042 max = 27.4 h = ÿ8 ! 8 k = ÿ8 ! 9 l = ÿ12 ! 13 For the preparation of compounds (I)±(III), ®nely ground mixtures containing equimolar quantities of succinic anhydride and the appropriate nitroaniline were heated in an oil bath at 473 K until effervescence ceased. The resulting solids were cooled to ambient temperature and dissolved in chloroform. Activated charcoal was added and the mixtures were then heated under re¯ux for 10 min; this process was followed by ®ltration of the hot mixtures. After removal of the solvent under reduced pressure, crystallization of the solid products from ethanol gave crystals suitable for single-crystal X-ray diffraction. For the preparation of compounds (I)±(III), ®nely ground mixtures containing equimolar quantities of succinic anhydride and the appropriate nitroaniline were heated in an oil bath at 473 K until effervescence ceased. The resulting solids were cooled to ambient temperature and dissolved in chloroform. Activated charcoal was added and the mixtures were then heated under re¯ux for 10 min; this process was followed by ®ltration of the hot mixtures. After removal of the solvent under reduced pressure, crystallization of the solid products from ethanol gave crystals suitable for single-crystal X-ray diffraction. Compound (I) Crystal data C10H8N2O4 Mr = 220.18 Monoclinic, P21=c a = 8.3703 (2) AÊ b = 8.2500 (1) AÊ c = 14.1375 (3) AÊ = 101.0185 (10) V = 958.27 (3) AÊ 3 Z = 4 Dx = 1.526 Mg mÿ3 Mo K radiation Cell parameters from 2192 re¯ections = 3.5±27.5 = 0.12 mmÿ1 T = 120 (2) K Block, yellow 0.15 0.15 0.10 mm Data collection Nonius KappaCCD diffractometer ' and ! scans Absorption correction: multi-scan (SADABS; Sheldrick, 2003) Tmin = 0.976, Tmax = 0.988 12 771 measured re¯ections 2192 independent re¯ections 1846 re¯ections with I > 2(I) Rint = 0.031 max = 27.5 h = ÿ10 ! 10 k = ÿ10 ! 10 l = ÿ18 ! Experimental 18 Re®nement Re®nement on F 2 R[F 2 > 2(F 2)] = 0.043 wR(F 2) = 0.114 S = 1.10 2192 re¯ections 146 parameters H-atom parameters constrained w = 1/[2(F 2 o) + (0.0646P)2 + 0.2225P] where P = (F2 o + 2F2 c)/3 (/)max < 0.001 max = 0.35 e AÊ ÿ3 min = ÿ0.35 e AÊ ÿ3 Extinction correction: SHELXL97 Extinction coef®cient: 0.081 (7) Compound (II) Crystal data C10H8N2O4 Mr = 220.18 Monoclinic, P21 a = 6.6318 (2) AÊ b = 7.0944 (3) AÊ c = 10.4260 (5) AÊ = 108.234 (2) V = 465.90 (3) AÊ 3 Z = 2 Dx = 1.570 Mg mÿ3 Mo K radiation Cell parameters from 1152 re¯ections = 3.2±27.4 = 0.12 mmÿ1 T = 120 (2) K Block, colourless 0.40 0.35 0.30 mm Nonius KappaCCD diffractometer ' scans, and ! scans with offsets Absorption correction: multi-scan (SORTAV; Blessing, 1995, 1997) Tmin = 0.947, Tmax = 0.964 5403 measured re¯ections 1152 independent re¯ections 1067 re¯ections with I > 2(I) Rint = 0.042 max = 27.4 h = ÿ8 ! 8 k = ÿ8 ! 9 l = ÿ12 ! 13 Re®nement Re®nement on F 2 R[F 2 > 2(F 2)] = 0.031 wR(F 2) = 0.078 S = 1.08 1152 re¯ections 146 parameters H-atom parameters constrained w = 1/[2(F2 o) + (0.0485P)2 + 0.043P] where P = (F2 o + 2F2 c)/3 (/)max < 0.001 max = 0.22 e AÊ ÿ3 min = ÿ0.21 e AÊ ÿ3 Extinction correction: SHELXL97 Extinction coef®cient: 0.112 (14) Compound (III) Crystal data C10H8N2O4 Mr = 220.18 Monoclinic, C2=c a = 10.3731 (19) AÊ b = 11.590 (2) AÊ c = 7.9761 (18) AÊ = 108.135 (16) V = 911.3 (3) AÊ 3 Z = 4 Dx = 1.605 Mg mÿ3 Mo K radiation Cell parameters from 1054 re¯ections = 3.3±27.6 = 0.13 mmÿ1 T = 120 (2) K Lath, colourless 0.25 0.11 0.03 mm Data collection Nonius KappaCCD diffractometer ' and ! scans Absorption correction: multi-scan (SADABS; Sheldrick, 2003) Tmin = 0.974, Tmax = 0.996 9627 measured re¯ections 1054 independent re¯ections 690 re¯ections with I > 2(I) Rint = 0.095 max = 27.6 h = ÿ13 ! 13 k = ÿ15 ! 15 l = ÿ10 ! Acta Cryst. (2005). C61, o216±o220 Re®nement w = 1/[2(F2 o) + (0.0485P)2 + 0.043P] where P = (F2 o + 2F2 c)/3 (/)max < 0.001 max = 0.22 e AÊ ÿ3 min = ÿ0.21 e AÊ ÿ3 Extinction correction: SHELXL97 Extinction coef®cient: 0.112 (14) Table 2 Hydrogen-bond geometry (AÊ , ) for (II). Table 1 Hydrogen-bond geometry (AÊ , ) for (I). Cg is the centroid of the C11±C16 ring. organic compounds analysis. All H atoms were located from difference maps and then treated as riding atoms, with CÐH distances of 0.95 (aromatic) or 0.99 AÊ (CH2) and Uiso(H) values of 1.2Ueq(C). In the absence of any signi®cant anomalous dispersion, the Flack (1983) parameter for isomer (II) was indeterminate (Flack & Bernardinelli, 2000). Hence, it was not possible to determine the absolute con®guration of the molecules in the crystal selected for study (Jones, 1986); however, this con®guration has no chemical signi®cance. Accordingly, the Friedel pairs were merged prior to the ®nal re®nements. The data-to-para- meter ratio for isomer (II) is thus rather low, only 7.89, although the data set is 99.8% complete to = 27.43. Supplementary data for this paper are available from the IUCr electronic archives (Reference: SK1817). Services for accessing these data are described at the back of the journal. Compound (II) For isomer (I), the space group P21/c was uniquely assigned from the systematic absences. For isomer (II), the systematic absences permitted P21 and P21/m as possible space groups; since the unit-cell volume suggested Z = 2, space group P21 was selected and subse- quently con®rmed by the successful structure analysis. For isomer (III), the systematic absences permitted C2/c and Cc as possible space groups; C2/c was selected and con®rmed by the successful structure o219 Glidewell et al. Three isomers of C10H8N2O4 Acta Cryst. (2005). C61, o216±o220 o220 Glidewell et al. Three isomers of C10H8N2O4 References Allen, F. H. (2002). Acta Cryst. B58, 380±388. Allen, F. H., Baalham, C. A., Lommerse, J. P. M. & Raithby, P. R. (1998). Acta Cryst. B54, 320±329. Batten, S. R. & Robson, R. (1998). Angew. Chem. Int. Ed. 37, 1460±1494. Bernstein, J., Davis, R. E., Shimoni, L. & Chang, N.-L. (1995). Angew. Chem Batten, S. R. & Robson, R. (1998). Angew. Chem. Int. Ed. 37, 1460±1494. B t i J D i R E Shi i L & Ch N L (1995) A Ch Int. Ed. Engl. 34, 1555±1573. Int. Ed. Engl. 34, 1555±1573. g Blessing, R. H. (1995). Acta Cryst. A51, 33±37. For compounds (I) and (III), data collection: COLLECT (Hooft, 1999); cell re®nement: DENZO (Otwinowski & Minor, 1997) and COLLECT; data reduction: DENZO and COLLECT. For compound (II), data collection: KappaCCD Server Software (Nonius, 1997); cell re®nement: DENZO±SMN (Otwinowski & Minor, 1997); data reduction: DENZO±SMN. For all compounds, structure solu- tion: OSCAIL (McArdle, 2003) and SHELXS97 (Sheldrick, 1997); structure re®nement: OSCAIL and SHELXL97 (Sheldrick, 1997); molecular graphics: PLATON (Spek, 2003); software used to prepare material for publication: SHELXL97 and PRPKAPPA (Ferguson, 1999). Blessing, R. H. (1997). J. Appl. Cryst. 30, 421±426. Cremer, D. & Pople, J. A. (1975). J. Am. Chem. Soc. 97, 1354 Ferguson, G. (1999). PRPKAPPA. University of Guelph, Canada. Flack, H. D. (1983). Acta Cryst. A39, 876±881. Flack, H. D. & Bernardinelli, G. (2000). J. Appl. Cryst. 33, 11 Fruk, L. & Graham, D. (2003). Heterocycles, 60, 2307±2313. Hooft, R. W. W. (1999). COLLECT. Nonius BV, Delft, The Netherlands. Jones, P. G. (1986). Acta Cryst. A42, 57. Kwiatkowski, W. & Karolak-Wojciechowska, J. (1992). Acta Cryst. C48, 204±206. Kwiatkowski, W. & Karolak-Wojciechowska, J. (1992). Acta McArdle, P. (2003). OSCAIL for Windows. Version 10. Crystallography Centre, Chemistry Department, NUI Galway, Ireland. Moreno-Fuquen, R., Valencia, H., Abonia, R., Kennedy, A. R. & Graham, D. (2003). Acta Cryst. E59, o1717±o1718. Nonius (1997). KappaCCD Server Software. Windows 3.11 Version. Nonius BV, Delft, The Netherlands. X-ray data were collected at the EPSRC X-ray Crystal- lographic Service, University of Southampton, England; the authors thank the staff for all their help and advice. JNL thanks NCR Self-Service, Dundee, for grants which have provided computing facilities for this work. JLW thanks CNPq and FAPERJ for ®nancial support. Otwinowski, Z. & Minor, W. (1997). Methods in Enzymology, Vol. 276, Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp. 307±326. Acta Cryst. (2005). C61, o216±o220 References New York: Academic Press. Otwinowski, Z. & Minor, W. (1997). Methods in Enzymology, Vol. 276, M l l C t ll h P t A dit d b C W C t J & R M Macromolecular Crystallography, Part A, edited by C. W. Carter Jr & R. M. Sweet, pp. 307±326. New York: Academic Press. Sheldrick, G. M. (1997). SHELXS97 and SHELXL97. University of GoÈttingen, Germany. Sheldrick, G. M. (2003). SADABS. Version 2.10. University of GoÈttingen, Germany. Spek, A. L. (2003). J. Appl. Cryst. 36, 7±13. o220 Acta Cryst. (2005). C61, o216±o220
https://openalex.org/W3097515795	https://www.granthaalayahpublication.org/journals/index.php/granthaalayah/article/download/IJRG20_B06_3497/464	English	null	ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES AGAINST NEISSERIA GONORRHOEAE AND CANDIDA ALBICANS	International journal of research - granthaalayah	2,020	cc-by	5,225	ISSN (Online): 2350-0530 ISSN (Print): 2394-3629 ABSTRACT This Research on the antibacterial and antifungal activity of nanosilver against Neisseria gonorrhoeae and Candida albincas fungi has been carried out. The purpose of this study was to determine antibacterial activity of nanosilver against Neisseria gonorrhoeae and antifungal activity against Candida albincas. Synthesis Nanosilver uses bottom up method and characterized using UV-Vis Spectrophotometer. Nanosliver concentrations used were 30, 40, 50, and 60 ppm. Antibacterial and antifungal activity tests using disk diffusion method. Observations obtained in form of the presence or absence of clear zones formed around paper discs indicate the inhibition of nanosilver on microbial growth. The results of testing the antifungal activity of Candida albicans on nanosilver with concentrations of 30, 40, 50 and 60 ppm resulted in clear zones of 9.73 nm, 11.46 nm, 11.93 nm, and 13 nm with fungal inhibition response categories is medium and strong. The results antibacterial activity test of Neisseria gonorrhoeae on nanosilver with concentrations of 30, 40, 50 and 60 ppm did not show any clear zone around the disc, it showed that nanosilver in this study did not have antibacterial activity against Neisseria gonorrhoeae. Article Citation: Tasha Anandya Tantyani, and Titik Taufikurohmah. (2020). ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES AGAINST NEISSERIA GONORRHOEAE AND CANDIDA ALBICANS. International Journal of Research - GRANTHAALAYAH, 8(6), 179-187. https://doi.org/10.29121/granthaa layah.v8.i6.2020.461 Article Citation: Tasha Anandya Tantyani, and Titik Taufikurohmah. (2020). ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES AGAINST NEISSERIA GONORRHOEAE AND CANDIDA ALBICANS. International Journal of Research - GRANTHAALAYAH, 8(6), 179-187. https://doi.org/10.29121/granthaa layah.v8.i6.2020.461 Received Date: 09 May 2020 Accepted Date: 29 June 2020 Keywords: Nanosilver Antibacterial Activity Antifungal Activity Disk Diffusion Neisseria Gonorrhoeae Candida Albicans ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF SILVER NANOPARTICLES AGAINST NEISSERIA GONORRHOEAE AND CANDIDA ALBICANS Tasha Anandya Tantyani 1, Titik Taufikurohmah 1 Tasha Anandya Tantyani 1, Titik Taufikurohmah 1 *1 Department of Chemistry, Faculty of Mathematic and Natural Science, Universitas Negeri Surabaya, Indonesia © 2020 The Author(s). 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. 179 © 2020 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution Licen nd reproduction in any medium, provided the original author and source are credited. 1. INTRODUCTION Thin (1983) states that the most common cause of vaginal candidiasis is candida albicans, which is 81%, the rest is 16% by torulopsis glabarata, while the other 3% is caused by Candida tropicalis, Candida pseudotropicalis, Candida krusei and Candida stellatoidea [8]. Candidiasis vaginalis can occur in women of all ages, especially at puberty. The most prominent complaint in patients with vaginal candidiasis is vaginal itching accompanied by discharge of the vaginal body (fluor albus). Sometimes also found irritation, burning and dyspareunia. In the acute situation, the body of the vagina is runny while the chronic is thicker. Candidiasis that has entered the bloodstream can spread to various organs such as the kidneys, spleen, heart, brain, and cause various diseases such as endocarditis, meningitis, endophthalmitis and pyelonephritis [9]. Various attempts have been made to reduce various infectious diseases of the vagina that require antimicrobial agents to inhibit or kill bacteria and fungi that cause vaginal infections. Now many new technologies have been developed that are used to overcome problems for microbes such as bacteria and fungi. One of them is nanotechnology. Nanotechnology is the science of technology that uses the properties of molecular structures or atomic structures or materials in nanometer sizes. Nanometer-sized materials have better properties than large- sized materials. A nanomaterial or nanoparticle is a particle with the size of a nanometer (1-100 nm) [10]. One of the nanoparticles that has been developed is silver or nanosilver. Nanosilver can be obtained by synthesis using the bottom-up method, which is a synthesis process that involves chemical reactions by growing nanoparticles from a number of starting materials so that other nanometer-sized materials are produced [11]. Nanosilver has a very broad antimicrobial spectrum including antiviral activity such as HIV-AID and HSV herpes virus [12]. Nanosilver is proven to have the ability, among others, as an antibacterial and antifungal [13]. Silver nanoparticles (AgNP) also have antifungal, anti-inflammatory, antiviral, and antiplatelet activity [14]. Nanosilver as a strong antibacterial because of its chemically reactive and easily ionized nature and antimicrobial ability of nanosilver can kill all pathogenic microorganisms and there have not been reported any silver-resistant microbes [15]. 1. INTRODUCTION In this era of globalization there have been many advances in all fields and aspects of science about the importance of hygiene and also the basic health of physical and organ hygiene. One of the organs that really needs special care is the reproductive organs. Reproductive organs are one of the important things in every human life. Reproductive organs are the subject of several diseases. Early knowledge and understanding of the health of reproductive organs can be used as prevention for men and women so that they will be better able to maintain the health of their reproductive organs.[1]. In women, the vaginal canal is very vulnerable to risk of infection from the outside. In addition, the wrong way to clean the vagina and leave the condition of the vagina moist also triggers the disease in female reproductive organs [2]. In women, there is a term called Vaginal Infection. Vaginal infection is one of the most frequent problems where women visit a doctor [3]. One example of vaginal infection is vaginitis. Vaginitis Antibacterial and Antifungal Activity of Silver Nanoparticles Against Neisseria Gonorrhoeae and Candida Albicans Antibacterial and Antifungal Activity of Silver Nanoparticles Against Neisseria Gonorrh is inflammation and vaginal infections commonly encountered in clinical medicine [4]. Bacterial vaginosis, candidiasis vaginalis (an infection caused by Candida spesies) is responsible for the majority of vaginal infections in women, especially in women of reproductive age [5]. An example of the most serious disease in bacterial vaginosis is Gonorrhea disease caused by the bacterium Neisseria gonorrhoeae is Sexually Transmitted Disease (STD). g Gonorrhea in Indonesia ranks highest of all STD. According to the World Health Organization (WHO, 2011) as many as 70% of female patients and some male patients infected with gonorrhoeae or chlamydia have asymptomatic symptoms. Sexually transmitted diseases are also the most common cause of infertility, especially in women. Several studies in Surabaya, Jakarta and Bandung in female sex workers show that the prevalence of gonorrhoeae ranges from 7.4% - 50% [6]. Besides gonorrhea, there are also candidiasis vaginalis which is one of the vaginal infections caused by fungus Candida species especially Candida albicans. Candida albicans is a normal flora that lives in oral mucrosa, respiratory tract, digestive tract and in the vagina [7]. Candida albicans with excessive amounts in the body can cause disease. One example is Candidiasis vaginalis. International Journal of Research -GRANTHAALAYAH International Journal of Research -GRANTHAALAYAH MATERIALS This research was carried out using several materials including 250 ml beaker glass, 10 ml beaker glass, 50 ml beaker glass, hot plate and stirrer, Ohauss analytical balance, spatula, 100 ml measuring flask, Shimadzu UV-Vis Spectrophotometer 1800, micropipette, autoclave, incubator, calipers, ose needles, tweezers, vortex, petri dishes, AgNO3 (≥99%, Merck), sodium citrate (99%, Merck), aquades (PT Bratachem), aquabides (PT Bratachem), liquid media Potato Dextrosa Agar (DPA), ketocenazole, dimethyl sufoxide (DMSO), Neisseria gonnorhoeae stock colony, Candida albicans stock colony, filter paper, paper discs, Ciprofloxacin. PREPARATION OF AgNO3 SOLUTION 1000 ppm AgNO3 solution was obtained by dissolving 1.57 g of AgNO3 crystal (colored white) which was put into a 1000 ml volumetric flask, then diluted with aquabides to mark limits.Then the mixture is homogeneous and a 1000 ppm AgNO3 solution is formed which is ready to be used as material in nanosilver synthesis. 1000 ppm AgNO3 is used for synthesis. ANTIFUNGAL ASSAY OF CANDIDA ALBICANS USING DISC DIFFUSION METHOD The steps of make a specific culture media for Candida albicans are embed Candida albicans stock on SDB (Sabour Dextrosa Broth) media by applying 1 ose needle pure culture Candida albicans then incubated for 24 hours at 37°C. Antifungal assay use sterile paper discs dropped with 10 microliter nanosilver, then the paper discs containing nanosilver are placed on the surface of SDB media and incubated for 48 hours. The positive control used ketokenazol and negative control used aquadest. The results of observations can be measured use calipers. 1. INTRODUCTION The antibacterial activity of silver nanoparticles is influenced by several things, such as the concentration of silver nanoparticles, the shape of silver nanoparticles, the size of silver nanoparticles, the type of bacteria, the number of bacterial colonies and the contact time of silver nanoparticles with bacteria [16].The antibacterial ability of silver nanoparticles include damaging bacterial cell walls, disrupting cell metabolism, and inhibiting bacterial cell synthesis. Nanosilver can approach the microbial cell membrane and penetrate into the microbe. Furthermore, the nanosilver diffuses and attacks the respiratory chain of the microbes, so it can kill the microbes [17]. Nanosilver is a natural mineral that is non-toxic and safe for daily use. The combined minerals are also found in groundwater or natural water sources [18]. In this research, basic ingredients of nanosilver in the form of AgNO3 by reducing sodium citrate in aquo media [19]. Synthesis of nanosilver was carried out by bottom-up method using sodium citrate as a reducing agent. Several variations of nanosilver concentrations were performed by 30, 40, 50, 60 ppm. Nanosilver with several variations of concentration will be analyzed by UV-Vis spectrophotometer at a wavelength of 400-513 nm to determine the peak absorbance. In the antibacterial assay of Neisseria gonorrhoeae and antifungal assay of Candida albicans used disc diffusion method. The observation results obtained are the presence or absence of clear areas formed around the disc paper which shows inhibition zones on microbial growth. International Journal of Research -GRANTHAALAYAH 180 SYNTHESIS OF NANOSILVER Synthesis of nanisilver was carried out by the bottom up method by heating 200 ml of distilled water to boiling, then adding sodium gratrate 0.4 g and 1000 ppm 4 ml AgNO3 solution for a concentration of 20 ppm. Warming is continued until the solution turns from colorless to yellow then grayish yellow for about 15-20 minutes. Furthermore, the colloid is cooled at room temperature. Synthesis was continued with variations of concentrations of 30, 40, 50, 60 ppm with a volume of 1000 ppm AgNO3 solution of 6, 8, 10, 12 ml. The synthesized nanosilver measured its maximum wavelength on a UV-Vis spectrophotometer in the wavelength range of 400-513 nm. ANTIBACTERIAL ASSAY OF NEISSERIA GONORRHOEAE USING DISC DIFFUS The steps taken in this antibacterial assay are make a suspension of the bacteria Neisseria gonorrhoeae with a turbidity of 1mc farland then insert a sterile swab into the suspension of the Neisseria gonorrhoeae and rubbed evenly into the MH plate media then saturate the disc paper in nanosilver for 15 minutes and insert the saturated disc paper into the MH plate media. The positive control used 5 mcg ciprofloxacin antibiotic disk and negative control used aquadest. Incubation at 35°-37°C for 24 hours. The results of observations can be measured use calipers. International Journal of Research -GRANTHAALAYAH SYNTHESIS AND CHARACTERIZAION OF NANOSILVER Synthesis of Nanosilver use bottom up method which is reducing chemicals by using sodium citrate. In synthesis of nanosilver with this method produces a change in the color of solution from initially colorless to stable yellow, so that heating can be stopped. The colorless solution indicates that there is no interaction with each other between Ag 181 Antifungal Activity of Silver Nanoparticles Against Neisseria Gonorrhoeae and Candida Albican Antibacterial and Antifungal Activity of Silver Nanoparticles Against Neisseria Gonorrhoeae a atoms, while the stable yellow color indicates that Ag particles enter nano size and the growth of particle size (cluster) is getting bigger. The results synthesis are brownish-yellow colloids as shown in Figure 1. Figure 1: Nanosilver colloids with concentrations of 30, 40, 50, 60 ppm Figure 1: Nanosilver colloids with concentrations of 30, 40, 50, 60 ppm There is a difference in the intensity of the brownish yellow color that occurs in the results of nanosilver synthesis, the greater the concentration of nanosilver shows the more concentrated brownish yellow color. This happens because there are differences in the size of the cluster produced. If the concentration of nanosilver is greater then more clusters will form so that the color intensity results will be stronger. Silver atoms will interact with their fellow metal bonds and produce large numbers of nano clusters. Reaction that occurs in synthesis of nanosilver is written in equation (1). 4Ag+ + C6H5O7Na3 + 2H2O → 4Ag0 +C6H5O7H3 + 3Na+ + H+ + O2 4Ag+ + C6H5O7Na3 + 2H2O → 4Ag0 +C6H5O7H3 + 3Na+ + H+ + O2 (1) Nanosilver was then characterized by a UV-Vis Spectrophotometer in the wavelength range of 300-450 nm to determine the maximum wavelength of nanosilver used to measure the cluster diameter. Cluster diameter of nanosilver was calculated using the Brush equation. The results of the characterization are shown in Table 1. Nanosilver was then characterized by a UV-Vis Spectrophotometer in the wavelength range of 300-450 nm to determine the maximum wavelength of nanosilver used to measure the cluster diameter. Cluster diameter of nanosilver was calculated using the Brush equation. The results of the characterization are shown in Table 1. Table 1: Maximum wavelength and diameter of nanosilver cluster Concentration of Nanosilver λ (nm) Diameter of nanosilver cluster 30 425 18,14 40 421 17,10 50 416 16,93 60 410 16,69 Table 1: Maximum wavelength and diameter of nanosilver cluster International Journal of Research -GRANTHAALAYAH ANTIBACTERIAL ASSAY OF NEISSERIA GONORRHOEAE USING DISC DIFFUSION METHOD Nanosilver with concentration of 30,40,50 and 60 ppm then tested for antibacterial activity against Neisseria gonorrhoeae. In this study, antibacterial assay use disc diffusion method that’s make a suspension of Neisseria gonorrhoeae with a turbidity of 1mc farland then insert a sterile swab into the suspension of the Neisseria gonorrhoeae and rubbed evenly into the MH plate media then saturate the disc paper in nanosilver for 15 minutes and insert the saturated disc paper into the MH plate media. The positive control used 5 mcg ciprofloxacin antibiotic disk and negative control used aquadest. Incubation at 35°-37°C for 24 hours. The results of research using this method are the clear zone diameters that occur around the paper disk. The results of the qualitative test antibacterial activity of nanosilver against Neisseria gonorrhoeae as shown in Figure 2. and the results of the qualitative test antibacterial activity of positive control (Ciprofloxacin) against Neisseria gonorrhoeae as shown in Figure 3. 182 Tasha Anandya Tantyani, and Titik Taufikurohmah Figure 2: Result of antibacterial assay of nanosilver with concentration 30,40,50, and 60 ppm against Neisseria gonorrhoeae. (a) Replication I (b) Replication II Tasha Anandya Tantyani, and Titik Taufikurohmah Figure 2: Result of antibacterial assay of nanosilver with concentration 30,40,50, and 60 ppm against Neisseria gonorrhoeae. (a) Replication I (b) Replication II Figure 3: Results of the qualitative test antibacterial activity of positive control (Ciprofloxacin) against Neisseria gonorrhoeae Figure 3: Results of the qualitative test antibacterial activity of positive control (Ciprofloxacin) against Neisseria gonorrhoeae The quantitative test results of the antibacterial assay of nanosilver which is diameter of clear zone can be sured using the calipers shown in Table 2. The quantitative test results of the antibacterial assay of nanosilver which is diameter of clear zone can be measured using the calipers shown in Table 2. ANTIBACTERIAL ASSAY OF NEISSERIA GONORRHOEAE USING DISC DIFFUSION METHOD ne diameter of the antibacterial test on nanosilver against Neisseria gonorrhoeae S l Cl di ( ) Table 2: Clear zone diameter of the antibacterial test on nanosilver against Nei Table 2: Clear zone diameter of the antibacterial test on nanosilver against Neisseria gonorrhoeae Sample Clear zone diameter (mm) I II III IV Nanosilver 30 ppm 6 6 6 6 Nanosilver 40 ppm 6 6 6 6 Nanosilver 50 ppm 6 6 6 6 Nanosilver 60 ppm 6 6 6 6 Control (+) 48 - - - Control (-) 6 - - - In this study, four replications were carried out and from the results of the clear zone diameter it can be stated that the nanosilver in this study does not have antibacterial activity against Neisseria gonorrhoeae, it can be known because there is no clear zone diameter that occurs around the disc paper that has been saturated on the nanosilver. The criteria for antibacterial activity according to Muharni (2016) [20] were clear zone diameters <10 nm included in the weak category, 10-16 nm included in the moderate category and >16 nm included in the strong category. In this study, four replications were carried out and from the results of the clear zone diameter it can be stated that the nanosilver in this study does not have antibacterial activity against Neisseria gonorrhoeae, it can be known because there is no clear zone diameter that occurs around the disc paper that has been saturated on the nanosilver. The criteria for antibacterial activity according to Muharni (2016) [20] were clear zone diameters <10 nm included in the weak category, 10-16 nm included in the moderate category and >16 nm included in the strong category. International Journal of Research -GRANTHAALAYAH 183 Antibacterial and Antifungal Activity of Silver Nanoparticles Against Neisseria Gonorrhoeae and Candida Albicans Antibacterial and Antifungal Activity of Silver Nanoparticles Against Neisseria Gonorrhoeae and Candida Albicans Nanosilver has good antibacterial activity but there is no research that states that nanosilver can inhibit the growth of the bacteria Neisseria gonorrhoeae. In this study, nanosilver used concentrations of 30, 40, 50 and 60 ppm and nanosilver was allowed to stand for 2 weeks after the synthesis process, after that testing was carried out on the Neisseria gonorrhoeae bacteria, it also could reduce the antibacterial properties of the nanosilver because it was not directly tested after the process synthesis is done. ANTIBACTERIAL ASSAY OF NEISSERIA GONORRHOEAE USING DISC DIFFUSION METHOD The absence of antibacterial activity in this study can also occur because the concentration of nanosilver used is too small or not optimal to inhibit the growth of Neisseria gonorrhoeae. N. gonorrheae is a gram-negative bacterium that has a selection system for certain substances. According to Muharini (2017) [21], the structure of gram negative bacteria also affects the results of bacterial inhibition zones because it has a more complex structure with three layers, namely the outer layer in the form of lipoprotein, the middle layer in the form of lipopolysaccharide and the inner layer in the form of peptidoglycan, which causes Gram-negative bacterial cell walls are more difficult to penetrate by antibacterial compounds and gram-negative bacteria have the property of less susceptible to some antibacterial compounds. In negative control area (aquades) no clear zone appears. The absence of this clear zone indicates that aquades do not have antibacterial properties and can not inhibit the growth of Neisseria gonorrhoeae bacteria while in the positive control area (ciprofloxacin) shows the presence of a clear zone diameter of 48 mm which indicates that the antibiotic ciprofloxacin has a strong antibacterial ability against Neisseria bacteria gonorrhoeae. ANTIFUNGAL ASSAY OF CANDIDA ALBICANS USING DISC DIFFUSION METH Table 3: Clear zone diameter of the antifungal test on nanosilver against Candida albicans Sample Clear zone diameter (mm) I II III IV Rata-rata Nanosilver 30 ppm 9,70 9,85 9,85 9,0 9,73 Nanosilver 40 ppm 11,40 11,25 10,95 12,25 11,46 Nanosilver 50 ppm 11,70 13,25 10,80 11,95 11,93 Nanosilver 60 ppm 13,30 12,95 12,70 13,05 13 Control (+) 15,65 16,25 13,10 19,65 16,16 Control (-) - - - - - Figure 6: Graphs of nano silver concentrations with clear zone diameters 9.73 11.46 11.93 13 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70 diameter clear zone Concentration Graphs of nano silver concentrations with clear zone diameters Table 3: Clear zone diameter of the antifungal test on nanosilver against Candida albicans Sample Clear zone diameter (mm) I II III IV Rata-rata Nanosilver 30 ppm 9,70 9,85 9,85 9,0 9,73 Nanosilver 40 ppm 11,40 11,25 10,95 12,25 11,46 Nanosilver 50 ppm 11,70 13,25 10,80 11,95 11,93 Nanosilver 60 ppm 13,30 12,95 12,70 13,05 13 Control (+) 15,65 16,25 13,10 19,65 16,16 Control (-) - - - - - Figure 6: Graphs of nano silver concentrations with clear zone diameters 9.73 11.46 11.93 13 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70 diameter clear zone Concentration Graphs of nano silver concentrations with clear zone diameters Figure 6: Graphs of nano silver concentrations with clear zone diameters In this study, four replications were carried out and it can be seen from the results of the diameter of the clear zone that occurs around the disc that the nanosilver in this study has inhibitory properties as an antifungal against Candida albicans. The greater the concentration of nanosilver, the greater the diameter of the clear zone that occurs. In the nanosilver with a concentration of 30 ppm the clear zone diameter that occurred was 9.73 mm, the nanosilver with a concentration of 40 ppm the clear zone diameter that occurred was 11.46 nm, the nanosilver with a concentration of 50 ppm the clear zone diameter that occurred was 11.93 nm, and nanosilver with a concentration of 60 ppm diameter of a clear zone that occurs by 13 mm. International Journal of Research -GRANTHAALAYAH ANTIFUNGAL ASSAY OF CANDIDA ALBICANS USING DISC DIFFUSION METH Nanosilver with concentration of 30,40,50 and 60 ppm then tested for antifungal activity against Candida albicans. In this study, antifungal assay use disc diffusion method that’s make a specific culture media for Candida albicans are embed Candida albicans stock on SDB (Sabour Dextrosa Broth) media by applying 1 ose needle pure culture Candida albicans then incubated for 24 hours at 37°C. Antifungal assay use sterile paper discs dropped with 10 microliter nanosilver, then the paper discs containing nanosilver are placed on the surface of SDB media and incubated for 48 hours. The positive control used ketokenazol and negative control used aquadest. The results of research using this method are the clear zone diameters that occur around the paper disk. The results of the qualitative test antifungal activity of nanosilver against Candida albicans as shown in Figure 4. and the results of the qualitative test antifungal activity of positive control (ketokenazol) against Candida albicans as shown in Figure 5. Figure 4: Result of antifungal assay of nanosilver with concentration 30,40,50,60 ppm against Candida albicans Figure 4: Result of antifungal assay of nanosilver with concentration 30,40,50,60 ppm against Candida albicans Figure 4: Result of antifungal assay of nanosilver with concentration 30,40,50,60 pp Figure 5: Result of antifungal assay of Ketokenazol against Candida albicans Figure 5: Result of antifungal assay of Ketokenazol against Candida albicans 184 International Journal of Research -GRANTHAALAYAH International Journal of Research -GRANTHAALAYAH International Journal of Research -GRANTHAALAYAH Tasha Anandya Tantyani, and Titik Taufikurohmah The quantitative test results of the antifungal assay of nanosilver which is diameter of clear zone can be measured using the calipers shown in Table 3. and graphs of nano silver concentrations with clear zone diameters are shown in Figure 6. ANTIFUNGAL ASSAY OF CANDIDA ALBICANS USING DISC DIFFUSION METH According to Mandey and Londok (2014) [22], there are categories of antifungal activity based on inhibition zone diameter that occurs namely <5 mm is weak category, 5- 10 mm is moderate category, 10-20 mm is strong category, and >20 is very strong category. Nanosilver in this study included in medium and strong antifungal because at the concentration of 30 ppm the diameter of the inhibitory zone produced was in the range <10 mm or in the category of moderate antifungal and at concentrations of 40, 50, and 60 ppm the diameter of the inhibitory zone produced was included in the range of 10-20 mm or a strong antifungal category against Candida albicans. In the research of Keuk-jun et al (2008) [23], states that the mechanism by which nano-Ag breaks down the membrane permeability barrier, it is possible that nano-Ag perturbs the membrane lipid bilayers, causing the leakage of ions and other materials as well as forming pores and dissipating the electrical potential of the membrane and also the interaction between nano-Ag and the membrane structure. C. albicans cells, during nano-Ag exposure, show significant changes to their membranes, which are recognized by the formation of 'pits' on their surfaces, and finally, the result in the formation of pores and cell death. So nanosilver can inhibit the growth of Candida albicans through destruction of membrane integrity; therefore, it was concluded that nano-Ag has strong antifungal activity against Candida albicans. 185 Antibacterial and Antifungal Activity of Silver Nanoparticles Against Neisseria Gonorrhoeae and Candida Albicans The positive control used in this study was ketocenazole which produced clear zone diameter at 16.16 mm. Ketocenazole is also included in a powerful antifungal to inhibit the growth of Candida albicans. While the negative control used in this study is aquadest, in negative control (aquades) no clear zone appears around the disc. The absence of this clear zone indicates that aquades has no antifungal activity and can not inhibit the growth of Candida albicans. SOURCES OF FUNDING None. 4. CONCLUSION Based on the results of the study it can be seen that nanosilver in this study does not have antibacterial activity against Neisseria gonorrhoeae and can not inhibit the growth of Neisseria gonorrhoeae as indicated by the absence of clear zone that occur around the discs, but nanosilver in this study has a strong antifungal activity in inhibiting growth Candida albicans, with an increase in concentration on nanosilver can increase the growth inhibition of Candida albicans shown by a wider diameter of the clear zone that occurs around the disk. The clear zone diameters that occur in Candida albicans were at a conmcentration of 30 ppm at 9.73 mm, 40 ppm at 11.46 mm, 50 ppm at 11.93 nm, and 60 ppm at 13 mm. CONFLICT OF INTEREST None. ACKNOWLEDGMENT Thank you is conveyed to Prof. Dr. Titik Taufikurohmah, M.Si., who has funded all of activities in this study including nanosilver, antifungal and antibacterial activity test and UV-Vis Spectrophotometer analysis. Thank you is conveyed to Prof. Dr. Titik Taufikurohmah, M.Si., who has funded all of activities in this study including nanosilver, antifungal and antibacterial activity test and UV-Vis Spectrophotometer analysis. REFERENCES [1] Barbara Nash and Patricia Gilbert, Panduan Kesehatan Seksual. Penerjemah Khotibul Uman. Jakarta: Prestasi Pustaka, 2006. [1] Barbara Nash and Patricia Gilbert, Panduan Kesehatan Seksual. Penerjemah Khotibul Uman. Jakarta: Prestasi Pustaka, 2006. [2] Samini, Faktor-faktor yang Berhubungan dengan Kejadian Kandidiasis Vaginalis Pada Wanita. Surabaya: FKM Universitas Airlangga, 2001. [3] R.A Rosenblatt, D.C. Cherkin, R. Schneweiss, The structure and content of family practice: current status and future trends.: J Fam Pract, 1982. [3] R.A Rosenblatt, D.C. Cherkin, R. Schneweiss, The structure and content of family practice: current status and future trends.: J Fam Pract, 1982. [4] H. Hacer, B. Reyhan, and Y. Sibel, to determine of the prevalence of Bacterial Vaginosis, Candida sp, mixed infections (Bacterial Vaginosis + Candida sp), Trichomonas Vaginalis, Actinomyces sp in Turkish women from Ankara, Turkey. Turkey: Ginekol Pol, 2012. [4] H. Hacer, B. Reyhan, and Y. Sibel, to determine of the prevalence of Bacterial Vaginosis, Candida sp, mixed infections (Bacterial Vaginosis + Candida sp), Trichomonas Vaginalis, Actinomyces sp in Turkish women from Ankara, Turkey. Turkey: Ginekol Pol, 2012. y y [5] A. Spinillo, A.M. Bernuzzi, C. Cevini, R. Gulminetti, S. Luzi, A. Santolo, "The relationship of bacterial vaginosis, candida and trichomonas infection to symptomatic vaginitis in postmenopausal women attending a vaginitis clinic," Maturitas, p. 253, 1997. [5] A. Spinillo, A.M. Bernuzzi, C. Cevini, R. Gulminetti, S. Luzi, A. Santolo, "The relationship of bacterial vaginosis, candida and trichomonas infection to symptomatic vaginitis in postmenopausal women attending a vaginitis clinic," Maturitas, p. 253, 1997. [6] Ministry of Health of the Republic Indonesia. (2011, December) KEMKES. [Online]. https://www.kemkes.go.id/resources/download/profil/PROFIL_KES_PROV_2011/P.Prov.JATIM_11.pdf [6] Ministry of Health of the Republic Indonesia. (2011, December) KEMKES. [Online]. https://www.kemkes.go.id/resources/download/profil/PROFIL_KES_PROV_2011/P.Prov.JATIM_11.pdf [7] J.C.O. Sardi, L. Scorzoni, T. Bernardi, A.A.M. Fusco, and G.M.J.S. Mendes, "Candida species: current [6] Ministry of Health of the Republic Indonesia. (2011, December) KEMKES. [Online]. https://www.kemkes.go.id/resources/download/profil/PROFIL_KES_PROV_2011/P.Prov.JATIM_11.pdf [7] J.C.O. Sardi, L. Scorzoni, T. Bernardi, A.A.M. Fusco, and G.M.J.S. Mendes, "Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options.," l f d l b l https://www.kemkes.go.id/resources/download/profil/PROFIL_KES_PROV_2011/P.Prov.JATIM_11.pdf [7] J.C.O. Sardi, L. Scorzoni, T. Bernardi, A.A.M. Fusco, and G.M.J.S. Mendes, "Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options.," Journal of Medical Microbiology, pp. 10-24, 2013. J gy pp [8] S.G. Saydam, Waspadai Penyakit Reproduksi Anda. Bandung: Pustaka Reka Cipta, 2012. [9] F.H. Kayser, K.A. Bienz, J. Eckert, and R.M Zinkernagel, Medical Microbiology. N [10] S. Raj, S. Jose, U.S. International Journal of Research -GRANTHAALAYAH REFERENCES Sumod, and M Sabitha, "Nanotechnology in cosmetics: opportunities and challenges," Journal of Pharmacy and Bioallied Sciences, vol. 4, pp. 186-193, 2012. 186 Tasha Anandya Tantyani, and Titik Taufikurohmah [11] M. Abdullah, Virgius, Yudistira, Nirmin, and Khairurrijal, "Sintesis Nanomaterial," Jurnal Nanosains dan Nanoteknologi, pp. 33-57, 2008. [12] T. Taufikurohmah, D Soepardjo, and Rusmini, "Utilization of Nanogold And Nanosilver To Treat Herpes Disease: Case Study Of Herpes Transmission In Islamic Cottage Schools," Atlantis Highlights in Chemistry and Pharmaceutical Sciences, p. 88, 2019. [13] J.S. Kim, E. Kuk, K. Yu, J. Kim, S. Park, "Antimicrobial effects of silver nanoparticles," Nanomedicine, pp. 95-101, 2007. [14] L. Ge, Q. Li, M. Wang, J. Ouyang, M. Xing, "Nanosilver particles in medical applications: synthesis, performance, and toxicity," International Journal of Nanomedicine, vol. 9, pp. 2399-2407, 2014. [15] S. Prabhu and E.K. Poulose, "Review: Silver Nanoparticles: Mechanism of Antimicrobial Action, Synthesis, Medical Applications, and Toxicity Effects," International Nano Letters, vol. 02, pp. 1-10, 2012. [16] I. Sondi and B.S. Sondi, "Silver Nanoparticle as Antimicrobacterial Agent: a Case Study on E. coli as a Model for Gram-Negative Bacteria," J. ColloidInterface Sci., pp. 177-182, 2004. [17] M. Khalandi, N. Asadi, and M. Milani, "A review on potential role of silver nanoparticles and possible mechanisms of their actions on bac¬teria.," Drug Res (Stuttg), pp. 70-76, 2017. [18] Titik Taufikurohmah, Djodjok Soepardjo, Hari Armadianto, and Rusmini, "Synthesis and Characterization of Nanogold and Nanosilver as Leprosy Drug Candidates and Their Activity Tests in Leprosy Patients; Case Study," Advances in Computer Science Research, p. 23, 2019. [19] Titik Taufikurohmah, Djodjok Soepardjo, Hari Armadianto, and Rusmini, "Synthesis and Characterization of Nanogold-Nanosilver Cluster Diameter Using UV Visible Instruments and TEM Electron Microscope Transform Instruments," Advances in Social Science, Education and Humanities Research, p. 146, 2019. p d P Emil,. Palembang, Indonesia: Kab. Ogan Ilir: Universitas Sriwijaya, 2016. [20] Muharni, Elfita, and P Emil,. Palembang, Indonesia: Kab. Ogan Ilir: Universitas [21] Muharini, Fitrya, and S Farida, "Uji Aktivitas Antibakteri Ekstrak Etanol Tanaman Obat Suku Musi di Kabupaten Musi Banyuasin, Sumatera Selatan," Jurnal Kefarmasian Indonesia, pp. 127-135, 2017. [22] J.S. Mandey and J.J.M.R dan Londok, "Fitokimia dan aktivitas antimikroba daun sirsak (Annona muricata linn.) Sebagai kandidat bahan pakan ayam pedaging," Jurnal LPPM Bidang Sains dan Teknologi, 2014. [23] K Keukjun, S. Woosang, S. Bokyoung, M. Seokki, C. Jongsoo, K. Jongguk, L. REFERENCES Donggun, "Antifungal Activity and Mode of Action of Silver Nano-particles on Candida albicans," Journal Biology of Metals, 2008. [23] K Keukjun, S. Woosang, S. Bokyoung, M. Seokki, C. Jongsoo, K. Jongguk, L. Donggun, "Antifungal Activity and Mode of Action of Silver Nano-particles on Candida albicans," Journal Biology of Metals, 2008. 187 International Journal of Research -GRANTHAALAYAH
https://openalex.org/W4361805868	https://zenodo.org/records/7787506/files/IJISRT23MAR1248.pdf	English	null	Clinical Therapy of UTI in Children Under the Age of Five Varies Widely	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	8,902	Clinical Therapy of UTI in Children Under the Age of Five Varies Widely 2Dr. P.S.V.Lakshman Sai, Consultant Paediatrician, Rainbow Hospital, Andhrapradesh 1Dr.Venugopal Reddy.I, Medical Director and Consultant Paediatrician, Ovum Hospital, Bangalore Abstarct:- Urinary tract infection ( UTI) is a common bacterial infection in babies and children, counting for four to ten percent of febrile children admitted to sanitarium. It's frequently delicate to honor UTI in babies and youthful children due to the presenting symptoms and signs.E.coli is the most common bacterial cause of UTI, and its vulnerability patterns vary with geographical region. This study aims to identify the clinical, bacteriological, and radiological biographies of UTI and reduce the variability of clinical practise in the operation of UTI in children under the age of five. The study actors' age and coitus distribution, with 39(41.1) in the age group between 1 months and 12 months, 32(33.7) between 13 months and 48 months, and 24(25.3) between 49 months and 60 months, influences the frequence of UTI. UTI affects women more constantly than men across all age orders, with dysuria, frequence, abdominal pain, pungent urine, and fever being the most common symptoms.67.4 of babies with UTI had substantial pyuria, and 8 distinct species of bacteria were discovered in 95 societies. Colistin and amikacin were the two medicines that Pseudomonas SPP that causes UTI was most sensitive to, followed by CIP. 18 of the 95 kiddies had serious anomalies. This study set up that urinary tract infections( UTI) are more current in babies( 1- 12 months) and decline with age. The most common clinical symptom was fever, followed by dysuria and stomach pain.E. coli was the most common bacterium causing UTI, followed by klebsiella and proteus. After collecting urine for culture, a suspected UTI can be treated empirically with an aminoglycoside or a third generation cephalosporin, but there's a significant frequence of in- vitro resistance to amoxicillin and trimethoprim- sulfamethoxazole. When children were submitted to ultrasonography,18.9 of them displayed radiological abnormalities, with cystitis, pyelonephritis, and vesicourethral influx being the most frequent findings. MCU is needed to exclude VUR. frequently delicate to honor UTI in babies and youthful children because the presenting symptoms and signs are minimum or frequentlynon-specific. The typical trio of abdominal pain, puking and fever with chills, rigor or supra- pubic pain are common donations. UTI is one of the causes of serious bacterial illness in babies taking sanitarium admissions and has been associated with significant morbidity. Keywords: Paediatric Uti, Urinary Tract Infection, Infants, Abnormalities, Affected, Condition, Fever. Volume 8, Issue 3, March – 2023 Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Clinical Therapy of UTI in Children Under the Age of Five Varies Widely It has also been allowed to be get or contribute to, the development of renal scarring and latterly leading on to renal failure, hypertension and end stage renal complaint. Some children are at threat of developing UTI due to certain anatomic and physiologic factors, similar as vesicoureteric kickback( VUR).E.coli is the most common bacterial cause of UTI, and its vulnerability patterns vary with geographical region. The aetiology of pediatric UTI and antibiotic vulnerability of urinary pathogens in both the community and hospitals have been changing, and medicine resistance has come a major problem. It's important to diagnose this condition at the applicable time as it's a preventable cause of renal damage. In our position, no attestation has been done and this dearth of information could limit clinicians consideration for UTI while assessing children under five with fever. This study aims to ascertain the common presenting features, laboratory and radiological abnormalities generally seen.  Inclusion Criteria: Any child who is suspected of having a urinary tract infection and whose infection is later confirmed by a positive urine culture is between the ages of 1 month and 5 years.  Exclusion Criteria:  Exclusion Criteria:  Age <1 month and > 5 years.  Children with history of antibiotic intake less than 7 days to the day of enrolment.  Children under went urological manipulation such as catheterisation or with urinary tract anomaly.  Children with chronic illness such as severe PEM, malignancies, nephrotic syndrome, glomerulonephritis, chronic renal failure and HIV/ acquired immunodeficiency will also be excluded.  Microbiological And Radiological Methods: per Indian Society of Pediatric Nephrology (2010). Urine bitsy examination of a centrifuged sample for White blood cells was done. Urine instance were invested on Cysteine lysine electrolyte deficient( CLED) medium using a standard 1microml circle and incubated aerobically at 37 °C for 72 hours. After 72 hours of incubation, bacterial Depending on how the urine is collected, a certain number of bacteria must be present in order to diagnose UTI. Table 1 Criteria for the Diagnosis of UTI(3) Method of collection Colony count Probability of infection Suprapubic aspiration Any number of pathogens 99% Urethral catheterization >5×104 CFU/ml 95% Midstream clean catch >105 CFU/ml 90-95% Leukocyturia: Presence of > 5 WBCs/high power field in a centrifuged urine sample or more than 10 WBCs/mm3 in uncentrifuged urine. I. Urinary tract infection( UTI) is a common bacterial infection in babies and children. It's the third most common infection in the paediatric age group, counting for four to ten percent of febrile children admitted to sanitarium. It's  Children with definitive source of fever on examination. 1317 IJISRT23MAR1248 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023  Descriptive Maneuver:  Sample Collection: All childrens were subjected to Ultrasonographic examination of the abdomen of soon after the diagnosis of UTI. The Micturating cystourethrogram (MCU) was done before discharge of the child from hospital, while DMSA scan is carried out 2-3 months after treatment(3). For children under the age of two, clean catheterization was used to collect urine samples; for older children, midstream clean catch collection was used. Within an hour of collection, sterile vials were utilized to collect urine samples, which were then used for microscopy, culture, and sensitivity testing.  Study Definitions: As per Indian Society of Pediatric Nephrology (2010).  Descriptive Maneuver: colonies were linked grounded on characteristic social morphology, gram stain appearance and standard commercially set biochemical tests. Antimirobial vulnerability pattern of insulated bacterial pathogens were determined by Kirby Bauer prolixity system as per the Clinical Laboratory norms Institute( CLSI). The results were reported as sensitive, intermediate or repel ant to the agents that had been tested. A written consent from each child's parent or legal guardian was obtained before include any children who met the study's eligibility requirements. Background data on the patient's demographics, prior incidence of UTI, clinical presentation, family history of UTI/VUR drug use, prior interventions, and any additional complaints were gathered from the patient's guardian or parent.  Statistical Tools: The data were analysed using SPSS (Statistical Package for Social Science) Ver 20. Continuous data were analyzed for its mean, median and standard deviation (summary statistics). Categorical variable were analyzed using chi-square test and ‘p’ value of ≤ 0.05 will be considered as statistically significant. III. OBSERVATION AND RESULTS Table 2 Age Distribution of Study Participants Age Groups Frequency Percentage 1 - 12 months 39 41.1 13 – 48 months 32 33.7 49 – 60 months 24 25.3 Total 95 100 1318 IJISRT23MAR1248 IJISRT23MAR1248 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 ISSN No:-2456-2165 Fig 1 Age Distribution of Study Participants Fig 1 Age Distribution of Study Participants Fig 1 Age Distribution of Study Participants Out of the total 95 children, 39 (41%) were in the age group of 1-12 months, 32 (33.7%) in 13-48 months and 24 (25.3%) were in the age group of 49-60 months. Out of the total 95 children, 39 (41%) were in the age group of 1-12 months, 32 (33.7%) in 13-48 months and 24 (25.3%) were in the age group of 49-60 months. Table 3 Distribution of Sex Among Study Participants Sex Frequency Percentage Male 45 47.4 Female 50 52.6 Total 95 100 Fig 2 Distribution of Sex Among Study Participants Among these 95 children, 50 (52.6%) were females and 45 (47.4%) were males. Table 3 Distribution of Sex Among Study Participants Sex Frequency Percentage Male 45 47.4 Female 50 52.6 Total 95 100 Fig 2 Distribution of Sex Among Study Participants Among these 95 children 50 (52 6%) were females and 45 (47 4%) were males Fig 2 Distribution of Sex Among Study Participants Among these 95 children, 50 (52.6%) were females and 45 (47.4%) were males. Among these 95 children, 50 (52.6%) were females and 45 (47.4%) were males. 1319 www.ijisrt.com IJISRT23MAR1248 Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Table 4 Age and Gender Distribution Age Groups Sex Total Male Female 1 - 12 months 18 21 39 13 – 48 months 15 17 32 49 – 60 months 12 12 24 Total 45 50 95 Fig 3 Age and Gender Distribution Among the 95 study participants, 45(47.4%) were males and 50 (52.6%) were females.  Statistical Tools: According to age wise distribution 39 were in the age group of 1-12 months ,out of which 18 (46.2%) were males and 21 (53.8%) were females. In the 13-48 months age group 32 children were there, among that 15 (46.9%) were males and 17(53.1%) were females. Out of the 24 children in 49-60 age group, there was equal distribution of 12(50%) males and 12(50%) females. Table 5 Distribution of Fever Among Study Participants Volume 8, Issue 3, March – 2023 Fig 3 Age and Gender Distribution Fig 3 Age and Gender Distribution Among the 95 study participants, 45(47.4%) were males and 50 (52.6%) were females. According to age wise distribution 39 were in the age group of 1-12 months ,out of which 18 (46.2%) were males and 21 (53.8%) were females. In the 13-48 months age group 32 children were there, among that 15 (46.9%) were males and 17(53.1%) were females. Out of the 24 children in 49-60 age group, there was equal distribution of 12(50%) males and 12(50%) females. Among the 95 study participants, 45(47.4%) were males and 50 (52.6%) were females. According to age wise distribution 39 were in the age group of 1-12 months ,out of which 18 (46.2%) were males and 21 (53.8%) were females. In the 13-48 months age group 32 children were there, among that 15 (46.9%) were males and 17(53.1%) were females. Out of the 24 children in 49-60 age group, there was equal distribution of 12(50%) males and 12(50%) females. Table 5 Distribution of Fever Among Study Participants Fever Frequency Percentage Present 64 67.4 Absent 31 32.6 Total 95 100 Fig 4 Distribution of Fever Among Study Participants Out of 95 children, 64 (67.4%) presented with fever and 31 (32.6%) did not have fever at the time of the study. Table 5 Distribution of Fever Among Study Participants Fever Frequency Percentage Present 64 67.4 Absent 31 32.6 Total 95 100 Table 5 Distribution of Fever Among Study Participants Fever Frequency Percentage Present 64 67.4 Absent 31 32.6 Total 95 100 Fig 4 Distribution of Fever Among Study Participants Fig 4 Distribution of Fever Among Study Participants f 95 children, 64 (67.4%) presented with fever and 31 (32.6%) did not have fever at the time of the study. Out of 95 children, 64 (67.4%) presented with fever and 31 (32.6%) did not have fever at the time of the study.  Statistical Tools: 1320 IJISRT23MAR1248 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Table 6 Distribution of Abdominal Pain Among Study Participants Abdominal Pain Frequency Percentage Present 35 36.8 Absent 60 63.2 Total 95 100 Fig 5 Distribution of Abdominal Pain Among Study Participants Among the 95 study participants 35 (36 8%) were reported to be having abdominal pain and 60 (63 2%) without abdominal International Journal of Innovative Science and Research Technology Volume 8, Issue 3, March – 2023 ISSN No:-2456-2165 Table 6 Distribution of Abdominal Pain Among Study Participants Abdominal Pain Frequency Percentage Present 35 36.8 Absent 60 63.2 Total 95 100 Fig 5 Distribution of Abdominal Pain Among Study Participants Fig 5 Distribution of Abdominal Pain Among Study Participants Fig 5 Distribution of Abdominal Pain Among Study Participants Among the 95 study participants, 35 (36.8%) were reported to be having abdominal pain and 60 (63.2%) without abdominal i Among the 95 study participants, 35 (36.8%) were reported to be having abdominal pain and 60 (63.2%) without abdominal pain. p Table 7 Distribution of Nausea/Vomitting Among Study Participants Nausea/Vomiting Frequency Percentage Present 20 21.1 Absent 75 78.9 Total 95 100 Fig 6 Distribution of Nausea/Vomitting Among Study Participants Table 7 Distribution of Nausea/Vomitting Among Study Participants Nausea/Vomiting Frequency Percentage Present 20 21.1 Absent 75 78.9 Total 95 100 Table 7 Distribution of Nausea/Vomitting Among Study Participants Nausea/Vomiting Frequency Percentage Present 20 21.1 Absent 75 78.9 Total 95 100 Fig 6 Distribution of Nausea/Vomitting Among Study Participants Fig 6 Distribution of Nausea/Vomitting Among Study Participants When asked about the presence of Nausea/Vomitting, 20 (21%) were reported to have Nausea/Vomitting and 75 (79%) with out Nausea/Vomitting. When asked about the presence of Nausea/Vomitting, 20 (21%) were reported to have Nausea/Vomitting and 75 (79%) with out Nausea/Vomitting.  Statistical Tools: Table 8 Distribution of Smelly Urine Among Study Participants Smelly Urine Frequency Percentage Present 8 8.4 Absent 87 91.6 Total 95 100 www.ijisrt.com 1321 IJISRT23MAR1248 Volume 8, Issue 3, March – 2023 International Journal of Innovative Scienc Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 ISSN No:-2456-2165 Fig 7 Distribution of Smelly Urine Among Study Participants Fig 7 Distribution of Smelly Urine Among Study Participants Out of 95 children, 8 (8.4%) were found to be having smelly urine and 87 (91.6%)were not having so. ut of 95 children, 8 (8.4%) were found to be having smelly urine and 87 (91.6%)were not having so. Table 9 Distribution of Increased Frequency of Urine Among Study Participants Increased Frequency Frequency Percentage Present 11 11.6 Absent 84 88.4 Total 95 100 Fig 8 Distribution of Increased Frequency of Urine Among Study Participants Fig 8 Distribution of Increased Frequency of Urine Among Study Participants Fig 8 Distribution of Increased Frequency of Urine Among Study Participants Out of 95 children, presence of increased frequency of urine was reported in 11 (11.6%)and 84 (88.4%) with absence of increased frequency. his study showed that among 95 children, 83 (87.4%) children had pyuria and 12 (12.6%) had not.  Statistical Tools: Table 12 Distribution of Bacterial Growth Among Study Participants BACTERIAL GROWTH Frequency Percentage E.Coli 63 66.3 Klebsiella 13 13.7 Proteus 6 6.3 Pseudomonas 4 4.2 Coagulase Negative Staphylococci(Cons) 3 3.2 Enterobacter 3 3.2 Enterococcus Fecalis 2 2.1 Morganella 1 1.1 Total 95 100 Table 12 Distribution of Bacterial Growth Among Study Participants 1323 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Fig 11 Distribution of Bacterial Growth Among Study Participants Among the study participants, 63 (66.3%) were reported to be infected with E.coli, 13 (13.7%) with Klebsiella, 6 (6.3%) with Proteus, 4 (4.2%) with Pseudomonas, 3 (3.1%) with Coagulase Negative Staphylococci (Cons), 3 (3.1%) with Enterobacter, 2(2.1%) with Enterococcus Fecalis, and 1 (1.1%) with Morganella. ISSN No:-2456-2165 Fig 11 Distribution of Bacterial Growth Among Study Participants Among the study participants, 63 (66.3%) were reported to be infected with E.coli, 13 (13.7%) with Klebsiella, 6 (6.3%) with Proteus, 4 (4.2%) with Pseudomonas, 3 (3.1%) with Coagulase Negative Staphylococci (Cons), 3 (3.1%) with Enterobacter, 2(2.1%) with Enterococcus Fecalis, and 1 (1.1%) with Morganella. Among the study participants, 63 (66.3%) were reported to be infected with E.coli, 13 (13.7%) with Klebsiella, 6 (6.3%) with Proteus, 4 (4.2%) with Pseudomonas, 3 (3.1%) with Coagulase Negative Staphylococci (Cons), 3 (3.1%) with Enterobacter, 2(2.1%) with Enterococcus Fecalis, and 1 (1.1%) with Morganella.  Statistical Tools: Table 10 Distribution of Dysuria Among Study Participants Dysuria Frequency Percentage Present 22 23.2 Absent 73 76.8 Total 95 100 1322 IJISRT23MAR1248 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Scien Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN N 2456 2165 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 Fig 9 Distribution of Dysuria Among Study Participants Fig 9 Distribution of Dysuria Among Study Participants Fig 9 Distribution of Dysuria Among Study Participants The presence and absence of dysuria among 95 study participants showed that dysuria present in 22 (23.2%) and absent in 73 (76 8%) The presence and absence of dysuria among 95 study participants showed that dysuria present in 22 (23.2%) and absent in 73 (76 8%) bsence of dysuria among 95 study participants showed that dysuria present in 22 (23.2%) and absent in 73 The presence and absence of dysuria among 95 study participants showed that dysuria present in 22 (23.2%) and absent in 73 (76.8%) The presence and absence of dysuria among 95 study participants showed that dysuria present in 22 (23.2%) and absent in 73 (76.8%) Table 11 Distribution of Pyuria Among Study Participants Pyuria Frequency Percentage Present 83 87.4 Absent 12 12.6 Total 95 100 Fig 10 Distribution of Pyuria Among Study Participants Table 11 Distribution of Pyuria Among Study Participants Pyuria Frequency Percentage Present 83 87.4 Absent 12 12.6 Total 95 100 Fig 10 Distribution of Pyuria Among Study Participants Fig 10 Distribution of Pyuria Among Study Participants tudy showed that among 95 children, 83 (87.4%) children had pyuria and 12 (12.6%) had not.  Statistical Tools: Table 13 Distribution of Sensitive, Intermediate and Resistance Pattern in Different Antibiotics used Antibiotics Sensitive Intermediate Resistance Amoxicillin 25(26.3) 2(2.1) 68 (71.6) Amikacin 70(73.7) 1(1.1) 24(25.3) Gentamycin 77(81.1) 3(3.2) 15(15.8) Ceftazidime 75(78.9) 1(1.1) 19(20) Ciprofloxacin 69(72.6) 3(3.2) 23(24.2) Cefoperazone 72(75.8) 10(10.5) 13(13.7) Nitrofurantoin 66(69.3) 8(8.4) 21(22.1) Septran 59(62.1) 3(3.2) 33(34.7) Piptaz 73(76.8) 7(7.4) 15(15.8) Meropenem 70(73.7) 9(9.5) 16(16.8) Colistin 95(100) 0 0 Fig 12 Distribution of Sensitive, Intermediate and Resistance Pattern in Different Antibiotics used Table 13 Distribution of Sensitive, Intermediate and Resistance Pattern in Different Antibiotics used Antibiotics Sensitive Intermediate Resistance Amoxicillin 25(26.3) 2(2.1) 68 (71.6) Amikacin 70(73.7) 1(1.1) 24(25.3) Gentamycin 77(81.1) 3(3.2) 15(15.8) Ceftazidime 75(78.9) 1(1.1) 19(20) Ciprofloxacin 69(72.6) 3(3.2) 23(24.2) Cefoperazone 72(75.8) 10(10.5) 13(13.7) Nitrofurantoin 66(69.3) 8(8.4) 21(22.1) Septran 59(62.1) 3(3.2) 33(34.7) Piptaz 73(76.8) 7(7.4) 15(15.8) Meropenem 70(73.7) 9(9.5) 16(16.8) Colistin 95(100) 0 0 Fig 12 Distribution of Sensitive, Intermediate and Resistance Pattern in Different Antibiotics used Fig 12 Distribution of Sensitive, Intermediate and Resistance Pattern in Different Antibiotics used 1324 IJISRT23MAR1248 IJISRT23MAR1248 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Among the antibiotics, colistin had showed the highest sensitivity of 100% followed by Gentamycin 81.1% and Ceftazidime 78.9%. In view with resistance , these organisms showed lowest resistance to colistin (0%). Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No: 2456 2165 Volume 8, Issue 3, March – 2023 Volume 8, Issue 3, March – 2023 gy ISSN No:-2456-2165 Among the antibiotics, colistin had showed the highest sensitivity of 100% followed by Gentamycin 81.1% and Ceftazidime 78.9%. In view with resistance , these organisms showed lowest resistance to colistin (0%). Among the antibiotics, colistin had showed the highest sensitivity of 100% followed by Gentamycin 81.1% and Ceftazidime 78.9%. In view with resistance , these organisms showed lowest resistance to colistin (0%). Table 14 Distribution of Ultra Sound Finding of Study Participants. USG Frequency Percentage Bladder Wall Thickening (Cystitis) 9 9.5 Pyelonephritis 5 5.3 Vur 3 3.2 Urolithiasis 1 1.1 Normal 77 81.1 Total 95 100 Fig 13 Distribution of Ultra Sound Finding f Study Participants. Among the 95 children, 77 (81%) were found to be normal, 9 (9.4%) had bladder wall thickening, 5 (5.2%) had pyelonephritis, 3 (3.1%) had VUR and only 1(1%) had urolithiasis. Table 15 Distribution of MCU Finding of Study Participants.  Statistical Tools: Among the antibiotics, colistin had showed the highest sensitivity of 100% followed by Gentamycin 81.1% and Ceftazidime 78.9%. In view with resistance , these organisms showed lowest resistance to colistin (0%). Among the antibiotics, colistin had showed the highest sensitivity of 100% followed by Gentamycin 81.1% and Ceftazidime 78.9%. In view with resistance , these organisms showed lowest resistance to colistin (0%). Table 14 Distribution of Ultra Sound Finding of Study Participants. USG Frequency Percentage Bladder Wall Thickening (Cystitis) 9 9.5 Pyelonephritis 5 5.3 Vur 3 3.2 Urolithiasis 1 1.1 Normal 77 81.1 Total 95 100 Fig 13 Distribution of Ultra Sound Finding f Study Participants. Fig 13 Distribution of Ultra Sound Finding f Study Participants. Among the 95 children, 77 (81%) were found to be normal, 9 (9.4%) had bladder wall thickening, 5 (5.2%) had pyelonephritis, 3 (3.1%) had VUR and only 1(1%) had urolithiasis. Among the 95 children, 77 (81%) were found to be normal, 9 (9.4%) had bladder wall thickening, 5 (5.2%) had pyelonephritis, 3 (3.1%) had VUR and only 1(1%) had urolithiasis. Table 15 Distribution of MCU Finding of Study Participants. MCU Frequency Percentage VUR 14 14.7 PUV 3 3.2 Normal 78 82.1 Total 95 100 Fig 14 Distribution of MCU Finding of Study Participants. Table 15 Distribution of MCU Finding of Study Participants. MCU Frequency Percentage VUR 14 14.7 PUV 3 3.2 Normal 78 82.1 Total 95 100 Table 15 Distribution of MCU Finding of Study Participants. MCU Frequency Percentage VUR 14 14.7 PUV 3 3.2 Normal 78 82.1 Total 95 100 Fig 14 Distribution of MCU Finding of Study Participants. Fig 14 Distribution of MCU Finding of Study Participants. 1325 IJISRT23MAR1248 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 The MCU findings revealed that out of 95, 78 (82.1%) were normal, 4 (4.2%) had VUR and 3 (3.1%) had PUV. Table 16 Distribution of DMSA Finding of Study Participants. DMSA Frequency Percentage Multiple scars 8 8.4 Normal 56 58.9 Test could not be done 31 32.6 Total 95 100 Fig 15 Distribution of DMSA Finding of Study Participants. The DMSA scan was done in 64 study participants, among which 56 ( 59%) were normal, 8 (8.4%) had multiple scars and the test could not be done for 31(32.6%).  Statistical Tools: International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 Volume 8, Issue 3, March – 2023 The MCU findings revealed that out of 95, 78 (82.1%) were normal, 4 (4.2%) had VUR and 3 (3.1%) had PUV. Table 16 Distribution of DMSA Finding of Study Participants. DMSA Frequency Percentage Multiple scars 8 8.4 Normal 56 58.9 Test could not be done 31 32.6 Total 95 100 Fig 15 Distribution of DMSA Finding of Study Participants. Fig 15 Distribution of DMSA Finding of Study Participants. The DMSA scan was done in 64 study participants, among which 56 ( 59%) were normal, 8 (8.4%) had multiple scars and the test could not be done for 31(32.6%). The DMSA scan was done in 64 study participants, among which 56 ( 59%) were normal, 8 (8.4%) had multiple scars and the test could not be done for 31(32.6%). The DMSA scan was done in 64 study participants, among which 56 ( 59%) were normal, 8 (8.4%) had multiple scars and the test could not be done for 31(32.6%). Table 17 Distribution of Bacterial Growth in Different Age Group BACTERIAL GROWTH Age groups Chi- Square P value 1 - 12 months 13 – 48 months 49 – 60months E.Coli 23 23 17 15.39 0.35 Klebsiella 5 4 4 Proteus 4 2 0 Pseudomonas 3 0 1 Coagulase Negative Staphylococci (Cons) 1 0 2 Enterobacter 2 1 0 Enterococcus Fecalis 0 2 0 Morganella 1 0 0 Total 39 32 24 Fig 16 Distribution of Bacterial Growth in Different Age Group Fig 16 Distribution of Bacterial Growth in Different Age Group Fig 16 Distribution of Bacterial Growth in Different Age Group 1326 IJISRT23MAR1248 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 In this study, among the 1- 12 months age group,58.9 were infected withE.coli,12.8 with klebsiella,10.3 with proteus,7.7 with pseudomonas,5.1 with enterococcus,2.6 with morganella and2.6 with coagulase negative staphylococcus. In 13-48 months,71.9 with E coli,12.6 with klebsiella,6.2 with proteus,3.1 with enterobacter,6.2 with enterococcus faecalis. In 49- 60 months age group, 70.8 with ecoli, 16.7 with klebsiella, 4.2 pseudomonas,8.3 with coagulase negative staphylococcus. Distribution of Bacterial growth in different age group showed no association. Table 18 Age Distribution and Sensitive Pattern in Amoxicillin Age Groups Amoxicillin Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 11 1 27 0.862 0.93 13 – 48 months 8 1 23 49 – 60 months 6 0 18 Total 25 2 68 Fig 17 Age Distribution and Sensitive Pattern in Amoxicillin Fig 17 Age Distribution and Sensitive Pattern in Amoxicillin 28.2% of people showed sensitivity between 1 and 12 months, 2.6% showed intermediate sensitivity, and 69.2% showed resistance. 13-48 month olds were 25% sensitive, 3.1% had moderate sensitivity, and 71.8% had resistance. Amoxicillin is largely resistant in the 49–60 age range (75%), and just 25% were susceptible. The relationship between the age distribution and the amoxicillin sensitivity pattern is not The relationship between the age distribution and the amoxicillin sensitivity pattern is not statistically significant. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Table 19 Age Distribution and Sensitive Pattern in Amikacin Age Groups Amikacin Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 23 1 15 9.278 0.05* 13 – 48 months 25 0 7 49 – 60 months 22 0 2 Total 70 1 24 Fig 18 Age Distribution and Sensitive Pattern in Amikacin Table 19 Age Distribution and Sensitive Pattern in Amikacin Age Groups Amikacin Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 23 1 15 9.278 0.05* 13 – 48 months 25 0 7 49 – 60 months 22 0 2 Total 70 1 24 Table 19 Age Distribution and Sensitive Pattern in Amikacin Age Groups Amikacin Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 23 1 15 9.278 0.05* 13 – 48 months 25 0 7 49 – 60 months 22 0 2 Total 70 1 24 Fig 18 Age Distribution and Sensitive Pattern in Amikacin Fig 18 Age Distribution and Sensitive Pattern in Amikacin IJISRT23MAR1248 1327 www.ijisrt.com IJISRT23MAR1248 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 Volume 8, Issue 3, March – 2023 39 people aged 1 to 12 months were tested, and out of those, 58.9% were amikacin susceptible, 2.6% showed intermediate sensitivity, and 38.5% were resistant. The sensitivity and resistance among children aged 13 to 48 months were 78.1% and 21.9%, respectively. In 49–60 months, amikacin has the highest sensitivity (91.7%), and the lowest resistance (8.3%). Age distribution and amikacin sensitivity have a strong statistical relationship. Age distribution and amikacin sensitivity have a strong statistical relationship. Table 20 Age Distribution and Sensitive Pattern in Gentamycin Age Groups Gentamycin Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 29 2 8 3.029 0.553 13 – 48 months 28 1 3 49 – 60 months 20 0 4 Total 70 3 15 Fig 19 Age Distribution and Sensitive Pattern in Gentamycin Fig 19 Age Distribution and Sensitive Pattern in Gentamycin Fig 19 Age Distribution and Sensitive Pattern in Gentamycin Among 1-12months, gentamycin has sensitivity, intermediate and resistance of 74.4%,5.1%and 20.5% respectively. Between 13 to 48 months, the sensitivity increased to 87.5% ,intermediate sensitivity of 3.1%and resistance of 9.4%. in 49-60 months, 83.3% sensitive and 16.7% resistant. There is no corelation between age distribution and sensitive pattern of gentamycin. Among 1-12months, gentamycin has sensitivity, intermediate and resistance of 74.4%,5.1%and 20.5% respectively. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Between 13 to 48 months, the sensitivity increased to 87.5% ,intermediate sensitivity of 3.1%and resistance of 9.4%. in 49-60 months, 83.3% sensitive and 16.7% resistant. There is no corelation between age distribution and sensitive pattern of gentamycin. Table 21 Age Distribution and Sensitive Pattern in Ceftazidime Age Groups Ceftazidime Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 26 1 12 8.364 0.079 13 – 48 months 26 0 6 49 – 60 months 23 0 1 Total 70 1 19 Fig 20 Age Distribution and Sensitive Pattern in Ceftazidime Fig 20 Age Distribution and Sensitive Pattern in Ceftazidime Fig 20 Age Distribution and Sensitive Pattern in Ceftazidime IJISRT23MAR1248 www.ijisrt.com IJISRT23MAR1248 1328 Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 In this study, ceftazidime showed the maximum sensitivity of 95.8% in the age group of 49–60 months, followed by 81.2% in the age group of 13–48 months and 66.7% in the age group of 1–12 months. Only 4.2%, 18.8%, and 30.8% of patients were found to be resistant in 49–60 months, 13–48 months, and 1–12 months, respectively.2.6% has moderate sensitivity between 1 and 12 months. There is no discernible connection between ceftazidime and age distribution. There is no discernible connection between ceftazidime and age distribution. Table 22 Age Distribution and Sensitive Pattern in Ciprofloxacin Age Groups Ciprofloxacin Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 26 1 12 4.50 0.342 13 – 48 months 22 1 9 49 – 60 months 21 1 2 Total 70 3 23 Fig 21 Age Distribution and Sensitive Pattern in Ciprofloxacin Fig 21 Age Distribution and Sensitive Pattern in Ciprofloxacin Between 1-12 months, 66.7% showed sensitivity ,2.6% with intermediate and 30.8%with resistance. In 13-48 months, 68.8% were sensitive with 3.1% showing intermediate and 28.2% showing resistance. Among 49-60 years, 87.5% were highly sensitive, intermediate showed by 4.2% and resistance by 8.3%. Distribution of age and sensitive pattern of ciprofloxacin has no significant relationship. Distribution of age and sensitive pattern of ciprofloxacin has no significant relationship. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Table 23 Age Distribution and Sensitive Pattern in Cefoperazone Age Groups Cefoperazone Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 29 5 5 1.724 0.786 13 – 48 months 23 3 6 49 – 60 months 20 2 2 Total 72 10 13 Fig 22 Age Distribution and Sensitive Pattern in Cefoperazone Fig 22 Age Distribution and Sensitive Pattern in Cefoperazone Fig 22 Age Distribution and Sensitive Pattern in Cefoperazone 1329 IJISRT23MAR1248 IJISRT23MAR1248 www.ijisrt.com International Journal of Innovative Science and Research Technology Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Among 39 children in 1-12 months ,74.4% were sensitive , intermediate and resistance showed by 12.8% each.71.9% of children among 32 in 13-48 months showed sensitivity, 9.3% being intermediate and 18.8% showing resistance. Out of 24 in 49- 60 age group, 83.4% showed sensitivity, intermediate and resistance showed by 8.3% each. There is no significant statistical corelation noted Table 24 Age Distribution and Sensitive Pattern in Nitrofurantoin Age Groups Nitrofurantoin Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 26 4 9 4.052 0.39 13 – 48 months 23 4 5 49 – 60 months 17 0 7 Total 66 8 21 Fig 23 Age Distribution and Sensitive Pattern in Nitrofurantoin Fig 23 Age Distribution and Sensitive Pattern in Nitrofurantoin Fig 23 Age Distribution and Sensitive Pattern in Nitrofurantoin Fig 23 Age Distribution and Sensitive Pattern in Nitrofurantoin Between 1 to 12 months , 66.7% showed sensitivity while 10.2 % showed intermediate and 23.1% showed resistance.79.9% among 13-48 months showed sensitivity with 12.5% showing intermediate and 15.6% resistance.in 49-60 months age group, 70.8% showed sensitivity and 29.2% showed resistance. There is no significance between nitrofurantoin sensitive pattern and age distribution. Table 25 Age Distribution and Sensitive Pattern in Septran Age Groups Septran Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 24 0 15 9.760 0.04* 13 – 48 months 20 0 32 49 – 60 months 15 3 6 Total 59 3 95 Fig 24 Age Distribution and Sensitive Pattern in Septran Fig 24 Age Distribution and Sensitive Pattern in Septran 1330 IJISRT23MAR1248 IJISRT23MAR1248 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Out of 39 among the 1-12months age, 61.5% showed sensitivity and 38.5% showed resistance. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Among 32 in 13-48 months, 62.5% were sensitive and 37.5% were resistant. In 49-60 months out of 24, 62.5% were sensitive,12.5% were intermediate and 25% were resistant. There is a significant statistical correlation between age distribution and septran sensitive pattern. Table 26 Age Distribution and Sensitive Pattern in Piptaz Age Groups Piptaz Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 26 4 9 5.581 0.233NS 13 – 48 months 26 1 5 49 – 60 months 21 2 1 Total 73 7 15 Fig 25 Age Distribution and Sensitive Pattern in Piptaz Fig 25 Age Distribution and Sensitive Pattern in Piptaz Fig 25 Age Distribution and Sensitive Pattern in Piptaz Among the study participants in the age group 1-12 months , 66.7% were sensitive to nitrofurantoin , 10.2% were showing intermediate resistant and 23.1% showing resistance. In 13-48months 81.3% were sensitive ,3.1% having intermediate and 15.6% having resistance. Among 49-60 months, 87.5% were sensitive ,8.3% intermediate and only 4.2% resistant. There is no significant corelation between age distribution and nitrofurantoin sensitive pattern. here is no significant corelation between age distribution and nitrofurantoin sensitive pattern. Table 27 Age Distribution and Sensitive Pattern in Meropenem Age Groups Meropenem Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 27 4 8 0.925 0.921NS 13 – 48 months 25 3 4 49 – 60 months 18 2 4 Total 70 9 16 Fig 26 Age Distribution and Sensitive Pattern in Meropenem Table 27 Age Distribution and Sensitive Pattern in Meropenem Age Groups Meropenem Chi-square P value Sensitive Intermediate Resistant 1 - 12 months 27 4 8 0.925 0.921NS 13 – 48 months 25 3 4 49 – 60 months 18 2 4 Total 70 9 16 Fig 26 Age Distribution and Sensitive Pattern in Meropenem Fig 26 Age Distribution and Sensitive Pattern in Meropenem IJISRT23MAR1248 1331 IJISRT23MAR1248 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology In the 1-12 months age group, 69.2% were sensitive to meropenam, 10.3 % show intermediate and 20.5% show resistance. Among 13-48months, 78.1% were sensitive with 9.3% showing intermediate and 12.5% showing resistance patterns. In 49-60 months age group, 75% showed sensitive, 8.3% intermediate and 16.7% resistance. www.ijisrt.com 1332 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 9) 4(30.7 6) 13(10 0) Proteus 6 (6.3) 3(50) 4(66.6) 3(50) 5(83.3 3) 2(33.3 3) 3(50) 3(50) 2(33.3 3) 3(50) 5(83.3 3) 6(10) Pseudomonas 4 (4.2) 1(25) 4(100) 2(50) 3(75) 3(75) 2(50) 1(25) 2(50) 1(25) 2(50) 4(100 ) Coagulase Negative Staphylococci( Cons) 3 (3.2) 0 2(66.66 ) 3(100 ) 2(66.6 6) 3(100 ) 2(66.6 6) 3(100 ) 2(66.6 6) 1(33.3 3) 1(33.3 3) 3(100 ) Enterobacter 3 (3.2) 1(33.3 3) 3(100) 2(66.6 6) 1(33.3 3) 2(66.6 6) 2(66.6 6) 2(66.6 6) 2(66.6 6) 2(66.6 6) 2(66.6 6) 3(66.6 6) Enterococcus Fecalis 2 (2.1) 0 2(100) 1(50) 2(100 ) 1(50) 2(100 ) 2(100 ) 1(50) 0 1(50) 2(100 ) Morganella 1 (1.1) 0 0 1(100 ) 1(100 ) 0 0 1(100 ) 1(100 ) 1(100 ) 1(100 ) 1(100 ) Table 29 Antibiotic Sensitivity Pattern of Isolated Uropathogens (% Sensitive) www.ijisrt.com 1332 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 ISSN No: 2456 2165 Fig 28 Antibiotic Sensitivity Pattern of Isolated Uropathogens (% Sensitive) The major uropathogens isolated were Escherichia coli followed by Klebsiella pneumonia and Proteus Majority of the Fig 28 Antibiotic Sensitivity Pattern of Isolated Uropathogens (% Sensitive) Fig 28 Antibiotic Sensitivity Pattern of Isolated Uropathogens (% Sensitive) iotic Sensitivity Pattern of Isolated Uropathogens (% The major uropathogens isolated were Escherichia coli, followed by Klebsiella pneumonia and Proteus. Majority of the E.coli and Klebsiella isolates were sensitive to colistin, meropenem, piptaz, cefoperazone-sulbactam, gentamycine, ceftazi followed by nitrofurantoin. The major uropathogens isolated were Escherichia coli, followed by Klebsiella pneumonia and Proteus. Majority of the E.coli and Klebsiella isolates were sensitive to colistin, meropenem, piptaz, cefoperazone-sulbactam, gentamycine, ceftazi followed by nitrofurantoin. The major uropathogens isolated were Escherichia coli, followed by Klebsiella pneumonia and Proteus. Majority of the E.coli and Klebsiella isolates were sensitive to colistin, meropenem, piptaz, cefoperazone-sulbactam, gentamycine, ceftazi followed by nitrofurantoin. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Table 28 Age Distribution and Sensitive Pattern in Colistin Col Age Group Sensitive 1 - 12 months 39 13 – 48 months 32 49 – 60 months 24 Total 95 Fig 27 Age Distribution and Sensitive Pattern in Colistin Colistin has the maximum sensitivity of 100% in all the age groups under this study Table 28 Age Distribution and Sensitive Pattern in Colistin Col Age Group Sensitive 1 - 12 months 39 13 – 48 months 32 49 – 60 months 24 Total 95 Table 28 Age Distribution and Sensitive Pattern in Colistin Fig 27 Age Distribution and Sensitive Pattern in Colistin Fig 27 Age Distribution and Sensitive Pattern in Colistin Fig 27 Age Distribution and Sensitive Pattern in Colistin Colistin has the maximum sensitivity of 100% in all the age groups under this study. Colistin has the maximum sensitivity of 100% in all the age groups under this study. Table 29 Antibiotic Sensitivity Pattern of Isolated Uropathogens (% Sensitive) Numb er Amox i Amika cin Genta Cefta zi Cipro Cefop Nitro septr an Pipta z Mero p Colist in E.coli 63 (63.3) 19 (30.1) 45 (71.4) 53 (84.1) 51 (80.9) 52 (82.5) 54 (85.7) 49 (77.7) 41 (65.0) 55 (87.3) 54 (85.7) 63 (100) Klebsiella 13 (13.7) 1 (7.69) 10 (76.9) 12 (92.3) 10 (76.9) 6 (46.15 ) 7 (53.8) 5 (38.4) 8 (61.5) 10(76. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Table 30 Antibiotic Sensitivity Pattern of Isolated Uropathogens (%Intermediate) Numb er Amo xi Amikac in Gent a Cefta zi Cipro Cefop Nitro Septr an Piptaz Mero p Colist in E.coli 63 (63.3) 0 0 2(3.1 7) 1(1.5 8) 0 2(3.17 ) 3(4.76 ) 0 3(4.76 ) 3(4.76 ) 0 Klebsiella 13 (13.7) 0 1(7.69) 0 0 3(23.0 7) 5(28.4 6) 3(23.0 7) 2(15.3 8) 1(7.69 ) 4(30.7 6) 0 Proteus 6 (6.3) 0 0 0 0 0 1(16.6 6) 2(33.3 3) 0 1(16.6 6) 1(16.6 6) 0 Pseudomonas 4 (4.2) 1(25) 0 1(25) 0 0 0 0 1(25) 1(25) 0 0 Coagulase Negative Staphylococci( Cons) 3 (3.2) 0 0 0 0 0 1(33.3 3) 0 0 1(33.3 3) 1(33.3 3) 0 Enterobacter 3 (3.2) 1(33.3 3) 0 0 0 0 1(33.3 3) 0 0 0 0 0 Enterococcus Fecalis 2 (2.1) 0 0 0 0 0 0 0 0 0 0 0 Morganella 1 (1.1) 0 0 0 0 0 0 0 0 0 0 0 1333 IJISRT23MAR1248 www.ijisrt.com International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 Fig 29 Antibiotic Intermediate Pattern of Isolated Uropathogens (% Intermediate) Fig 29 Antibiotic Intermediate Pattern of Isolated Uropathogens (% Intermediate) Fig 29 Antibiotic Intermediate Pattern of Isolated Uropathogens (% Intermediate) Table 31 Antibiotic Resistant Pattern of Isolated Uropathogens (% Resistant) Num ber Amoxi Amika cin Genta Ceftaz i Cipro Cefop Nitr o Septra n Pipta z Mer op Colis tin E.coli 63 (63.3) 44(69. 84) 18(28. 57) 8(12.6 9) 11(17. 46) 11(17.46 ) 7(11. 11) 11(1 7.46) 22(34. 92) 5(7.9 3) 6(9.5 2) 0 Klebsiella 13 (13.7) 12(92. 3) 2(15.3 8) 1(7.69 ) 3(23.0 7) 4(30.76) 1(7.6 9) 5(38. 46) 3(23.0 7) 2(15. 3) 5(38. 4) 0 Proteus 6 (6.3) 3(50) 2(33.3 3) 3(50) 1(16.6 6) 4(66.66) 2(33. 33) 1(16. 66) 4(66.6 6) 2(33. 3) 0 0 Pseudomonas 4 (4.2) 2(50) 0 1(25) 1() 1() 2() 3() 1() 2() 2() 0 Coagulase Negative Staphylococci (Cons) 3 (3.2) 3(100) 1(33.3 3) 0 1(33.3 3) 0 0 0 1(33.3 3) 1(33. 3) 1(33. 3) 0 Enterobacter 3 (3.2) 1(33.3 3) 0 1(33.3 3) 2(66.6 6) 1(33.33) 0 1(33. 33) 1(33.3 3) 1(33. 3) 1(33. IV. DISCUSSION SPP that causes UTI was most sensitive to, followed by CIP. 95 kiddies with UTIs that tested positive on culture passed ultrasonography( USG) and micturating cystourethrogram( MCU). 18 of the 95 kiddies who had USGs had serious anomalies, including thickening of the bladder wall, pyelonephritis, vesicoureteral influx, urolithiasis, and cystitis. Indeed when there was no abnormalities on ultrasonography, MCU was carried out in all cases in agreement with the recommendations of the Indian Association of Pediatric Nephrology. 64 of 95 youths had a dimercaptosuccinic acid checkup( DMSA), of which 56(56.6) had no abnormalities and 8(8.1) showed signs of multitudinous scars. SPP that causes UTI was most sensitive to, followed by CIP. 95 kiddies with UTIs that tested positive on culture passed ultrasonography( USG) and micturating cystourethrogram( MCU). 18 of the 95 kiddies who had USGs had serious anomalies, including thickening of the bladder wall, pyelonephritis, vesicoureteral influx, urolithiasis, and cystitis. Indeed when there was no abnormalities on ultrasonography, MCU was carried out in all cases in agreement with the recommendations of the Indian Association of Pediatric Nephrology. 64 of 95 youths had a dimercaptosuccinic acid checkup( DMSA), of which 56(56.6) had no abnormalities and 8(8.1) showed signs of multitudinous scars. The operation of urinary tract infection( UTI), a frequent cause of acute sickness in babies and kiddies, is impacted by the circumstance of vague signs and symptoms. With the identification of the clinical, bacteriological, and radiological biographies of UTI, this study seeks to reduce the variability of clinical practise in the operation of UTI in children under the age of five. The study actors' age and coitus distribution, with 39(41.1) in the age group between 1 months and 12 months, 32(33.7) between 13 months and 48 months, and 24(25.3) between 49 months and 60 months, influences the frequence of UTI. UTI frequence peaked in immaturity( 1 month to 12 months) and peaked in puberty( 49- 60 months). Research conducted in the Philippines( 21) by Bay AG etal. revealed that women(53.9) were more affected than men(46.1). Males are more affected than ladies, according to Taneja et al( 23) who studied youths progressed 12 times. According to this study, UTI affects women more constantly than men across all age orders. generally, dysuria, frequence, abdominal pain, pungent urine, and fever are the clinical signs of UTI.  Limitations The sample size is small and hence may not be representative of the entire population. DMSA checkup couldnot be performed on all subjects due to loss of follow up or plutocrat constraint. We didnot follow up all children prospectively to see if they develop intermittent UTI. RECOMMENDATIONS A long- term follow up study of children with UTI, to determine which child will develop long term complications. Randomised placebo- controlled trials are needed to determine the effectiveness of antibiotic prophylaxis in precluding intermittent UTI or parenchymal damage. Other threat factors like ethinicity, socioeconomic status, circumcision status in manly children, and their unproductive relationship with UTI should be studied. Parents must be councelled regarding the significance of acceptable fluid input, restroom training and consequences of constipation. International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 3) 0 Enterococcus Fecalis 2 (2.1) 2(100) 0 1(50) 0 1(50) 0 0 1(50) 2(10 0) 1(50) 0 Morganella 1 (1.1) 1(100) 1(100) 0 0 1(100) 1(100 ) 0 0 0 0 0 Table 31 Antibiotic Resistant Pattern of Isolated Uropathogens (% Resistant) 1334 IJISRT23MAR1248 www.ijisrt.com IJISRT23MAR1248 Volume 8, Issue 3, March – 2023 Fig 30 Antibiotic Resistant Pattern Of Isolated Uropathogens (% Resistant) Fig 30 Antibiotic Resistant Pattern Of Isolated Uropathogens (% Resistant) IV. DISCUSSION The most frequent symptom, fever, was endured by67.4 of cases, who were also affected by abdominal discomfort(46.8), dysuria(23.2), nausea or vomiting(21.1), increased frequence(11.6), and ripe urine(8.4). Escherichia coli(E.coli,66.3), followed by Klebsiellaspp.(13.7), was the most current pathogen.67.3 of babies with UTI had substantial pyuria, according to urine microscopy, and 8 distinct species of bacteria were discovered in 95 societies. In their exploration in Dhaka, Sharmin S etal. (1990) discovered a analogous trend of antibiotic perceptivity, demonstrating low vulnerability ofE. coli to routinely used specifics as imipenem, ceftazidime, and amikacin. analogous trends were discovered by Nasim Kashef etal.( 1991) and Fakhrossadat etal.( 1992), who showed that Klebsiella was veritably susceptible to cefixime, nalidixic acid, and ciprofloxacin and largely resistant to ceftriaxone, gentamycin, and trimethoprim- sulfamethoxazole. Colistin and amikacin were the two medicines that Pseudomonas ISSN No:-2456-2165 which was followed by dysuria and stomach pain. In all age groups( 1 – 60 months),E. coli was the most common bacterium causing UTI, followed by klebsiella and proteus. Colistin had the loftiest overall perceptivity, followed by gentamycin and ceftazidime. After collecting urine for culture, a suspected UTI can be treated empirically with an aminoglycoside or a third generation cephalosporin, still there's a significant frequence of in- vitro resistance to amoxicillin and trimethoprim- sulfamethoxazole. When choosing treatment plans, it's important to keep in mind that the resistance pattern of uropathogens causing urinary tract infections to popular antimicrobial medicines is evolving. Not only will the condition be snappily cured with the right antibiotic tradition, but it'll also prop in precluding the development of rising resistance. When children were submitted to ultrasonography,18.9 of them displayed radiological abnormalities, with cystitis, pyelonephritis, and vesicourethral influx being the most frequent findings. MCU is needed to exclude VUR. 56 children were normal and 8 children have DMSA checkup substantiation of multitudinous scars. It's the duty of every Health care professional to insure that when a child was set up to have UTI, they're given applicable information about the need for treatment, the significance of completing the course of treatment, advice about forestallment, possibility of UTI recreating and understand the need for alert and to seek prompt treatment. [9]. Boyko EJ, Fihn SD, Scholes D, Abraham L, Monsey B. Risk of urinary tract infection and asymptomatic bacteriuria among diabetic and nondiabetic postmenopausal women. American Journal of Epidemiology. 2005 Mar 15;161(6):557-64. [10]. Shaw KN, Gorelick M, McGowan KL, Yakscoe NM, Schwartz JS. Prevalence of urinary tract infection in febrile young children in the emergency department. Pediatrics. 1998 Aug 1;102(2):e16-. g [11]. Sheinfeld J, Schaeffer AJ, Cordon-Cardo C, Rogatko A, Fair WR. Association of the Lewis blood-group phenotype with recurrent urinary tract infections in women. New England Journal of Medicine. 1989 Mar 23;320(12):773-7. [12]. Bagga A, Tripathi P, Jatana V, Hari P, Kapil A, Srivastava RN, Bhan MK. Bacteriuria and urinary tract infections in malnourished children. Pediatric nephrology. 2003 Apr 1;18(4):366-70. [13]. Scholes D, Hooton TM, Roberts PL, Stapleton AE, Gupta K, Stamm WE. Risk factors for recurrent urinary tract infection in young women. Journal of infectious diseases. 2000 Oct 1;182(4):1177-82. [14]. Chon CH, Lai FC, Shortliffe LM. Pediatric urinary tract infections. Pediatric clinics of North America. 2001 Dec;48(6):1441-59. [15]. Ramamurthy HR, Kanitkar M. ISSN No:-2456-2165 Recurrent urinary tract infection and functional voiding disorders. Indian pediatrics. 2008 Aug 1;45(8):689. V. According to this study, urinary tract infections( UTI) are more current in babies( 1 – 12 months) and decline with age. The most typical clinical symptom was fever, 1335 1335 www.ijisrt.com IJISRT23MAR1248 Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 REFERENCES Antibiotic prophylaxis in children with relapsing urinary tract infections. Journal of chemotherapy. 2000 Jan 1;12(2):115-23. [36]. Mellata M, Dho-Moulin M, Dozois CM, Curtiss III R, Brown PK, Arné P, Brée A, Desautels C, Fairbrother JM. Role of virulence factors in resistance of avian pathogenic Escherichia coli to serum and in pathogenicity. Infection and immunity. 2003 Jan 1;71(1):536-40. [24]. Watson AR. Pediatric urinary tract infection. EAU update Series. 2004 Sep 30;2(3):94-100. [37]. Trautner BW, Darouiche RO. Role of biofilm in catheter-associated urinary tract infection. American journal of infection control. 2004 May 31;32(3):177- 83. [25]. Kaper JB, Nataro JP, Mobley HL. Pathogenic escherichia coli. Nature Reviews Microbiology. 2004 Feb 1;2(2):123-40. [26]. Alteri CJ, Smith SN, Mobley HL. Fitness of Escherichia coli during urinary tract infection requires gluconeogenesis and the TCA cycle. PLoS pathogens. 2009 May 29;5(5):e1000448. [38]. Alexander C, Rietschel ET. Invited review: bacterial lipopolysaccharides and innate immunity. Journal of endotoxin research. 2001 Jun;7(3):167-202. [27]. Manges AR, Johnson JR, Foxman B, O'Bryan TT, Fullerton KE, Riley LW. Widespread distribution of urinary tract infections caused by a multidrug- resistant Escherichia coli clonal group. New England Journal of Medicine. 2001 Oct 4;345(14):1007-13. [39]. Heimer SR, Rasko DA, Lockatell CV, Johnson DE, Mobley HL. Autotransporter genes pic and tsh are associated with Escherichia coli strains that cause acute pyelonephritis and are expressed during urinary tract infection. Infection and immunity. 2004 Jan 1;72(1):593-7. [28]. Johnson JR, Orskov I, Orskov F, Goullet P, Picard B, Moseley SL, Roberts PL, Stamm WE. O, K, and H antigens predict virulence factors, carboxylesterase B pattern, antimicrobial resistance, and host compromise among Escherichia coli strains causing urosepsis. Journal of Infectious Diseases. 1994 Jan 1;169(1):119-26. [40]. Hooton TM. Pathogenesis of urinary tract infections: an update. Journal of Antimicrobial Chemotherapy. 2000 Aug 1;46(suppl_1):1-7. [29]. Guyer DM, Gunther IV NW, Mobley HL. Secreted proteins and other features specific to uropathogenic Escherichia coli. The Journal of infectious diseases. 2001 Mar 1;183(Supplement_1):S32-5. [30]. Snyder JA, Haugen BJ, Buckles EL, Lockatell CV, Johnson DE, Donnenberg MS, Welch RA, Mobley HL. Transcriptome of uropathogenic Escherichia coli during urinary tract infection. Infection and immunity. 2004 Nov 1;72(11):6373-81. y ( ) [31]. Gunther NW, Lockatell V, Johnson DE, Mobley HL. In vivo dynamics of type 1 fimbria regulation in uropathogenicEscherichia coli during experimental urinary tract infection. Infection and immunity. 2001 May 1;69(5):2838-46. [32]. Selvarangan R, Goluszko P, Singhal J, Carnoy C, Moseley S, Hudson B, Nowicki S, Nowicki B. REFERENCES [16]. Coppa GV, Gabrielli O, Giorgi P, Catassi C, Montanari MP, Varaldo PE, Nichols BL. Preliminary study of breastfeeding and bacterial adhesion to uroepithelial cells. The Lancet. 1990 Mar 10;335(8689):569-71. [1]. Chang SL, Shortliffe LD. Pediatric urinary tract infections. Pediatric Clinics of North America. 2006 Jun 30;53(3):379-400. [17]. Hanson LÅ, Korotkova M, Håversen L, Mattsby‐ Baltzer I, Hahn‐Zoric M, Silfverdal SA, Strandvik B, Telemo E. Breast‐feeding, a complex support system for the offspring. Pediatrics International. 2002 Aug 1;44(4):347-52. [2]. Sahsi RS, Carpenter CR. Does This Child Have a Urinary Tract Infection?. Annals of Emergency Medicine. 2009 May;53(5):6804. [3]. Vijayakumar M, Kanitkar M, Nammalwar BR, Bagga A. Revised statement on management of urinary tract infections. Indian pediatrics. 2011 Sep; 48(9):709-17. [18]. Mazzola BL, von Vigier RO, Marchand S, Tönz M, Bianchetti MG. Behavioral and functional abnormalities linked with recurrent urinary tract infections in girls. Journal of nephrology. 2003;16(1):133-8. [4]. Elder JS. Urinary tract infections In: Behrman RE, Kliegman RM, Tewson HB ,editors. Nelson Text Book of Pediatrics.20th ed. Philadelphia: Elsevier;2000:P. 2556-2566. [19]. Wan J, Kaplinsky R, Greenfield S. Toilet habits of children evaluated for urinary tract infection. The Journal of urology. 1995 Aug 31;154(2):797-9. [5]. Ghedira BL, Messaoudi A, Ben MC, Guediche MN. Profile of antimicrobial resistance of agents causing urinary tract infections in children. La Tunisie medicale. 2004 Mar;82(3):299-305. [20]. Bakker E, Sprundel MV, Auwera JV, Gool JV, Wyndaele JJ. Voiding habits and wetting in a population of 4332 Belgian schoolchildren aged between 10 and 14 years. Scandinavian journal of urology and nephrology. 2002 Jan 1;36(5):354-62. [6]. Heffner VA, Gorelick MH. Pediatric urinary tract infection. Clinical Pediatric Emergency Medicine. 2008 Dec 31;9(4):233-7. [7]. White B. Diagnosis and treatment of urinary tract infections in children. American family physician. 2011 Feb 15;83(4). [21]. Dulczak S, Kirk J. Overview of the evaluation, diagnosis, and management of urinary tract infections in infants and children. Urologic nursing. 2005 Jun 1;25(3):185. [8]. Wiswell TE. The prepuce, urinary tract infections, and the consequences. Pediatrics. 2000 Apr 1;105(4):860-2. [22]. Lazarević G, Petreska D, Pavlović S. Antibiotic sensitivity of bacteria isolated from the urine of children with urinary tract infections from 1986 to 1995. Srpski arhiv za celokupno lekarstvo. 1997 Dec;126(11-12):423-9. IJISRT23MAR1248 1336 www.ijisrt.com Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology International Journal of Innovative Science and Research Technology ISSN No:-2456-2165 [23]. Mangiarotti P, Pizzini C, Fanos V. REFERENCES Interaction of Dr adhesin with collagen type IV is a critical step in Escherichia coli renal persistence. Infection and immunity. 2004 Aug 1;72(8):4827-35. [33]. Barnhart MM, Chapman MR. Curli biogenesis and function. Annu. Rev. Microbiol.. 2006 Oct 13;60:131-47. [34]. Ramos HC, Rumbo M, Sirard JC. Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa. Trends in microbiology. 2004 Nov 30;12(11):509-17. gy [35]. Wright KJ, Seed PC, Hultgren SJ. Uropathogenic Escherichia coli flagella aid in efficient urinary tract colonization. Infection and immunity. 2005 Nov 1;73(11):7657-68. 1337 IJISRT23MAR1248 IJISRT23MAR1248 www.ijisrt.com
https://openalex.org/W3044277939	https://europepmc.org/articles/pmc7374621?pdf=render	English	null	Author Correction: Interferon-Dependent and Respiratory Virus-Specific Interference in Dual Infections of Airway Epithelia	Scientific reports	2,020	cc-by	265	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Manel Essaidi‑Laziosi, Johan Geiser, Song Huang, Samuel Constant, Laurent Kaiser & Caroline Tapparel Manel Essaidi‑Laziosi, Johan Geiser, Song Huang, Samuel Constant, Laurent Kaiser & Caroline Tapparel Correction to: Scientific Reports https://doi.org/10.1038/s41598-020-66748-6, published online 24 June 202 This Article contains an error in the Results section under the subheading “H1N1 and RSV-A interfere with RV-A16 replication, while RV-A16 does not interfere with either of these viruses” where, “Co- or sequential infection (with a two-day interval) between RV-A16 and each of the other viruses (using a MOI of around 0.01 viral particles per accessible cell)” should read: “Co- or sequential infection (with a two-day interval) between RV-A16 and each of the other viruses (using a MOI of around 0.001 viral particles per accessible cell)” Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 \| https://doi.org/10.1038/s41598-020-69600-z Scientific Reports \| (2020) 10:12523
https://openalex.org/W2745144279	https://portal.findresearcher.sdu.dk/files/134244382/Long_term_follow_up_in_primary_Sj_gren_s_syndrome_reveals_differences_in_clinical_presentation_between_female_and_male_patients.pdf	English	null	Long-term follow-up in primary Sjögren’s syndrome reveals differences in clinical presentation between female and male patients	Biology of sex differences	2,017	cc-by	7,574	Long-term follow-up in primary Sjögren's syndrome reveals differences in clinical presentation between female and male patients Ramírez Sepúlveda, Jorge I; Kvarnström, Marika; Eriksson, Per; Mandl, Thomas; Norheim, Katrine Brække; Johnsen, Svein Joar; Hammenfors, Daniel; Jonsson, Malin V; Skarstein, Kathrine; Brun, Johan G; Rönnblom, Lars; Forsblad-d'Elia, Helena; Magnusson Bucher, Sara; Baecklund, Eva; Theander, Elke; Omdal, Roald; Jonsson, Roland; Nordmark, Gunnel; Wahren-Herlenius, Marie; DISSECT consortium; Diederichsen, Louise C. Pyndt Raun Published in: Biology of Sex Differences Document version: Final published version Document license: CC BY Citation for pulished version (APA): Ramírez Sepúlveda, J. I., Kvarnström, M., Eriksson, P., Mandl, T., Norheim, K. B., Johnsen, S. J., Hammenfors, D., Jonsson, M. V., Skarstein, K., Brun, J. G., Rönnblom, L., Forsblad-d'Elia, H., Magnusson Bucher, S., Baecklund, E., Theander, E., Omdal, R., Jonsson, R., Nordmark, G., Wahren-Herlenius, M., ... Diederichsen, L. C. P. R. (2017). Long-term follow-up in primary Sjögren's syndrome reveals differences in clinical presentation between female and male patients. Biology of Sex Differences, 8, Article 25. https://doi.org/10.1186/s13293-017- 0146-6 Citation for pulished version (APA): Ramírez Sepúlveda, J. I., Kvarnström, M., Eriksson, P., Mandl, T., Norheim, K. B., Johnsen, S. J., Hammenfors, D., Jonsson, M. V., Skarstein, K., Brun, J. G., Rönnblom, L., Forsblad-d'Elia, H., Magnusson Bucher, S., Baecklund, E., Theander, E., Omdal, R., Jonsson, R., Nordmark, G., Wahren-Herlenius, M., ... Diederichsen, L. C. P. R. (2017). Long-term follow-up in primary Sjögren's syndrome reveals differences in clinical presentation between female and male patients. Biology of Sex Differences, 8, Article 25. https://doi.org/10.1186/s13293-017- 0146-6 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: g y y y Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply: • You may download this work for personal use only You may not further distribute the material or use it for any profit making activity or • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to puresupport@bib.sdu.dk University of Southern Denmark Long-term follow-up in primary Sjögren's syndrome reveals differences in clinical presentation between female and male patients Citation for pulished version (APA): Ramírez Sepúlveda, J. I., Kvarnström, M., Eriksson, P., Mandl, T., Norheim, K. B., Johnsen, S. J., Hammenfors, D., Jonsson, M. V., Skarstein, K., Brun, J. G., Rönnblom, L., Forsblad-d'Elia, H., Magnusson Bucher, S., Baecklund, E., Theander, E., Omdal, R., Jonsson, R., Nordmark, G., Wahren-Herlenius, M., ... Diederichsen, L. C. P. R. (2017). Long-term follow-up in primary Sjögren's syndrome reveals differences in clinical presentation between female and male patients. Biology of Sex Differences, 8, Article 25. https://doi.org/10.1186/s13293-017- 0146-6 * Correspondence: Marie.Wahren@ki.se 1Unit of Experimental Rheumatology, Department of Medicine, Karolinska University Hospital, Karolinska Institutet, SE-171 76 Stockholm, Sweden Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Long-term follow-up in primary Sjögren’s syndrome reveals differences in clinical presentation between female and male patients Jorge I. Ramírez Sepúlveda1, Marika Kvarnström1, Per Eriksson2, Thomas Mandl3, Katrine Brække Norheim4, Svein Joar Johnsen4, Daniel Hammenfors5,6, Malin V. Jonsson5,7, Kathrine Skarstein8,9, Johan G. Brun5,6, the DISSECT consortium, Lars Rönnblom10, Helena Forsblad-d’Elia11, Sara Magnusson Bucher12, Eva Baecklund10, Elke Theander3, Roald Omdal4, Roland Jonsson5,6, Gunnel Nordmark10 Marie Wahren-Herlenius1* Download date: 24. Oct. 2024 Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 DOI 10.1186/s13293-017-0146-6 Open Access Background Brandt et al. [3], some authors have identified differences in extraglandular manifestations and serological markers, with a focus on female prevalence. However, there is no clear consensus on whether male sex is associated with a more severe disease. Our group has described that at diagnosis, male patients more frequently present with extraglandular manifestations, more concomitant extraglandular manifes- tations, and higher anti-Ro52 levels by investigation in two independent cohorts [19]. To understand if there are sex differences in the clinical presentation of pSS also after a long-standing disease and whether risk for comorbidities vary between the sexes, we assessed glandular and extra- glandular manifestations, serological parameters, and co- morbidities of pSS in men and women years after diagnosis in a large Scandinavian cohort. It has been widely established that women are more prone to develop autoimmune diseases [1]. Primary Sjögren’s syndrome (pSS) is a systemic autoimmune dis- ease characterized by inflammation of the salivary and lacrimal glands, causing a reduction in exocrine secre- tion that ultimately leads to the clinical presentation of sicca symptoms. The reported population-based female to male ratio is 14:1 [2]. Many hypotheses have been proposed to explain the overall marked sex bias in auto- immunity and pSS [3], including genetic and epigenetic factors [4, 5], sex hormones [6], and X-chromosome ab- errances [7, 8]. However, the molecular mechanisms that drive this sex skewing still remain elusive. g Interestingly, the differences between the sexes like- wise extend to the clinical manifestations, where female and male patients differ in disease presentation and se- verity. Despite being less prone to develop autoimmune diseases, male patients have been reported to have a more severe disease and a worse prognosis. In systemic lupus erythematosus (SLE), men present more renal dis- ease [9] and serositis [10]. Further, Andrade et al. [11] identified male sex as a strong predictor for poorer long- term prognosis due to accelerated damage accrual, while Manger et al. [12] reported male sex as a risk factor for increased SLE mortality. Male sex is also deemed as a factor for accelerated disease progression in multiple sclerosis (MS) [13] and associated with a significantly higher prevalence of comorbidities [14] such as diabetes, epilepsy, depression, and anxiety. Background Although less clear, sex differences in rheumatoid arthritis (RA) severity and extra-articular manifestations have also been described; women appear more prone to present sicca symptoms and men to have erosive joint disease, rheumatoid nod- ules, and interstitial lung disease [15, 16]. Abstract Background: Despite men being less prone to develop autoimmune diseases, male sex has been associated with a more severe disease course in several systemic autoimmune diseases. In the present study, we aimed to investigate differences in the clinical presentation of primary Sjögren’s syndrome (pSS) between the sexes and establish whether male sex is associated with a more severe form of long-term pSS. Methods: Our study population included 967 patients with pSS (899 females and 68 males) from Scandinavian clinical centers. The mean follow-up time (years) was 8.8 ± 7.6 for women and 8.5 ± 6.2 for men (ns). Clinical data including serological and hematological parameters and glandular and extraglandular manifestations were compared between men and women. Results: Male patient serology was characterized by more frequent positivity for anti-Ro/SSA and anti-La/SSB (p = 0. 02), and ANA (p = 0.02). Further, men with pSS were more frequently diagnosed with interstitial lung disease (p = 0. 008), lymphadenopathy (p = 0.04) and lymphoma (p = 0.007). Conversely, concomitant hypothyroidism was more common among female patients (p = 0.009). Conclusions: We observe enhanced serological responses and higher frequencies of lymphoma-related extraglandular manifestations in men with pSS. Notably, lymphoma itself was also significantly more common in men. These observations may reflect an aggravated immune activation and a more severe pathophysiological state in male patients with pSS and indicate a personalized managing of the disease due to the influence of the sex of patients with pSS. : Sjögren’s syndrome, Autoimmunity, Sex difference, Disease severity, Extraglandular manifestations Keywords: Sjögren’s syndrome, Autoimmunity, Sex difference, Disease severity, Extraglandular manifestations Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Page 2 of 9 Page 2 of 9 Methods P i DISSECT—“Dissecting disease mechanisms in three sys- temic inflammatory autoimmune diseases with an inter- feron signature”—is a multicenter consortium comprising the Scandinavian Sjögren’s syndrome research network, the Swedish SLE network and the Swedish Myositis net- work linked to the European Myositis network. All 967 patients with pSS and fulfilling the American–European consensus criteria [20] in the DISSECT cohort were in- cluded in this study. Out of these, 899 were females and 68 were males (Table 1). The patients were diagnosed and followed at the Departments of Rheumatology at the Uni- versity Hospitals in Gothenburg, Skåne, Linköping, Örebro, and Uppsala, as well as the Karolinska University Hospital in Stockholm, Sweden, and the Department of Rheumatology at Haukeland University Hospital, Bergen, and the University Hospital in Stavanger, Norway. Of the 205 patients from the Karolinska University Hospital, 127 were included in a previous analysis of clinical manifesta- tions in female and male patients at diagnosis [19]. Regarding pSS, several studies have addressed sex differ- ences in clinical presentation [17, 18]. As reviewed by Table 1 Demographic and basic characteristics of the cohort Women n = 899% (frequency) Men n = 68% (frequency) p value Basic characteristics Sex 93% (899/967) 7% (68/967) <0.0001 Age at symptom onset (years, mean ± SD) 46.16 ± 14.79 47.88 ± 14.71 0.50 Age at diagnosis (years, mean ± SD) 52.65 ± 13.75 52.63 ± 13.52 0.96 Follow-up time from diagnosis (years, mean ± SD) 8.76 ± 7.62 8.48 ± 6.15 0.68 Item IV. Histopathologya Salivary gland biopsy Performed 87% (760/871) 79% (52/66) 0.06 Positive (focus score ≥1) 90% (616/685) 86% (42/49) 0.35 Germinal centers 21% (75/351) 33% (8/24) 0.20 Bold values indicate statistically significant findings (p < 0.05) aAccording to the 2002 Revised American-European Consensus Group criteria for Sjögren’s syndrome Table 1 Demographic and basic characteristics of the cohort Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Page 3 of 9 Clinical data with regard to autoantibody status and clin- ical manifestations were retrieved from the patient’s med- ical records. This included information on sicca symptom onset, age at diagnosis, histopathological examination of minor labial salivary gland biopsies, and serological ana- lysis of ANA, Ro/SSA, and La/SSB autoantibodies. Methods P i ANA was determined by indirect immunoflourescence of Hep2 cells for the vast majority of patients, while methods for determining Ro/SSA and La/SSB autoantibodies at the re- spective accredited Clinical Immunology department var- ied over time and between the centers and included indirect immunofluorescence of transfected cells, im- munoblotting, ELISA, and multiplex technologies. Infor- mation on extraglandular manifestations according to doctors’ clinical assessments included articular, pulmon- ary, renal, cutaneous, muscular, endocrine, and lymphoid systems. The study was approved by the local ethical com- mittee for respective study center, and patients gave in- formed written consent. We also compared the histopathological parameters of the salivary gland biopsy from all the included patients (Table 1). Although women tended to more frequently undergo a salivary gland biopsy than men (p = 0.06), the histological findings revealed no significant differences in terms of a positive focus score or presence of germi- nal center-like structures. Serological differences between female and male patients with pSS Autoantibody profiles were also analyzed in a sex- specific manner (Table 2). Autoantibody positivity was defined as presenting both or either of anti-Ro/SSA or anti-La/SSB. Accordingly, 72% of the patients from this cohort were autoantibody positive; 71% of the female and 81% of the male patients had either Ro/SSA and/or La/SSB antibodies. Anti-Ro/SSA positivity was observed in 68% of the women and 76% of the men; SSB autoantibodies were detected in 41% of the women and 57% of the men (p = 0.01) and positivity for both anti-Ro/SSA and anti- La/SSB was found in 38% of the women and 52% of the men (p = 0.02) (Table 2). Furthermore, ANA positivity was significantly more frequent in male patients (p = 0.02). Thus, the stratified analysis indicated that the male group presents significantly higher frequencies of positivity towards ANA, La/SSB, and Ro/SSA + La/SSB. Statistical analysis For the comparison of continuous variables, the Mann– Whitney U test was used. The chi-square test was used when analyzing categorical data, and Fisher’s exact test was employed if the observed frequency of any given cell was <5 and/or the total number of analyzed individuals in any group was <40. Data was analyzed with GraphPad Prism 6, and p values <0.05 were considered statistically significant. Sex hormones have been suggested to influence the immune system, especially in terms of antibody produc- tion [21]. To evaluate whether the number or percentage of autoantibody positive individuals diagnosed was re- lated to menopause, we further stratified the female and male patients with and without autoantibodies based on age at diagnosis (Fig. 1). We observed an increasing number of autoantibody-positive women being diag- nosed up to 60 years of age, and that at the same time, a steadily increasing number of autoantibody negative women receiving the diagnosis (Fig. 1a). The male group displayed a comparable pattern (Fig. 1b). Consistently, also when analyzed as percent autoantibody positive (Fig. 1c), the trend was similar in both the female and male groups. Already in the late thirties/early forties the Basic characteristics of the cohort The cohort consisted of 967 pSS patients, of which 899 were women (93%) and 68 were men (7%) (Table 1). The female/male ratio was 13:1. The mean age at symptom onset for the female group was 46 years ± 14.8 (95% CI) and 48 years ± 14.7 (95% CI) for the male group. There was no significant difference with regard to the age at diagnosis between women and men (52.6 and 52.6 years, respectively), or the follow-up time from diagnosis be- tween female and male patients (8.8 years ± 7.6, 95% CI and 8.5 years ± 6.1, 95% CI, respectively). Table 2 Serological characteristics in female and male patients with pSS Women n = 899% (frequency) Men n = 68% (frequency) p value Item VI.a Autoantibodies Ro/SSA and/or La/SSB positive 71% (640/899) 81% (54/67) 0.1 Ro/SSA positive 68% (612/897) 76% (51/67) 0.18 La/SSB positive 41% (367/894) 57% (38/67) 0.01 Ro/SSA and La/SSB positive 38% (342/892) 52% (35/67) 0.02 ANA positive 74% (660/895) 87% (58/67) 0.02 Italicized values indicate statistically significant findings (p < 0.05) aAccording to the 2002 Revised American-European Consensus Group criteria for Sjögren’s syndrome Table 2 Serological characteristics in female and male patients with pSS Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Page 4 of 9 20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 66-70 71-75 76-80 >80 0 50 100 150 Age at diagnosis Number diagnosed Women Autoab+ Women Autoab- 20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 66-70 71-75 76-80 >80 0 5 10 15 20 25 30 Age at diagnosis Number diagnosed Men Autoab+ Men Autoab- a b c 24 25-34 35-44 45-54 55-64 65-74 75-84 >85 0 50 100 150 Age at diagnosis % of Autoab positive Men Women Mean age at diagnosis 1 Ro/SSA- and/or La/SSB-positive and negative women and men diagnosed with Sjögren’s syndrome at different age intervals. a Number o/SSA- and/or La/SSB-positive and negative women diagnosed with Sjögren’s syndrome. b Number of Ro/SSA and/or La/SSB positive and ative men diagnosed with Sjögren’s syndrome. Basic characteristics of the cohort c Percentage of Ro/SSA and/or La/SSB positive women and men diagnosed with ren’s syndrome 20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 66-70 71-75 76-80 >80 0 50 100 150 Number diagnosed Women Autoab+ Women Autoab- a Age at diagnosis 20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 66-70 71-75 76-80 >80 0 5 10 15 20 25 30 Number diagnosed Men Autoab+ Men Autoab- b b Age at diagnosis c 24 25-34 35-44 45-54 55-64 65-74 75-84 >85 0 50 100 150 Age at diagnosis % of Autoab positive Men Women Mean age at diagnosis c Men Women Age at diagnosis Fig. 1 Ro/SSA- and/or La/SSB-positive and negative women and men diagnosed with Sjögren’s syndrome at different age intervals. a Number of Ro/SSA- and/or La/SSB-positive and negative women diagnosed with Sjögren’s syndrome. b Number of Ro/SSA and/or La/SSB positive and negative men diagnosed with Sjögren’s syndrome. c Percentage of Ro/SSA and/or La/SSB positive women and men diagnosed with Sjögren’s syndrome Fig. 1 Ro/SSA- and/or La/SSB-positive and negative women and men diagnosed with Sjögren’s syndrome at different age intervals. a Number of Ro/SSA- and/or La/SSB-positive and negative women diagnosed with Sjögren’s syndrome. b Number of Ro/SSA and/or La/SSB positive and negative men diagnosed with Sjögren’s syndrome. c Percentage of Ro/SSA and/or La/SSB positive women and men diagnosed with Sjögren’s syndrome Frequencies of clinical manifestations differ between female and male pSS patients Frequencies of clinical manifestations differ between female and male pSS patients Frequencies of clinical manifestations differ between female and male pSS patients percentage of autoantibody-positive patients diagnosed with pSS started to decline and did so steadily until the mid-seventies. Very few males were diagnosed after the age of 75 (n = 2), making the last point of the curve less relevant to consider. Altogether, the data show a consist- ent higher percentage of autoantibody-positive men. Neither the female nor male group did show any obvious change specific for age 50 or 55, which is commonly used as a proxy for menopause. The presence and type of extraglandular manifestations were obtained in order to evaluate differences in frequen- cies among the sexes. There were significant sex differ- ences in the frequencies of several extraglandular manifestations (Table 3). Interstitial lung disease was more frequent in male patients (p = 0.008), as well as lymph- adenopathy (p = 0.04) and, notably, lymphoma Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Page 5 of 9 Table 3 Frequency of pSS-associated clinical manifestations and comorbidities in female and male patients with pSS Women n = 899% (frequency) Men n = 68% (frequency) p value Table 3 Frequency of pSS-associated clinical manifestations and comorbidities in female and male patients with pSS Women n = 899% (frequency) Men n = 68% (frequency) p value Table 3 Frequency of pSS-associated clinical manifestations and comorbidities in female and male patients with pSS Women n = 899% (frequency) Men n = 68% (frequency) p value Classificationa Articular Arthritis 20% (169/859) 14% (9/65) 0.25 Pulmonary Interstitial lung disease 6% (40/619) 17% (8/48) 0.008 Renal Interstitial nephritis 3% (19/607) 2% (1/48) 1.00 Cutaneous Dermal vasculitis 11% (91/836) 8% (5/62) 0.49 Lymphadenopathy and lymphoma Enlarged lymph nodes 8% (69/825) 16% (10/62) 0.04 Lymphoma 4% (32/889) 10% (7/68) 0.007 Muscular Myositis 0.9% (7/772) 3% (2/61) 0.14 Glandular Major salivary gland swelling 29% (225/767) 40% (21/53) 0.11 Presence of EGM 34% (304/899) 41% (28/68) 0.22 Number of EGM (mean + SD) 0.44 ± 0.69 0.51 ± 0.74 0.30 Other common comorbidities and clinical manifestations Hypothyroidism 24% (175/739) 8% (4/51) 0.009 Raynaud’s phenomenon 29% (247/851) 30% (20/66) 0.83 Italicized values indicate statistically significant findings (p < 0.05) aAvailable data on the extraglandular manifestations evaluated to estimate the EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI) are included EGM extraglandular manifestations Hypothyroidism to women (Table 3), we analyzed the difference between histopathologically verified subtypes of lymphoma. Discussion Primary Sjögren’s syndrome represents the autoimmune disease with the highest female bias, ranging from a ratio of 10–20:1 [22]. Besides the overwhelming sex bias ob- served in disease susceptibility, previous studies have also aimed to investigate whether the disease manifests differently between female and male patients. Earlier observations have not reached a clear consensus as to whether male pSS patients have a distinct clinical course and a more severe presentation of the disease [3]. In a recent study, though, we reported that men with pSS, from a population-based incident case cohort, displayed significantly higher levels of anti-Ro52, were diagnosed at an earlier age than their female counterparts, pre- sented more concomitant extraglandular manifestations, and had a higher frequency of pulmonary complications and cutaneous vasculitis. Similarly, an Italian cohort re- vealed a significant male propensity towards extragland- ular manifestation presentation [19]. These findings strongly suggest that men affected by pSS have a more severe disease at time of diagnosis. In the present study, we addressed whether differences between the sexes are also present several years after diagnosis. Interstitial lung disease has been extensively studied in the context of pSS [29, 30]. Male sex is widely recog- nized as a risk factor for developing interstitial lung dis- ease [26, 31]. In accordance with more recent studies [19], our extended cohort shows that male patients with pSS are indeed more prone to develop interstitial lung disease. The reasons for this male preponderance are poorly understood; however, this might be due to in- creased seropositivity, environmental exposure to certain pollutants [32] and smoking [31] in the male pSS group. In fact, idiopathic pulmonary fibrosis, which represents a usual interstitial pneumonia histopathological pattern, is one of the most common forms of interstitial lung dis- ease detected in pSS [29] and has a higher prevalence in men, as epidemiological studies have previously described [33, 34]. In other words, regardless of pSS diagnosis, men in general are more frequently affected by a type of interstitial lung disease that is associated with a worse prognosis. This susceptibility, thus, might be augmented in pSS, driven by other pathophysiological factors that enhance this propensity to develop pulmon- ary disease. We identified significant sex differences in terms of serological parameters and frequencies of some organ in- volvement. Autoantibody-positive pSS female and male patients differ in terms of clinical manifestations present more often with La/SSB, Ro/SSA + La/SSB and ANA positivity. This increased immune activity observed among the male patients is of special interest since in a healthy state, men mount a lower antibody response in comparison with women [23–25]. Although the patho- genic effect of autoantibodies has not been clearly estab- lished, the presence of certain autoantibodies has been associated with organ manifestations. Noteworthy, SSA antibodies are related to pulmonary diseases [26, 27], an extraglandular manifestation we observed overrepresented in the male patients from our cohort. Further, a recent study proposed that anti-La/SSB antibodies are a risk fac- tor associated with increased mortality in pSS patients [28]. Thus, even though the pathogenic role of pSS- associated autoantibodies remains unknown, seropositivity has a strong correlation with organ involvement and worse prognosis, supporting the conclusion that the dis- ease course is more severe in male patients than in female patients. We further assessed whether presentation between autoantibody-positive female and male patients differed from the unstratified analysis described above, focusing on the extraglandular manifestations and other clinical manifestations that had significantly differed between female and male patients regardless of serology-status. Interstitial lung disease and lymphoma were significantly more frequent in men (p = 0.01 and p = 0.03, respect- ively) also when including only seropositive cases in the analysis, and hypothyroidism was more common in women (p = 0.03) (Table 5). Lymphadenopathy had a higher observed frequency among male patients (Table 3), which was however not statistically significant when considering only the autoantibody positive cases. Frequencies of clinical manifestations differ between female and male pSS patients Mucosa-associated lymphoid tissue (MALT) lymphoma and diffuse large B cell lymphoma (DLBCL) were the most common subtypes, but no significant difference be- tween the occurrence between women and men was found (Table 4). (p = 0.007). There was also a tendency for men to present more often with major salivary glands swelling (p = 0.11), as well as myositis (p = 0.14), while hypothyroidism was more common in female patients, present in 24% of the women as opposed to 8% of the male patients (p = 0.009). Considering the observation that men with pSS present an increased risk for lymphoma when compared Table 4 Frequencies of subtypes of lymphoma in women and men with pSS Women n = 32% (frequency) Men n = 7% (frequency) p valuea MALT lymphoma 56% (18/32) 57% (4/7) 1.00 DLBCL 16% (5/32) 0% (0/7) 0.56 Follicular lymphoma 6% (2/32) 14% (1/7) – Myeloma 3% (1/32) 0% (0/7) – CLL 3% (1/32) 0% (0/7) – Lymphoplasmacytic lymphoma 0% (0/32) 14% (1/7) – Other NHL 13% (4/32) 14% (1/7) – Hodgkin lymphoma 3% (1/32) 0% (0/7) – According to the WHO 2016 classification aCalculated when n ≥5 for either group MALT mucosa-associated lymphoid tissue, DLBCL diffuse large B cell lymphoma, CLL Chronic lymphatic leukemia, NHL non-Hodgkin lymphoma Table 4 Frequencies of subtypes of lymphoma in women and men with pSS aCalculated when n ≥5 for either group MALT mucosa-associated lymphoid tissue, DLBCL diffuse large B cell lymphoma, CLL Chronic lymphatic leukemia, NHL non-Hodgkin lymphoma Page 6 of 9 Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Discussion with rheumatic disease has been seldom studied, mainly due to the inclusion of mostly female patients. Neverthe- less, Ansell et al. have reported a significantly higher in- cidence of lymphoma in male RA patients. Despite the increased association of autoimmune diseases and lymphoma in men [39], earlier studies of sex differences in lymphoproliferative malignancies in pSS have not shown a clear sex-specific predominance [40, 41]. In contrast, our present study is the largest pSS cohort to report a significantly increased risk for male patients to present lymphoma in comparison with female patients. This is in accordance with the results from a smaller patient sample from which an increased risk for men affected with pSS, SLE, RA, and autoimmune hemolytic anemia to develop lymphoma was reported [42]. with rheumatic disease has been seldom studied, mainly due to the inclusion of mostly female patients. Neverthe- less, Ansell et al. have reported a significantly higher in- cidence of lymphoma in male RA patients. Despite the increased association of autoimmune diseases and lymphoma in men [39], earlier studies of sex differences in lymphoproliferative malignancies in pSS have not shown a clear sex-specific predominance [40, 41]. In contrast, our present study is the largest pSS cohort to report a significantly increased risk for male patients to present lymphoma in comparison with female patients. This is in accordance with the results from a smaller patient sample from which an increased risk for men affected with pSS, SLE, RA, and autoimmune hemolytic anemia to develop lymphoma was reported [42]. Since the male bias observed in pSS-associated malig- nancies has only recently been described, it is not fully understood. However, considering the reported predict- ive factors of lymphoma development, an increased risk of lymphoma in male patients is logical. As reviewed by Nocturne and Mariette [43], the main clinical manifesta- tions and parameters associated with this type of cancer are swelling of salivary glands, lymphadenopathy, palp- able purpura, cryoglobulinemia, lymphopenia, low com- plement levels, and a monoclonal component in serum or urine. Interestingly, the male pSS patients from our cohort presented more frequently with lymphadenop- athy. Although not included in the data analysis due to the high amount of missing data, cryoglobulinemia was also more commonly observed in the male patients (20/ 151 in females vs 3/7 in males, p = 0.03). Discussion p ) The only clinical manifestation that was more signifi- cantly represented in the female pSS patients from our cohort was hypothyroidism. Endocrine problems are not uncommon in patients with autoimmune disorders [44], and the female bias towards thyroid diseases has been extensively documented [45–48]. Furthermore, since the predominantly female incidence of hypothyroidism cor- responds with the female susceptibility to autoimmune diseases, the thyroid gland has been proposed as a de- cisive organ to explain the sex skewness in autoimmune diseases. The effect of adipokines, which comprise a number of different cytokines including, e.g., leptins, adiponectins, TNF-α, and IL-6, on thyroid tissue has been suggested as a triggering mechanism for auto- immune thyroiditis, which probably precedes or coincides with the diagnosis of another systemic autoimmune dis- ease such as pSS and SLE. [44]. This emphasizes the need for a comprehensive screening and close monitoring of thyroid function in suspected patients because it might be an important marker for autoimmune disease develop- ment. As for the thyroid gland being responsible for female-preponderant diseases, further studies should be performed to clarify its role. Conclusions In summary, our findings provide compelling evidence that the clinical presentation of pSS differs between women and men. The sex-specific preference of some clinical manifestations hints at divergent pathophysio- logical mechanisms between women and men with pSS. Consequently, management of the disease will benefit from sex-specific tailored clinical programs to address complications that are common or expected in the respective sex. Discussion Our results indicate that the humoral response between women and men is different; particularly, men It is well known that pSS patients have an increased risk for developing non-Hodgkin lymphoma [35–38]. Sex-specific risk for lymphoma development in patients Table 5 Frequency of comorbidities, extraglandular, and other common clinical manifestations in SSA- and/or SSB-positive female and male patients with pSS Women n = 640% (frequency) Men n = 54% (frequency) p value Interstitial lung disease 7% (31/444) 19% (7/37) 0.01 Enlarged lymph nodes 10% (57/584) 16% (8/49) 0.15 Hypothyroidism 23% (117/517) 8% (3/40) 0.03 Lymphoma 4% (28/634) 11% (6/54) 0.03 Italicized values indicate statistically significant findings (p < 0.05) Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Page 7 of 9 Page 7 of 9 The studied cohort offers a valuable large group of clinically carefully characterized patients with pSS, allowing for analysis of parameters that differ between men and women affected by the syndrome. The long follow-up time is essential for identifying clinical mani- festations at different time points of the disease course. However, the patients included in this cohort were mostly included at tertiary referral centers of university hospital clinics. A possible limitation of the study is that the study population might therefore not mirror the general pSS patient population and that the patients described in this study represent cases with an overall more severe disease phenotype, both female and male patients. A further possibility is that male patients with mild symptoms and less severe disease are less often referred, as the primary health care doctor may not be as likely to suspect Sjögren’s syndrome due to its rarity in men, resulting in only men with more severe disease being included in the study. As the evaluation of extra- glandular manifestations was dependent on doctors’ clin- ical assessments and did not include specific laboratory or physiologic tests unless the patient had symptoms, it is also possible that subclinical extraglandular manifesta- tions may have been missed. However, the mean number of extraglandular manifestations diagnosed did not differ significantly between centers, nor did the proportion of men and women contributed. A further limitation is the lack of EULAR primary Sjögren’s syndrome disease ac- tivity (ESSDAI) and patient-reported (ESSPRI) indexes [49] at diagnosis, as well as information on other com- mon extraglandular manifestations such as neurological diseases. Acknowledgements Acknowledgements DISSECT consortium members: Agneta Zickert, Stockholm; Albin Björk, Stockholm; Anders A. Bengtsson, Lund; Andreas Jönsen, Lund; Andrei Alexsson, Uppsala; Anna Tjärnlund, Stockholm; Ann-Christine Syvänen, Uppsala; Antonella Notarnicola, Stockholm; Argyri Mathioudaki, Uppsala; Åsa Karlsson, Uppsala; Carin Backlin, Uppsala; Christine Bengtsson, Umeå; Christopher Sjöwall, Linköping; Dag Leonard, Uppsala; Daniel Hammenfors, Bergen; Elisabet Svenungsson, Stockholm; Elke Theander, Malmö; Eva Baecklund, Uppsala; Eva Murén, Uppsala; Fabiana Farias, Uppsala; Gerli Pielberg, Uppsala; Guðný Ella Thorlacius, Stockholm; Gunnel Nordmark, Uppsala; Hector Chinoy, Manchester; Helena Andersson, Oslo; Helena Enocsson, Linköping; Helena Forsblad-d’Elia, Gothenburg; Ingrid E. Lundberg, Stockholm; Iva Gunnarsson, Stockholm; Janine Lamb, Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 Received: 29 April 2017 Accepted: 26 July 2017 Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 15. Weyand CM, Schmidt D, Wagner U, Goronzy JJ. The influence of sex on the phenotype of rheumatoid arthritis. Arthritis Rheum. 1998;41:817–22. 16. Gossec L, Baro-Riba J, Bozonnat MC, et al. Influence of sex on disease severity in patients with rheumatoid arthritis. J Rheumatol. 2005;32:1448–51. Availability of data and materials Not applicable. 8. Harris VM, Sharma R, Cavett J, et al. Klinefelter's syndrome (47,XXY) is in excess among men with Sjogren's syndrome. Clin Immunol. 2016;168:25–9. Publisher’s Note 17. Gondran G, Fauchais A, Lambert M, et al. Primary Sjogren's syndrome in men. Scand J Rheumatol. 2008;37:300–5. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 18. Horvath IF, Szodoray P, Zeher M. Primary Sjogren's syndrome in men: clinical and immunological characteristic based on a large cohort of Hungarian patients. Clin Rheumatol. 2008;27:1479–83. Received: 29 April 2017 Accepted: 26 July 2017 Manchester; Jennifer Meadows, Uppsala; Jessika Nordin, Uppsala; Johan G Brun, Bergen; Johanna Dahlqvist, Uppsala; Johanna K. Sandling, Uppsala; John Mo, Gothenburg; Jonas Carlsson Almlöf, Uppsala; Jonas Wetterö, Linköping; Jorge I. Ramírez Sepúlveda, Stockholm; Juliana Imgenberg-Kreuz, Uppsala; Karin Bolin, Uppsala; Karin Hjorton, Uppsala; Karl A. Brokstad, Bergen; Karolina Tandre, Uppsala; Kathrine Skarstein, Bergen; Katrine Brække Norheim, Stavanger; Kerstin Lindblad-Toh, Uppsala; Lars Rönnblom, Uppsala; Leonid Padyukov, Stockholm; Lilian Vasaitis, Uppsala; Lina Hultin-Rosenberg, Uppsala; Louise Pyndt Diederichsen, Odense; Maija-Leena Eloranta, Uppsala; Malin V. Jonsson, Bergen; Marie Wahren-Herlenius, Stockholm; Marika Kvarnström, Stockholm; Maryam Dastmalchi, Stockholm; Matteo Bianchi, Uppsala; Niklas Hagberg, Uppsala; Outi Vaarala, Gothenburg; Øyvind Molberg, Oslo; Per Eriksson, Linköping; Roald Omdal, Stavanger; Robert G. Cooper, Liverpool; Roland Jonsson, Bergen; Sara Magnusson Bucher, Örebro; Sergey Kozyrev, Uppsala; Silke Appel, Bergen; Simon Rothwell, Manchester; Solbritt Rantapää- Dahlqvist, Umeå; Svein Joar Johnsen, Stavanger; Thomas Mandl, Malmö. References h 1. Whitacre CC. Sex differences in autoimmune disease. Nat Immunol. 2001;2: 777–80. 2. Kvarnstrom M, Ottosson V, Nordmark B, Wahren-Herlenius M. Incident cases of primary Sjogren's syndrome during a 5-year period in Stockholm County: a descriptive study of the patients and their characteristics. Scand J Rheumatol. 2015;44:135–42. 3. Brandt JE, Priori R, Valesini G, Fairweather D. Sex differences in Sjogren's syndrome: a comprehensive review of immune mechanisms. Biol Sex Differ. 2015;6:19. 3. Brandt JE, Priori R, Valesini G, Fairweather D. Sex differences in Sjogren's syndrome: a comprehensive review of immune mechanisms. Biol Sex Differ. 2015;6:19. 4. Lessard CJ, Li H, Adrianto I, et al. Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjogren's syndrome. Nat Genet. 2013;45:1284–92. 4. Lessard CJ, Li H, Adrianto I, et al. Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjogren's syndrome. Nat Genet. 2013;45:1284–92. Authors’ contributions 9. Boodhoo KD, Liu S, Zuo X. Impact of sex disparities on the clinical manifestations in patients with systemic lupus erythematosus: a systematic review and meta-analysis. Medicine (Baltimore). 2016;95:e4272. 9. Boodhoo KD, Liu S, Zuo X. Impact of sex disparities on the clinical manifestations in patients with systemic lupus erythematosus: a systematic review and meta-analysis. Medicine (Baltimore). 2016;95:e4272. JIRS and MWH conceived and designed the study. DISSECT consortium: MK, PE, KBN, SJJ, DH, MVJ, KS, HFdE, EB, SMB, ET, TM, RO, RJ, and GN managed the study participant recruitment and clinical data acquisition. Data was analyzed by JIRS and GN. JIRS and MWH wrote the first draft of the manuscript, and all authors participated in the revision until its final stage. All authors read and approved the final manuscript. 10. Voulgari PV, Katsimbri P, Alamanos Y, Drosos AA. Gender and age differences in systemic lupus erythematosus. A study of 489 Greek patients with a review of the literature. Lupus. 2002;11:722–9. 11. Andrade RM, Alarcon GS, Fernandez M, et al. Accelerated damage accrual among men with systemic lupus erythematosus: XLIV. Results from a multiethnic US cohort. Arthritis Rheum. 2007;56:622–30. Consent for publication Consent for publication Not applicable. 14. Marrie RA, Patten SB, Tremlett H, et al. Sex differences in comorbidity at diagnosis of multiple sclerosis: a population-based study. Neurology. 2016. [Epub ahead of print] Ethics approval and consent to participate The study was approved by the regional ethical committee for respective study center, and patients gave informed written consent. 12. Manger K, Manger B, Repp R, et al. Definition of risk factors for death, end stage renal disease, and thromboembolic events in a monocentric cohort of 338 patients with systemic lupus erythematosus. Ann Rheum Dis. 2002;61:1065–70. 13. Tremlett H, Paty D, Devonshire V. Disability progression in multiple sclerosis is slower than previously reported. Neurology. 2006;66:172–7. 12. Manger K, Manger B, Repp R, et al. Definition of risk factors for death, end stage renal disease, and thromboembolic events in a monocentric cohort of 338 patients with systemic lupus erythematosus. Ann Rheum Dis. 2002;61:1065–70. 13. Tremlett H, Paty D, Devonshire V. Disability progression in multiple sclerosis is slower than previously reported. Neurology. 2006;66:172–7. Funding Th d 5. Imgenberg-Kreuz J, Sandling JK, Almlöf JC, Nordlund J, Signér L, Norheim KB, Omdal R, Rönnblom L, Eloranta ML, Syvänen AC, Nordmark G. Genome- wide DNA methylation analysis in multiple tissues in primary Sjögren's syndrome reveals regulatory effects at interferon-induced genes. Ann Rheum Dis. 2016;75(11):2029–36. doi:10.1136/annrheumdis-2015-208659. g The study was supported by grants from the Swedish Research Council, the Heart-Lung Foundation, the Stockholm County Council, Karolinska Institutet, the Swedish Rheumatism association, and the King Gustaf the Vth 80-year foundation. The DISSECT study is supported by an AstraZeneca Science for Life Laboratory Research Collaboration grant. ; ( ) 6. Cutolo M, Capellino S, Sulli A, et al. Estrogens and autoimmune diseases. Ann N Y Acad Sci. 2006;1089:538–47. 6. Cutolo M, Capellino S, Sulli A, et al. Estrogens and autoimmune diseases. Ann N Y Acad Sci. 2006;1089:538–47. 7. Liu K, Kurien BT, Zimmerman SL, et al. X chromosome dose and sex bias in autoimmune diseases: increased prevalence of 47,XXX in systemic lupus erythematosus and Sjogren's syndrome. Arthritis Rheumatol. 2016;68:1290–300. 7. Liu K, Kurien BT, Zimmerman SL, et al. X chromosome dose and sex bias in autoimmune diseases: increased prevalence of 47,XXX in systemic lupus erythematosus and Sjogren's syndrome. Arthritis Rheumatol. 2016;68:1290–300. 7. Liu K, Kurien BT, Zimmerman SL, et al. X chromosome dose and sex bias in autoimmune diseases: increased prevalence of 47,XXX in systemic lupus erythematosus and Sjogren's syndrome. Arthritis Rheumatol. 2016;68:1290–300. 8. Harris VM, Sharma R, Cavett J, et al. Klinefelter's syndrome (47,XXY) is in excess among men with Sjogren's syndrome. Clin Immunol. 2016;168:25–9. 9. Boodhoo KD, Liu S, Zuo X. Impact of sex disparities on the clinical manifestations in patients with systemic lupus erythematosus: a systematic review and meta-analysis. Medicine (Baltimore). 2016;95:e4272. Author details 1 1Unit of Experimental Rheumatology, Department of Medicine, Karolinska University Hospital, Karolinska Institutet, SE-171 76 Stockholm, Sweden. 2Division of Rheumatology, Department of Clinical Experimental Medicine, Linköping University, Linköping, Sweden. 3Department of Rheumatology, Skåne University Hospital, Malmö, Sweden. 4Clinical immunology unit, Department of Internal Medicine, Stavanger University Hospital, Stavanger, Norway. 5Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Bergen, Norway. 6Department of Rheumatology, Haukeland University Hospital, Bergen, Norway. 7Section for Oral and Maxillofacial Radiology, Department of Clinical Dentistry, University of Bergen, Bergen, Norway. 8Gade Laboratory for Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway. 9Department of Pathology, Haukeland University Hospital, Bergen, Norway. 10Department of Medical Sciences, Rheumatology and Science for Life Laboratory, Uppsala University, Uppsala, Sweden. 11Department of Public Health and Clinical Medicine, Rheumatology, Umeå University, Umeå, Sweden. 12Department of Rheumatology, Faculty of Medicine and Health, Örebro University, Örebro, Sweden. 19. Ramirez Sepulveda JI, Kvarnstrom M, Brauner S, Baldini C, Wahren-Herlenius M. Difference in clinical presentation between women and men in incident primary Sjogren's syndrome. Biol Sex Differ. 2017;8:16. 20. Vitali C, Bombardieri S, Jonsson R, et al. Classification criteria for Sjogren's syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis. 2002;61:554–8. 21. Sakiani S, Olsen NJ, Kovacs WJ. Gonadal steroids and humoral immunity. Nat Rev Endocrinol. 2013;9:56–62. 22. Garcia-Carrasco M, Mendoza-Pinto C, Jimenez-Hernandez C, et al. Serologic features of primary Sjogren's syndrome: clinical and prognostic correlation. Int J Clin Rheumtol. 2012;7:651–9. 23. Klein SL, Jedlicka A, Pekosz A. The Xs and Y of immune responses to viral vaccines. Lancet Infect Dis. 2010;10:338–49. 24. Furman D. Sexual dimorphism in immunity: improving our understanding of vaccine immune responses in men. Expert Rev Vaccines. 2015;14:461–71. 25. Klein SL, Marriott I, Fish EN. Sex-based differences in immune function and responses to vaccination. Trans R Soc Trop Med Hyg. 2015;109:9–15. Page 9 of 9 26. Boitiaux JF, Debray MP, Nicaise-Roland P, et al. Idiopathic interstitial lung disease with anti-SSA antibody. Rheumatology (Oxford). 2011;50:2245–50. 27. Ghillani P, Andre C, Toly C, et al. Clinical significance of anti-Ro52 (TRIM21) antibodies non-associated with anti-SSA 60kDa antibodies: results of a multicentric study. Autoimmun Rev. 2011;10:509–13. multicentric study. Autoimmun Rev. 2011;10:509–13. 28. Singh AG, Singh S, Matteson EL. Rate, risk factors and causes of mortality in patients with Sjogren's syndrome: a systematic review and meta-analysis of cohort studies. Rheumatology (Oxford). 2016;55:450–60. cohort studies. Rheumatology (Oxford). 2016;55:450–60. 29. Author details 1 Mira-Avendano IC, Abril A. Pulmonary manifestations of Sjogren syndrome, systemic lupus erythematosus, and mixed connective tissue disease. Rheum Dis Clin N Am. 2015;41:263–77. 30. Flament T, Bigot A, Chaigne B, et al. Pulmonary manifestations of Sjogren's syndrome. Eur Respir Rev. 2016;25:110–23. 31. Yazisiz V, Arslan G, Ozbudak IH, et al. Lung involvement in patients with primary Sjogren's syndrome: what are the predictors? Rheumatol Int. 2010; 30:1317–24. 32. Johannson KA, Balmes JR, Collard HR. Air pollution exposure: a novel environmental risk factor for interstitial lung disease? Chest. 2015;147:1161–7. 33. Ley B, Collard HR, King TE Jr. Clinical course and prediction of survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2011;183:431–40. pathic pulmonary fibrosis. Am J Respir Crit Care Med. 2011;183:431–40 34. Hopkins RB, Burke N, Fell C, Dion G, Kolb M. Epidemiology and survival of idiopathic pulmonary fibrosis from national data in Canada. Eur Respir J. 2016;48:187–95. 35. Kassan SS, Thomas TL, Moutsopoulos HM, et al. Increased risk of lymphoma in sicca syndrome. Ann Intern Med. 1978;89:888–92. 36. Theander E, Henriksson G, Ljungberg O, et al. Lymphoma and other malignancies in primary Sjogren's syndrome: a cohort study on cancer incidence and lymphoma predictors. Ann Rheum Dis. 2006;65:796–803. 37. Dias C, Isenberg DA. Susceptibility of patients with rheumatic diseases to B- cell non-Hodgkin lymphoma. Nat Rev Rheumatol. 2011;7:360–8. 38. Nishishinya MB, Pereda CA, Munoz-Fernandez S, et al. Identification of lymphoma predictors in patients with primary Sjogren's syndrome: a systematic literature review and meta-analysis. Rheumatol Int. 2015;35:17– 26. 39. Ansell P, Simpson J, Lightfoot T, et al. Non-Hodgkin lymphoma and autoimmunity: does gender matter? Int J Cancer. 2011;129:460–6. 40. Mellemkjaer L, Pfeiffer RM, Engels EA, et al. Autoimmune disease in individuals and close family members and susceptibility to non-Hodgkin's lymphoma. Arthritis Rheum. 2008;58:657–66. 41. Smedby KE, Askling J, Mariette X, Baecklund E. Autoimmune and inflammatory disorders and risk of malignant lymphomas–an update. J Intern Med. 2008;264:514–27. 42. Fallah M, Liu X, Ji J, et al. Hodgkin lymphoma after autoimmune diseases by age at diagnosis and histological subtype. Ann Oncol. 2014;25:1397–404. 43. Nocturne G, Mariette X. Sjogren syndrome-associated lymphomas: an update on pathogenesis and management. Br J Haematol. 2015;168:317–27. 44. Merrill SJ, Mu Y. Thyroid autoimmunity as a window to autoimmunity: an explanation for sex differences in the prevalence of thyroid autoimmunity. J Theor Biol. 2015;375:95–100. 45. Lu MC. Yin WY, Tsai TY, Koo M. 49. Seror R, Bootsma H, Saraux A, et al. Defining disease activity states and clinically meaningful improvement in primary Sjogren's syndrome with EULAR primary Sjogren's syndrome disease activity (ESSDAI) and patient- reported indexes (ESSPRI). Ann Rheum Dis. 2016;75:382–9. 48. Jara LJ, Navarro C, Brito-Zeron Mdel P, et al. Thyroid disease in Sjogren's syndrome. Clin Rheumatol. 2007;26:1601–6. Ramírez Sepúlveda et al. Biology of Sex Differences (2017) 8:25 47. Appenzeller S, Pallone AT, Natalin RA, Costallat LT. Prevalence of thyroid dysfunction in systemic lupus erythematosus. J Clin Rheumatol. 2009;15: 117–9. Author details 1 Lai NS Increased risk of primary Sjogren's syndrome in female patients with thyroid disorders: a longitudinal population-based study in Taiwan PLoS One. 2013;8:e77210. population-based study in Taiwan PLoS One. 2013;8:e77210. 46. Mavragani CP, Fragoulis GE, Moutsopoulos HM. Endocrine alterations in primary Sjogren's syndrome: an overview. J Autoimmun. 2012;39:354–8. 47. Appenzeller S, Pallone AT, Natalin RA, Costallat LT. Prevalence of thyroid dysfunction in systemic lupus erythematosus. J Clin Rheumatol. 2009;15: 117–9. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: 48. Jara LJ, Navarro C, Brito-Zeron Mdel P, et al. Thyroid disease in Sjogren's syndrome. Clin Rheumatol. 2007;26:1601–6. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit y p and we will help you at every step: 49. Seror R, Bootsma H, Saraux A, et al. Defining disease activity states and clinically meaningful improvement in primary Sjogren's syndrome with EULAR primary Sjogren's syndrome disease activity (ESSDAI) and patient- reported indexes (ESSPRI). Ann Rheum Dis. 2016;75:382–9.
https://openalex.org/W3036251122	https://zenodo.org/records/3952322/files/IJRR0036.pdf	English	null	Elaboration of a Cement Matrix Composite, Improved With the Pozzolan of Rice Husk Ash, Reinforced With Sugar Cane Bagasse Fibers	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	4,236	ABSTRACT The building sector has been proven as a vector of environmental pollution, through the consumption of natural resources (rocks, wood, water, etc.) and energy (construction process, transport, heating, lighting, etc.) during all phases of its evolution: construction, use, rehabilitation and destruction. Philippe. Deshayes (2012) showed that, the building represented around 40% of CO2 emissions from developed countries, 37% of energy consumption and 40% of the waste produced. Also faced with an exhaustion of fossil resources having non-renewable petro-sourced constituents, the researchers were interested in the valorization of renewable vegetable or natural materials. Bio-composite materials, reinforced of animal or vegetable origin, thus meet ecological and economic challenges. Solid waste management is one of the main concerns worldwide due to the increasing quantities of industrial waste and by- products. Therefore, for a few years, scientiﬁc interest has being turned to tropical fibers, such as sisal, alpha and jute fibers. A very few research where permormed on sugarcane bagasse reinforcements. To cater for the concern of valorization of the waste of the sugar cane, the husks of rice husk and also the reduction of greenhouse gases and for the civil engineer, the lightening of the structures, a solution was on the one hand, to study the In this work it’s developed a multifunctional eco-material based on short fibers of sugar cane with a cement matrix improved with the rice husk pozzolana. The properties of the sand were analyzed by particle size, fineness modulus and sand equivalence tests. The rice husk pozzolan was obtained by calcination at 600 ° C, the temperature at which the fire loss becomes constant, until calcination of the ash, which was determined by Thermogravimetric Analysis. The characterization of the ash was determined by the X-ray Diffraction and Fluorexence X tests to obtain its mineralogical constituents, in particular the silica content of 98.78%, in preponderance which had characterized its pozzolanicity. The pozzolanic activity of 0.71 was in the active range of 0.67 to 1. The composite material was tested for maneuverability with a water / cement ratio E = 0.7. Elaboration of a Cement Matrix Composite, Improved with the Pozzolan of Rice Husk Ash, Reinforced with Sugar Cane Bagasse Fibers ACODJI Vodouhè. Pamphile, OLODO T. Emmanuel, DOKO K. Valery Elaboration of a Cement Matrix Composite, Improved with the Pozzolan of Rice Husk Ash, Reinforced with Sugar Cane Bagasse Fibers ACODJI Vodouhè. Pamphile, OLODO T. Emmanuel, DOKO K. Valery Corresponding Author: ACODJI Vodouhè. Pamphile International Journal of Research and Review Vol.7; Issue: 6; June 2020 Website: www.ijrrjournal.com E-ISSN: 2349-9788; P-ISSN: 2454-2237 International Journal of Research and Review Vol.7; Issue: 6; June 2020 Website: www.ijrrjournal.com E-ISSN: 2349-9788; P-ISSN: 2454-2237 Research Paper ABSTRACT Substitution of the sand by sugarcane fibers, up to a rate of 5%, had improved the behavior in compression, in bending, which were tested by the 3-point bending test, combining good mechanical properties in compression of resistance of 4.82 MPa, in flexion of 28.83 MPa, ductile and very light for its use to reduce the weight of the structures and fight against global warming, due to the quantity of cement less, source of production of significant quantities of polluting CO2. Keywords: eco-material; matrix; maneuverability; fibers, mechanical; compression; resistance. International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 263 ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers after this treatment are dried in the open air for seven days. They are stoved at 105 ° C in the laboratory for 24 hours. possibility of using ecological materials produced from these wastes, respecting scientific standards, of very low weight. In Benin, SUCOBE, a factory that transforms sugar cane into industrial products (sugar, ethanol, etc.), produces a large amount of sugar cane waste, an industrial residue, eliminated by burning. The husks of the rice are also wastes from husking of rice from industries located in Zangnalando in central Benin. The originality of the study was to develop a material with a cementitious matrix, improved with an artificial pozzolan rich in amorphous silica and reinforced by short bagasse fibers from sugar cane, randomly arranged which gives a property of anisotropy to this material. Figure 1: Sugarcane bagasse Figure 2 : Particle distribution of sugarcane bagasse fibers 2.1.1 Bagasse fibers from sugar cane: According to the literature, the bagasse of sugar cane is a residue generated largely by agro-industries for the production of sugar or ethanol. Sandra. Luz, Adilson. Roberto. Goncalves, and al, (2007) showed that this residue, obtained after extraction of the sugar cane juice, is mainly made up of fibers usable for composite reinforcements. Davina. Michel, (2013) proved that this crude residue consists of 50-60% in the dry base of useful fibers. The main production plant in Benin is the Complant Sugar Factory of Benin (SUCOBE). Today, in the SUCOBE factory, the residues are used partly as fuel for boilers, and the rest eliminated by uncontrolled burning. In the context of the present study, preliminary treatments are carried out, consisting in separating the organic matter and the marrow, from the fibers of the bagasse itself, after immersion in water. The fibers, For made-up composites, we are interested in the granular class of [2 ;2,5 ;5]. 2. MATERIALS, EQUIPMENT AND METHODS Materials Materials In this study, several materials are used for the confection of composites including: Figure 1: Sugarcane bagasse Figure 2 : Particle distribution of sugarcane bagasse fibers - sugarcane bagasse fibers; The size distribution of crushed sugarcane bagasse fibers, is shown in Figure 2. This curve is similar to that obtained from Mohamed. El-Sayed and Taher. El-Samni, (2006), on the granulometry of the bagasse of sugarcane. - rice husk; - sand; - sand; - water; - cement. 2.1.2 Rice husk Ash / Artificial Pozzolan 2.1.2 Rice husk Ash / Artificial Pozzolan Anissa. Benredouane, Larbi. Kacimi, and al (2014) worked on artificial pozzolans and claimed that they are minerals from factories, thermal power stations and even from the calcination of agricultural by- products rich in reactive silica and alumina. Anissa. Benredouane, Larbi. Kacimi, and al (2014) worked on artificial pozzolans and claimed that they are minerals from factories, thermal power stations and even from the calcination of agricultural by- products rich in reactive silica and alumina. An artificial rice husk pozzolana was obtained by calcining the husks of rice, at various temperatures ranging from 400 ° C and 850 ° C. The optimal calcination temperature is given to us from the stabilization temperature of the mass of the ash. Figure 3 gives the loss on ignition as a function of variation in the calcination temperature. A ‘m’ mass of 10 g was taken for analysis. An artificial rice husk pozzolana was obtained by calcining the husks of rice, at various temperatures ranging from 400 ° C and 850 ° C. The optimal calcination temperature is given to us from the stabilization temperature of the mass of the ash. Figure 3 gives the loss on ignition as a function of variation in the calcination temperature. A ‘m’ mass of 10 g was taken for analysis. International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 264 ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers Figure 3. Variation of loss on ignition as a function of temperature. ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers Figure 3. Variation of loss on ignition as a function of temperature. Figure 3. Variation of loss on ignition as a function of temperature. The heating speed of the oven was adjusted to 5 ° C / min to obtain a calcined and homogeneous ash, according to the protocol, for 5 hours of time. The temperature was then kept constant for 2 hours. The calcination temperature was that from which the loss on ignition became constant. 2.1.2 Rice husk Ash / Artificial Pozzolan As shown in Figure 3, the target calcination temperature for one of this rice husk reactive pozzolana was 600 ° C. Lavie Arsène and Mango-Itulamya (2018) in; Jesse. Kigozi, Moses Kiggundu. and al (2015) obtained the same calcination temperature on the rice husk, and determined a reactive pozzolan. (a) (b) Figure 4 : (a) Rice husk - (b) rice husk ash obtained after calcination ( ) (a) Figure 4 : (a) Rice husk - (b) rice husk ash obtained after calcination 2.2 Equipment The tests are carried out in the laboratories: • Laboratory equipment “LEMA”, Department of Civil Engineering, Doctoral School of Engineering Sciences, Abomey Calavi, Benin; Table 1. The physical characteristics of cement Apparent density 1,073 Volumic mass 3,01 Beginning of setting 3h05 End of setting 4h39 Specific surface (Blaine) 3155 Expansion 1,5 Refusal on sieve 0.08 10,92 Refusal on sieve 0.16 0,8 Table 1. The physical characteristics of cement • Equipment of the laboratory of the School of Sciences and Techniques of Building and Roads (ESTBR) of Abomey in Benin; y • Isa Yakubu, Department of Chemical Engineering Ahmadu Bello, University Zaria Kaduna State. Figure 6: TGA Apparatus 4000 BY Perkin Elmer Figure 7: Tensile and compression testing machine Figure 6: TGA Apparatus 4000 BY Perkin Elmer Figure 7: Tensile and compression testing machine The principle of the formulation of the composite was that in Figure 8: 2.1.5 Cement The hydraulic binder used was the Cement Portlant with physical characteristics given in Table 1. 2.1.4 water The water used for the formulation of the composite was produced by ‘the Société Nationale des Eaux du Bénin’ (SONEB) with a pH of 6.7. Table 2. Mechanical characteristics of cement Number of days Compressive strength (MPa) 2days 13,2 7 days 24,2 28 days 33,0 2.1.3 Sand The sand used comes from the Dekoungbe quarry, of the MINEX company which was extracting on a large scale, sand, in Benin, of granulometry presented in figure5. International Journal of Research and Review (ijrrjournal.com) 265 Vol.7; Issue: 6; June 2020 Figure 5. Sand particle size analysis curve International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 265 ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers The mechanical characteristics of this cement are given in Table 2. 2.3 Methods 2.3.1 Molding of test pieces 2.3.1 Molding of test pieces 2.3.1 Molding of test pieces The control samples were obtained according to the cement / sand mass fraction of 1/3 and the water / cement ratio (W / C) fixed at 0.7, according to the protocol of the European Standard, “196–1, (1995). Different rates of substitution of sand with reinforcements of sugar cane bagasse (2.5%, 5%, 7.5% and 10%). The cement was substituded at a rate of 10% by the pozzolana of rice husk ash. Specimens of dimensions 4cm x 4cm x 16cm are made and kept in the laboratory. Figure 8: Schematization of the principle of formulation by volume substitution in the saturated state. Figure 8: Schematization of the principle of formulation by volume substitution in the saturated state. International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 266 ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers Figure 9 : Molding of test pieces Figure 10: stripped test pieces RESULTS The mineralogy of the ash from the rice husk produced was determined by X-ray diffractometry (XRD). The protocols of the DRX and FX tests that J. D. Martín-Ramos, J. L. Díaz-Hernández and all (2012); Khaled. Boughzala, Nabil. Fattah, and al, used, are based on linear correspondences between the intensities diffracted by each crystalline phase and their proportion in the powder of rice husk ash, are followed for the results of curve 6 and the determination of the constituents mineralogical content of the ash obtained, Table 3. ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers Figure 9 : Molding of test pieces Figure 10: stripped test pieces RESULTS ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers Figure 10: stripped test pieces RESULTS SU S The mineralogy of the ash from the rice husk produced was determined by X-ray diffractometry (XRD). The protocols of the DRX and FX tests that J. D. Martín-Ramos, J. L. Díaz-Hernández and all (2012); Khaled. Boughzala, Nabil. Fattah, and al, used, are based on linear correspondences between the intensities diffracted by each crystalline phase and their proportion in the powder of rice husk ash, are followed for the results of curve 6 and the determination of the constituents mineralogical content of the ash obtained, Table 3. The mineralogy of the ash from the rice husk produced was determined by X-ray diffractometry (XRD). The protocols of the DRX and FX tests that J. D. Martín-Ramos, J. L. Díaz-Hernández and all (2012); Khaled. Boughzala, Nabil. Fattah, and al, used, are based on linear correspondences between the intensities diffracted by each crystalline phase and their proportion in the powder of rice husk ash, are followed for the results of curve 6 and the determination of the constituents mineralogical content of the ash obtained, Table 3. Figure 11: DRX diagram of ash from the husk of elaborated rice Table 3. Chemical constituents of rice husk ash Chemical constituents (% by mass) SiO2 Al2O3 Fe2O3 CaO MgO S03 K2O Na2O Rates % 98,78 0,1 0,24 0,41 2,64 0,05 1,93 0 Figure 11: DRX diagram of ash from the husk of elaborated rice International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 267 ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers by volume substitution in the saturated state by the expression: The value of the activity index had been determined by Mouhamadou. Amar, Mahfoud. Benzerzour, and al, given by the ratio between the compression resistance of the standard reference mortar (M) at 28 days (NF EN 196-1) and the mortar of 25% substitution of the cement mass (25C) by the rice husk pozzolana. This formula is applied and gave us the value of the index for the elaborated rice husk pozzolana. 3 1 1 C eff SG FBCS m V E V W     (2) Where WFBCS is the water absorption coefficient, C the amount of cement and Eeff the effective amount of water. Table 4. Young's modules of the materials in function with variation in fiber content at 28 days of age. Materials Young's modulus (E) in MPa CFBC-2,5% 6,70 CFBC-5% 9,88 CFBC-7,5% 17,14 CFBC-10% 24,46 DISCUSSION fibrous materials. Table 4 shows Young's modulus values which increase with the increase in fiber content substitution, 25% - 6.70 MPa; 5% - 9.88 MPa; 7.5% - 17.14 MPa; 10% - 24.46MPa. We conclude that the bagasse fibers make the material more elastic. This is interpreted by a quasi-linear phase, which is similar to the behavior of pure cement, therefore a load mainly supported by the matrix, which is gradually transferred to the fibers. Once the maximum bending stress of 28.83 MPa, applied is reached, there is a sudden drop in load, delayed which reflects the resumption of the load by the fibers. We deduce by an approximation that the propagation of cracks in the material is limited by the fibers. Also a displacement which can be associated with a progressive rupture of the fiber-matrix interfaces followed by loosening, and a rupture of the fibers. fibrous materials. Table 4 shows Young's modulus values which increase with the increase in fiber content substitution, 25% - 6.70 MPa; 5% - 9.88 MPa; 7.5% - 17.14 MPa; 10% - 24.46MPa. We conclude that the bagasse fibers make the material more elastic. This is interpreted by a quasi-linear phase, which is similar to the behavior of pure cement, therefore a load mainly supported by the matrix, which is gradually transferred to the fibers. Once the maximum bending stress of 28.83 MPa, applied is reached, there is a sudden drop in load, delayed which reflects the resumption of the load by the fibers. We deduce by an approximation that the propagation of cracks in the material is limited by the fibers. Also a displacement which can be associated with a progressive rupture of the fiber-matrix interfaces followed by loosening, and a rupture of the fibers. The curve from the XRD test, Figure 6 and X-ray fluorescence spectrometry (2020), Table 3, showed the presence of an amorphous silica (Si), with a significant rate of 98.78%, which characterizes the pozzolanity of the rice husk ash. The resistance index determined for the husk of rice of 0.71 is between 0.67 and 1, located in the characterization range of pozzolanic products, according to ASTM Standard C618-08a (2008). RESULTS Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers Strain [mm] F o r c e ( k N ) According to the European Standard, “196– 1, (1995), the breaking strength in bending was given by the relation 3 1,5x x f F l Rf b  [14]. (4) According to the European Standard, “196– 1, (1995), the breaking strength in bending was given by the relation 3 1,5x x f F l Rf b  [14]. (4) (4) The values of the tensile strengths of the composites have been represented in Figure The values of the tensile strengths of the composites have been represented in Figure Strain [mm] Figure 13. stress-strain curves in bending [ ] Figure 13. stress-strain curves in bending Figure 13. stress-strain curves in bending Figure 14. Stress at break in bending of composites at 28 days of age. Figure 14. Stress at break in bending of composites at 28 days of age. RESULTS Standard NF EN 206-1 (1995) defined effective water as follows: eff gr adj cent abs E E E E E     (3) 28 28 ( ) (25 ) C j a C j R M i R C  (1) where:, Egr is the water from the aggregates; where:, Egr is the water from the aggregates; Eadj, the water provided by the adjuvants; Ecent , the water provided by the manufacturing unit; Eabs , the water absorbed by the aggregates. 0,67 ai  The composite was formulated in: The composite was formulated in: In accordance with standard EN 196-1, these test pieces were crushed at a speed of 50 N / s. fixing the W / C ratio at 0.7 which gave good handling of the material; Jonathaan. Page, (2017) ; Valery. Doko, Emmanuel. Olodo, (2016) and al, evaluated the volume of the granular skeleton (SG), in the saturated state of the fibers, in accordance with the principle of formulation The results of the compressive test of the manufactured test pieces, with the variation of the fiber rates (2.5%; 5%; 7.25% and 10%), have been shown by the respective stress-strain curves, plotted in Figure 12. Figure 12: Compression behavior curves for composites at 28 days of age. Strain S t r e s s M p a refer ence Figure 12: Compression behavior curves for composites at 28 days of age. The trend curves have been drawn for the study of the elastic domain of the material. The slopes of these curves were Young's modulus of 2.5% to 10% fiber substituted test pieces. The different values of Young's modules have been grouped in Table 4 below. Table 4. Young's modules of the materials in function with variation in fiber content at 28 days of age. Table 4. Young's modules of the materials in function with variation in fiber content at 28 days of age. Materials Young's modulus (E) in MPa CFBC-2,5% 6,70 CFBC-5% 9,88 CFBC-7,5% 17,14 CFBC-10% 24,46 The three-point bending test on the mortars made it possible to draw the different curves in Figure13. International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 268 ACODJI Vodouhè. Pamphile et.al. DISCUSSION The results of compressive crushing tests on test pieces made at fiber substitution rates of (2.5% ; 5% ; 7.25% and 10%), given by the stress- strain curves in Figure 1., show that the increase in the percentage of fibers causes the material's breaking stress to drop considerably. The three-point bending tests have shown that, up to a substitution rate of 5%, the bending resistance, of 28.83 MPa, is higher than that of the other substitutions, which decreases at a breaking stress in bending 7.71 MPa for a 10% fiber absorption rate. This is confirmed by David Sedan's conclusions on the behavior of Compression tests showed the anisotropy of the material. The substitution of sand by fibers makes it possible to obtain International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 269 ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers ACODJI Vodouhè. Pamphile et.al. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers », J. Glob. Ecol. Environ., vol. 2, no 4, p. 205‑208, 2015. a multi-functional material for civil engineering works available to the engineer, the use of which would make it possible to lighten the structures of constructions. The use of this material would reduce global warming. Finally, we note that this elaborate composite of materials from renewable resources, obtained with low energy demand, contributes to the reduction of greenhouse gas emissions and also to the lightening of the structures incorporating these plant reinforcements. 8. José. Daniel. Martín-Ramos, José. Luis. Díaz- Hernández, Jane. Scarrow, et Antonio. López- Galindo, « Pathways for Quantitative Analysis by X-Ray Diffraction », InTech Croat., p. 22, 2012. 9. Khaled. Boughzala, Nabil. Fattah, Khaled. Bouzouita, et Habib. Ben Hassine, « Etude minéralogique et chimique du phosphate naturel d’Oum El Khecheb (Gafsa, Tunisie) », Rev. Sci. Matér. Lab. LARHYSS, vol. 6, p. 11‑29, 2015. 10. Mouhamadou. Amar, Mahfoud. Benzerzour, Nor.-Edine. Abriak, et Yannick. Mamindy- Pajany, « Study of the pozzolanic activity of a dredged sediment from Dunkirk harbour », Powder Technol., vol. 320, p. 748‑764, oct. 2017, doi: 10.1016/j.powtec.2017.07.055. REFERENCES 1. Philippe. Deshayes, « Le secteur du bâtiment face aux enjeux du développement durable : logiques d’innovation et/ou problématiques du changement. », De Boeck Supérieur, France, p. 219‑236, 2012. 11. Jonathan. Page, « Formulation et caractérisation d’un composite cimentaire biofibré pour des procédés de construction préfabriquée », Normandine Université, 2017. 2. Sandra. Luz, Adilson. Roberto. Goncalves, et Antônio. Del’Arco, « Mechanical behavior and microstructural analysis of sugarcane bagasse fibers reinforced polypropylene composites », Compos. Part Appl. Sci. Manuf., vol. 38, p. 1455‑1461, 2007, doi: 10.1016/j.compositesa.2007.01.014. 12. Edem. Chabi, Valery. Doko, E. Agoua, Emmanuel. Olodo, et Edmond. C. Adjovi, « Formulation du béton de balles de riz : étude du comportement au cisaillement et au poinçonnement-flexion », p. 11, 2016. 3. Davina. Michel, « Evaluation du potentiel fibreux et textile de la canne (Saccharum officinarum L.) », Université de haute alsace, 2013. 13. « NF EN 206/CN ». 14. E. Norme Européenne, « 196–1, 1995 », Méthodes D’essais Cim. Partie, vol. 1. 15. Spectrométrie de fluorescence X : définition et explications, Techno-Science.net. https://www.techno- science.net/definition/4638.html (consulté le avr. 27, 2020). 4. Mohamed. El-Sayed et Taher. El-Samni, « Physical and Chemical Properties of Rice Straw Ash and Its Effect on the Cement Paste Produced from Different Cement Types », J. King Saud Univ. - Eng. Sci., vol. 19, no 1, p. 21‑29, 2006, doi: 10.1016/S1018- 3639(18)30845-6. 16. ASTM Standard C618-08a, « Standard Specification for Fly Ash And Raw Or Calcined Natural Pozzolan For Use As A Mineral Admixture In Portland Cement Concrete ». 2008. 5. Anissa. Benredouane, Larbi. Kacimi, Olga. Rodriguez Largo, et Aurora. Lopez Delargo, « Elaboration d’une Pouzzolane Artificielle à Base de la Zéolithe X Synthétisée à partir de Kaolin Naturel », MATEC Web Conf., vol. 11, p. 5, 2014, doi: 10.1051/matecconf/20141101005. 17. David. Sedan, « Etude des interactions physico-chimiques aux interfaces fibres de chanvre/ciment: influence sur les propriétés mécaniques du composite », 2007 6. Lavie. Arsène. Mango-Itulamya, « Kwilu- Ngongo: Formulation des matériaux de construction à base de terre et de résidus de canne à sucre », 2018. How to cite this article: Pamphile AV, Emmanuel OT, Valery DK. Elaboration of a cement matrix composite, improved with the pozzolan of rice husk ash, reinforced with sugar cane bagasse fibers. International Journal of Research and Review. 2020; 7(6): 263-270. 7. Jesse. Kigozi, et Moses. REFERENCES Kiggundu, and all« Investigation of sugar cane bagasse ash as a binding material for the construction industry ****** 270 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020 International Journal of Research and Review (ijrrjournal.com) Vol.7; Issue: 6; June 2020
https://openalex.org/W3206625148	https://repository.ubn.ru.nl/bitstream/handle/2066/249590/1/249590.pdf	English	null	The Demographic Window of Opportunity and Economic Growth at Sub-National Level in 91 Developing Countries	Social indicators research	2,021	cc-by	10,553	The Demographic Window of Opportunity and Economic Growth at Sub-National Level in 91 Developing Countries Crombach, L.G.A.; Smits, J.P.J.M. 2022, Article / Letter to editor (Social Indicators Research, 161, (2022), pp. 171-189) Doi link to publisher: https://doi.org/10.1007/s11205-021-02802-8 Note: To cite this publication please use the final published version (if applicable). Social Indicators Research (2022) 161:171–189 https://doi.org/10.1007/s11205-021-02802-8 ORIGINAL RESEARCH The Demographic Window of Opportunity and Economic Growth at Sub‑National Level in 91 Developing Countries Lamar Crombach1 · Jeroen Smits2 Accepted: 15 September 2021 / Published online: 12 October 2021 © The Author(s) 2021 Accepted: 15 September 2021 / Published online: 12 October 2021 © The Author(s) 2021 Abstract Data for low- and middle- income countries (LMICs) are used to investigate the effect of the demographic transition on economic growth at sub-national level. We introduce a detailed classification of demographic window phases, determine how these phases are dis- tributed among and within LMICs, and analyze the relationship between the demographic window of opportunity (DWO) and economic growth for 1921 urban and rural areas of sub-national regions within 91 LMICs. Many areas in Asia, Latin America and the Middle East have entered the window, but most of Sub-Saharan Africa is still in the traditional or pre-window phase. Our analyses reveal higher growth rates in areas passing through the DWO. Positive growth effects are particularly strong in rural and more educated regions and in countries with lower levels of corruption. Policy measures aimed at effectively using the DW for achieving growth should combine investments in education and rural develop- ment with better governance. Keywords Demographic window · Economic growth · Subnational · Fertility reduction · Developing countries · Governance * Lamar Crombach crombach@kof.ethz.ch Jeroen Smits jeroen.smits@fm.ru.nl 1 ETH Zürich, KOF Swiss Economic Institute, Konjunkturforschungsstelle, LEE G224 Leonhardstrasse 21, 8092 Zürich, Switzerland 2 Institute for Management Research, Global Data Lab, Radboud University, PO.Box 9108, 6500HK Nijmegen, The Netherlands * Lamar Crombach crombach@kof.ethz.ch Jeroen Smits jeroen.smits@fm.ru.nl 1 Introduction A low or declining dependency ratio indicates that an area is pass- ing through the DWO. Due to issues of data availability, the current literature has not yet extensively analyzed the effects of the DWO on economic growth in low-income countries. These countries are often thought of as being stuck in a high-fertility high-mortality situation, i.e. not to have entered the DWO yet (Bloom et al., 2003). Consequently, the empirical basis of the litera- ture has been heavily biased towards growth in middle- income countries with a well-edu- cated workforce and good infrastructure—such as the East Asian Tigers—where the poten- tial for economic growth may have been better than in many of the current low-income countries (Bloom & Williamson, 1998; Radelet et al., 2001; Williamson, 2013). The empirical literature has also been very focused on the national level, whereas fertil- ity decline and economic growth may differ substantially between different areas within a country. For instance, cities may show entirely different patterns than rural areas (Sander & Charles-Edwards, 2017; Williamson, 2013). Furthermore, the national-level fertility transi- tion has been shown to lead to within-country increases in fertility inequality (Eloundou- Enyegue et al., 2017). By analyzing data at a sub-national level—as is done in the cur- rent study—the added variation allows for a more refined analysis of the variables at hand. Indeed, a simple multilevel model of our dependent variable reveals that about 71% of vari- ation occurs at the sub-national level, while 29% occurs at the national level.f There are a few published papers that have analyzed the effects of demographic varia- bles at a sub-national level, but they tend to be restricted to regions within a single country. Firstly, Wei and Hao (2010) analyze the impacts of the growth rate and level of the depend- ency ratio, the total population, and population density, on economic growth in Chinese regions. They also include interactions to analyze under which conditions the dependency ratio is more or less important. Their analyses do not reveal effects of the level of popu- lation, urbanization, or the growth rate of the dependency ratio, but they do find signifi- cant negative effects of the dependency ratio and population density. Secondly, Baerlocher et al. 1 Introduction The role of the population age structure as a potential source for economic growth is gaining importance in the economic literature (e.g. Canning et al., 2015; Groth & May, 2017; Kelley & Schmidt, 2005). Fertility reduction may potentially enable a region to have a period of rapid economic growth during the Demographic Window of Opportunity (DWO). The DWO is a period of several decades that countries go through when moving from a situation of high fertility and mortality to low fertility and mortality. This transition * Lamar Crombach crombach@kof.ethz.ch Jeroen Smits jeroen.smits@fm.ru.nl 1 ETH Zürich, KOF Swiss Economic Institute, Konjunkturforschungsstelle, LEE G224 Leonhardstrasse 21, 8092 Zürich, Switzerland 2 Institute for Management Research, Global Data Lab, Radboud University, PO.Box 9108, 6500HK Nijmegen, The Netherlands 123456789) 1 3 123456789) 1 3 L. Crombach, J. Smits 172 is generally characterized by a baby boom period, in which (child) mortality is already reduced, but fertility levels are still high. When later fertility also decreases, the baby boom generation moves up in the age distribution and after some time enters the working age population. During this period, more women may enter the labor force as less time needs to be spent on children (Aaronson et al., 2021; Bloom et al., 2009; Cristia, 2008). Given that there are still few elderly—as their generation suffered from higher mortality rates in the past—the region experiences a period in which the working age population is large and the dependent population—the green and the grey—is small. Hence, there is high potential for economic growth, which, if realized, is called the “demographic dividend” (Bloom et al., 2003, 2009; Groth & May, 2017) The demographic dividend is often used as an explanation for the rapid economic growth that occurred in East Asian economies in the end of the twentieth century, account- ing for as much as one third of the observed national economic growth (Bloom & William- son, 1998). The size of this demographic dividend, however, is found to depend on poli- cies regarding labor markets, financial markets, education and health (Bloom et al., 2017; Bloom & Williamson, 1998; Groth & May, 2017). Most research tends to focus on the level or growth rate of the dependency ratio, which is measured as the ratio of the share of the dependent population (those aged below 15 and above 65) to the working-age popula- tion (those aged 15–65). 1 Introduction Which factors are associated with a more effective use of the DWO in terms of economic growth Our study contributes to the literature in several important ways. It is the first to com- pare sub-national regions across the developing world in terms of the impact of the DWO on economic growth. We use a database with information for two points in time for 1921 sub-national areas, both urban and rural, spread across 91 different LMICs. Second, a data- set with such richness offers more possibilities for analyzing the role of context factors than the national data on—at most—some 200 countries that are usually used for growth models. Third, besides average effects, we also study interaction effects. Interaction effects help us to understand how the effect of the DWO depends on specific circumstances. Such insights allow policymakers to implement better-targeted policies. Fourth, economic growth at the sub-national level is indicated by the International Wealth Index (IWI), an asset-based wealth index that is comparable over time and across countries (Smits & Steendijk, 2015). Analyzing the effects of demographics on the IWI will provide valuable new insights, as IWI measures a household’s possession of durables, housing quality and access to basic services and is as such a broader indicator than income or expenditure. As an outcome-based measure, IWI does not need arbitrary adjustments in terms of purchas- ing power parities, common baskets of goods, or inflation, as is the case with GDP per capita. The remainder of this paper is structured as follows. The next section will outline the theoretical model, our measure of economic growth and the context factors that are poten- tial determinants of DWO effectiveness. The third section describes the data, the methodol- ogy and the control factors. The fourth section outlines the results, and the fifth section will discuss and conclude. 1 Introduction (2019) estimate the impacts of the participation rate, the working age population, the growth rates of the (working age) population, and the level of and change in the mean years of schooling in micro-regions in Brazil. They find that demographic variables do matter for economic growth, though predominantly through their effect on human capital The Demographic Window of Opportunity and Economic Growth at… 173 accumulation. Lastly, Kumar (2013) analyzes the effects of the level of, and growth in, the working age share of the population on state-level net GDP per capita in 16 Indian states for the period 1971–2001 and finds significant positive effects of both level and growth, thus pointing towards a favorable effect of the DWO on growth.i accumulation. Lastly, Kumar (2013) analyzes the effects of the level of, and growth in, the working age share of the population on state-level net GDP per capita in 16 Indian states for the period 1971–2001 and finds significant positive effects of both level and growth, thus pointing towards a favorable effect of the DWO on growth. f The aim of the current paper is threefold. First, we want to find out how the DWO has spread across subnational regions of low and middle-income countries. Second, we aim to determine whether the positive effect of the DWO on economic growth—which has been found for middle-income countries—is also present in low-income countries and in particular at the level of sub-national regions. Third, we want to determine under which circumstances the effect of the DWO is strongest to help policy makers develop contextual- ized measures that facilitate reaping the demographic dividend in specific areas that have already entered the DWO. Therefore, our central research questions sound: 1. What is the variation in DWO phases at the level of sub-national regions across the developing world? 1. What is the variation in DWO phases at the level of sub-national regions across the developing world? p g 2. To what extent does the DWO foster economic growth at the sub-national level 3. Which factors are associated with a more effective use of the DWO in terms of economic h 2. To what extent does the DWO foster economic growth at the sub-national levelf 2. To what extent does the DWO foster economic growth at the sub-national level 3. Which factors are associated with a more effective use of the DWO in terms of economic growth 3. 2 The Theoretical & Empirical Model We begin by defining a standard Cobb–Douglas aggregate production function for region i in country j in the year T2: 1 3 1 3 L. Crombach, J. Smits 174 (1) YT2 ij = AT2 ij KT2 ij 훼HT2 ij 1−훼 (1) where Yij is GDP, Aij is Total Factor Productivity (TFP), Kij is the capital stock, and Hij is the stock of efficient labor, which consists of the labor force, Lij , multiplied by the human capital per worker, hij . Human capital per worker is determined by the years of schooling, sij , and the return to schooling, 휃 , which is assumed to be constant, in the following func- tional format: (2) hT2 ij = esT2 ij 휃 (2) Dividing output by the labor force allows us to rewrite the production function in per worker terms: Dividing output by the labor force allows us to rewrite the production function in per worker terms: (3) yT2 ij = AT2 ij kT2 ij 훼hT2 ij 1−훼 (3) where yij is GDP per worker, and kij is the capital stock per worker. To simplify notation, the growth rate is assumed to refer to the growth in period T1-T2. Substituting Eq. 2 into Eq. 3, taking logs and the first difference gives us the following growth rate equation: where yij is GDP per worker, and kij is the capital stock per worker. To simplify notation, the growth rate is assumed to refer to the growth in period T1-T2. Substituting Eq. 2 into Eq. 3, taking logs and the first difference gives us the following growth rate equation: (4) gy ij = gA ij + (1 −훼)휃Δsij + 훼gk ij (4) We follow Baerlocher et al. (2019) by allowing TFP growth to depend on the level of mean years of schooling, with the argument that countries with higher levels of school- ing are better able to innovate and generate economic growth. Additionally, countries that are closer to the technological frontier, as proxied by GDP per worker in period T1, will have lower TFP growth (Barro, 1991): (5) gA ij = 훿+ 휓sT1 ij −휇ln ( yT1 ij ) (5) In our analysis, we will control for capital stock per worker growth at the national level by using a fixed effects country dummy model. 2 The Theoretical & Empirical Model Substituting TFP into the GDP per worker growth equation gives: (6) gy ij = 훿+ 휓sT1 ij −휇ln ( yT1 ij ) + (1 −훼)휃Δsij + 훼gk ij (6) To translate the model from GDP per worker to GDP per capita terms, we must adjust the neoclassical model and recognize that a population does not merely consist of workers, but also of dependents: To translate the model from GDP per worker to GDP per capita terms, we must adjust the neoclassical model and recognize that a population does not merely consist of workers, but also of dependents: (7) ( Y N ) ij = (Y L ) ij ( L N ) ij = ( Y WA ) ij (WA N ) ij (7) where Nij is the total population, and WAij is the working age share of the population, i.e., those aged between 15 and 65. Thus, GDP per capita consists of a productivity component, Y WA = y , and a demo- graphic component: WA N . Additionally, for reasons of data availability, we assume Lij = WAij , i.e., full employment. Moreover, as in Wei and Hao (2010), we express the demographic component in terms of the dependency ratio, Dij , as opposed to share of the working age population to the total population: Dij = Nij−WAij WAij , which gives: 1 3 1 3 1 The Demographic Window of Opportunity and Economic Growth at… The Demographic Window of Opportunity and Economic Growth at… 175 (8) ( Y N ) ij = yij = yij 1 + Dij (8) where yij is GDP per capita.if where yij is GDP per capita. j As before, we take logs and the first difference to obtain growth rates. Additionally, we substitute the TFP growth equation into the growth rate of GDP per capita equation. Moreover, note that TFP growth depends on the initial level of income per worker, which we now translate to the initial level of income per capita as well: (9) gy ij = 훿+ 휓sT1 ij −휇ln ( yT1 ij ) −휇ln ( 1 + DT1 ij ) + 훼gk ij+ (1 −훼)휃Δsij −Δ ln (1 + Dij ). 2 The Theoretical & Empirical Model (9) Transforming the above equation into an empirical specification with country-specific fixed effects (which absorb national capital stock per worker growth, gk ij ), γj , and a random error term at the sub-national level, 휖ij , gives: (10) gy ij = 훽0 + 훽1sT1 ij + 훽2 ln ( yT1 ij ) + 훽3 ln ( 1 + DT1 ij ) + 훽4Δsij + 훽5g1+Dij + 훾j+ ∈ij . (10) 2.1 Economic Growth at Sub‑National Level Smits 176 (11) gy ij = ln ( yT2 ij ) −ln ( yT1 ij ) ≈ΔIWIij = 훽0 + 훽1sT1 ij + 훽2IWIT1 ij + 훽3 ln ( 1 + DT1 ij ) + 훽4Δsij + 훽5g1+Dij + 훾j+ ∈ij . (11) 2.2 The Role of the Context The effect of the DWO on economic growth is not expected to be the same everywhere, but may depend on characteristics of the context in which it takes place (Bloom et al., 2017; Bloom & Williamson, 1998; Groth & May, 2017). According to Zuber et al. (2017), DWO effectiveness is influenced by three major factors: (i) job creation, (ii) human capital building and (iii) good governance. A relatively high working age share of the population can only translate into growth if the workers have sufficient employment opportunities. To achieve these opportunities prudent macroeconomic policies are supposed to be required, including a high level of financial market development, a high degree of economic open- ness and low levels of positive inflation (Collier & Dollar, 2001; Turbat, 2017). However empirical evidence is mixed. Whereas Easterly (2005) found the effect of good policies on growth to be small and not robust to different econometric specifications, Wei and Hao (2010) present data for China showing the effect of the DWO to be stronger in regions with higher levels of market openness. Regarding the role of human capital building there is not yet much empirical support available. Whereas Kelley and Schmidt (2005) found little effect of education on the size of the DWO effect, other studies (Baerlocher et al., 2019; Crespo Cuaresma et al., 2014) suggest that most gains of the DWO are related to education decisions that are the result of the demographic changes.i The third factor is good governance. We follow Kaufmann et al. (2011) in defining gov- ernance as “the traditions and institutions by which authority in a country is exercised”. We see governance as good when it is participatory, consensus-oriented, accountable, transpar- ent, responsive, effective and efficient, equitable and inclusive and follows the rule of the law (Zuber et al., 2017). Good governance implies that the government offers high-quality and affordable education and health care to everyone, both of which increase human capital building. Further, the increase in tax income resulting from the relative increase in workers must be used productively. We therefore expect that if the additional resources are wasted on corruption and inefficiencies, the demographic dividend may be substantially harmed. 2.1 Economic Growth at Sub‑National Level A key issue to overcome is that for most LMICs data on (changes in) GDP per capita at sub-national level is not available. Therefore, we will use (changes in) standard of living of households, as indicated by the International Wealth Index (IWI) (Smits & Steendijk, 2015) as an alternative. IWI is an asset-based wealth index that measures household wealth in LMICs on the basis of ownership of consumer durables (TV, fridge, phone, car, etc.), quality of housing and access to public services (clean water, electricity). IWI ranks house- holds from 0 to 100, with 0 meaning none of the durables, bad quality housing and no access to services and 100 meaning all durables, good quality housing and access to high quality services. Conveniently, one can aggregate IWI from the household level to higher levels, such as the level of sub-national areas and the country level. It then indicates the mean wealth level of households in terms of asset ownership in the sub-national areas or country, while increases in the IWI level of an area over a period of time mean that households in the area were able to increase their stock of assets during that period. An advantage compared to indices based on household income in the context of LMICs is that IWI is less volatile, as it is not very sensitive to income changes due to unstable employment or economic shocks, which are rather prevalent in poor regions (Loayza et al., 2007). Testing the performance of IWI as indicator of economic growth at the subnational level is not possible, as there is no other indicator available at that level for comparison. How- ever, as Figure S1 in the Supplementary Information shows, at the national level IWI is highly (0.86) correlated with the logarithm of GDP per capita and even more highly (0.92) with the Human Development Index, which suggests that also at subnational level it might be a reasonable indicator of the level of development. Thus, IWIijt ≈ln(yijt ) , i.e., we assume IWI to be an appropriate proxy for the logarithm of GDP per Capita. This changes the specification as follows: 1 3 3 L. Crombach, J. 3.2 Methods To address the fact that the 1921 sub-national regions, which are the units of analysis, are nested within the 91 countries, our regression model included the complete set of fixed effects country dummies. In this way the direct effects of all (measured and unmeasured) country-specific characteristics—including number of sub-national regions and variation in number of years between T1 and T2—are accounted for, while keeping the possibility to study cross-level interactions with the country-level explanatory variables. Given that within the regions only data for two years was used, a two-period panel data analysis could be performed (Wooldridge, 2013) and no further control for multiple observations within the regions was needed. The main independent variables are the dependency ratio and the growth rate of the dependency ratio. The dependency ratio is measured as the ratio of the share of the work- ing age population to the share of the dependent population. We define the working age population as all individuals aged between 15 and 64. We consider all individuals aged below 15 or above 64 as the dependent population. Besides the dependency ratios, the models contained independent variables at subna- tional and national level. At subnational level, these variables included the T1 value of IWI, education, the gender difference in education and urbanization. Education was meas- ured as the mean years of schooling of male individuals aged 20 and above in the region. We choose education for the 20 + population because the starting age of schooling is often high in LMICs (Huisman & Smits, 2009) and individuals under 20 might not yet have completed their education. Gender inequality was measured by the difference between the mean years of schooling for men and for women (education men minus education women). Urbanization was measured by a dummy variable with value zero in rural areas and value one in urban areas. The dummy’s value depends on the designation (urban or rural) given by the source surveys, which are based on the official national definition used in the coun- try (United Nations, 2018). Given this rather crude division in only two categories, it may be possible that there are households living in urban areas (e.g., slums) which are less well- off than rural households. 3.1 Data A subnational indicator database was constructed from data of the Database Developing World of the Global Data Lab (GDL) (www.globaldatalab.org), which contains harmo- nized data for over 30 million persons in 130 + LMICs. The data is derived from Demo- graphic and Health Surveys (DHS, www.dhsprogram.com), Unicef MICS Surveys (mics. unicef.org), IPUMS census data (international.ipums.org), Afrobarometer surveys (www. afrobarometer.org) and several stand-alone surveys (Smits, 2016). Variables obtained from these surveys were aggregated to the level of urban and rural areas of sub-national regions using the regional/provincial codes available in the datasets. In this way, a dataset 1 3 1 The Demographic Window of Opportunity and Economic Growth at… 177 was created with information for two points in time (T1 and T2) for 1921 urban and rural sub-national areas within 91 countries. For each country, data were selected from the last two available surveys that were at least four years apart. Because the surveys were held in different years for the different countries, the time period between the two surveys could vary between four and 16 years. To obtain a simple and comparative measure of economic growth, the average annual change in IWI between the two survey years (T1 and T2) was used. The other change variables in the model reflect the average annual change between times T2 and T2 as well. In this way a dataset was obtained with for each of the 1921 regions the value of the regional characteristics in the first survey (T1) and the annual changes in these characteristics between the first and the second survey (T2). 3.2 Methods The level of IWI at T1 was included as explanatory variable to control for the distance to the technological frontier (convergence) (Barro, 1991).li At the national level, quality of governance, inflation, market openness and financial development were included. To test for good governance, a multidimensional governance index was used, aggregated from the six World Bank worldwide governance indicators (Kaufmann et al., 2011): control of corruption, rule of law, political stability and violence, voice and accountability, government effectiveness and regulatory quality. The scores for these dimensions were all obtained through expert surveys. Each dimension, as well as the 1 1 3 3 L. Crombach, J. Smits 178 aggregated governance index, ranges from −2.5 (low) to + 2.5 (high). Inflation was taken from the World Development Indicators (World Bank, 2021) and measured as the annual %-change in consumer prices. For market openness, the KOF economic globalization index is used, which ranges from 0 (closed) to 100 (open) and measures the degree of finan- cial and trade openness (Dreher, 2006; Dreher et al., 2008; Gygli et al., 2019). For finan- cial development, the IMF financial development index is used, which ranges from 0 (low development) to 1 (high development) (Svirydzenka, 2016). Missing values on the national variables were addressed using dummy variable adjustment, whereby the mean of the valid cases was imputed and a dummy was added to nullify them (compare Allison, 2002).The national variables were only used to study their interactions with the dependency ratios, as the direct effects of these variables on economic growth are completely covered by the fixed effects approach. if Although the risk of endogeneity bias was restricted because the current growth of the working age population is based on fertility decisions taken in the past, it remains pos- sible that an omitted variable bias in our model was caused by migration. The classic push–pull migration model (Lee, 1966) postulates that migration is caused by the uneven processes of development. Economic opportunities in the form of wage differentials may drive migration streams. Additionally, the demographic transition will lead to a large labor supply (Preston et al., 1989), and mostly in urban areas (Sander & Charles-Edwards, 2017; Williamson, 2013), which may lead to immigration/emigration if there are many/few opportunities available in the area. 1 3 3.2 Methods Given that migration in low- and middle income areas consists mostly of young adults, as they are the most mobile group (Sander & Charles- Edwards, 2017), this may lead to a positive relationship between economic development and immigration. As a robustness test, we therefore repeated our analysis with an indicator for migration in the study period. Given that such a measure is not readily available at the subnational level, we developed a proxy of migration as follows: (12) Migrationij = (100 + Actual change working age share) (100 + Expected change working age share) (12) Substituting for the actual and expected changes in the working age share gives us: (13) Migration = 100 + ∑64 k=15 Δsharek 100 + ∑15 m=15−T shareT1 m −∑64 n=64−T shareT1 n −∑T p=0 Δsharep × WorkAgeT1 ElderlyT1 (13) where share is the share of the population of a certain age, T is the total amount of years in the time period of the subnational region, T1 indicates that the value is from the start of the period, WorkAge is the share of the population aged 15–64, and Elderly is the share of the population aged 65 or over. Simply put, the migration measure is the ratio of the actual changes of the 15–64 population shares, divided by the expected changes of the 15–64 population shares based on the T1 population structure, with an adjustment for fertility. This measure is expected to provide a reasonable indication of the role of migration, as the population dynamics in the younger age groups are appropriately included and labor migration is a process that involves mostly young people. As a second robustness test, we repeated the main analysis, but using data from the period before the T1–T2 setup, i.e., the T0–T1 period. This enables the reader to see to what extent the results depended on the used sample. In a third robustness test we perform the same analysis using data at the national level, but split up into a rural and urban part to 1 3 The Demographic Window of Opportunity and Economic Growth at… 179 maintain comparability with the other analyses. This setup may highlight the importance of using sub-national data to capture context factors that are important during the DWO. maintain comparability with the other analyses. This setup may highlight the importance of using sub-national data to capture context factors that are important during the DWO. 4.1 Descriptive Analyses Before the discussion of the regression results, this section offers a descriptive perspective on the status of the DWO across the developing world. Because a detailed classification scheme for the DWO phase of a region was lacking, we use a new one that takes a sim- pler scheme used by the United Nations (UN) Population Division (UN, 2004, p. 70) as a starting point (compare Smits, 2016). The UN scheme distinguishes three DW phases: a pre-window phase with 30 or more percent of the population under 15 years old, a window phase with less than 30 percent under 15 years old and less than 15 percent above 64 years old, and a post-window phase with 15 or more percent above 64 years old. To get a more refined picture of the window, the GDL added a traditional phase, for countries that show hardly any sign of fertility reduction, and further subdivided the first two phases of the UN classification to obtain the following scheme: 1. Traditional phase (> 40% under 15 and < 15% over 64), 2. Pre-window phase (30–40% under 15 and < 15% over 64), 3. Early-window phase (25–30% under 15 and < 15% over 64), 4. Mid-window phase (20–25% under 15 and < 15% over 64), 5. Late-window phase (< 20% under 15 and < 15% over 64), 6. Post-window phase (> 15% over 64). 1. Traditional phase (> 40% under 15 and < 15% over 64), 2. Pre-window phase (30–40% under 15 and < 15% over 64), 3. Early-window phase (25–30% under 15 and < 15% over 64), 4. Mid-window phase (20–25% under 15 and < 15% over 64), 5. Late-window phase (< 20% under 15 and < 15% over 64), 6. Post-window phase (> 15% over 64). Figure 1 displays the DWO phase for 1921 subnational regions in 91 LMICs. The map reveals a substantial amount of variation in terms of DW phases both between and within LMICs. China is the only country where some sub-national regions are already in the post- window phase, while its northern neighbor, Mongolia, is mostly in the pre-window phase. Fig. 1 Demographic Window Phases across the developing world. Source: Global Data Lab Fig. 1 Demographic Window Phases across the developing world. Source: Global Data Lab 3 3 L. Crombach, J. Smits 180 India, on the other hand, has a clear north–south divide. 4.1 Descriptive Analyses While most of the south is in the mid-window phase, the north is still mostly in the pre-window phase. The MENA region shows a diverse pattern, with many regions in Turkey, Tunisia, and Iran already in the early-window phase, while other countries are still in the pre-window or traditional phases. In South America, countries such as Brazil and Argentina are in the lead in terms of the demographic transition.f In Sub-Saharan Africa (SSA), the picture is rather different from the other continents, with much less variation between countries and regions. Although a few areas, mostly in the south, appear to be past the traditional or pre-window phases, the majority of the central African region remains in the traditional phase. In Fig. 2, which zooms in on the SSA region, the picture remains more or less the same as well, with only a limited number of areas in the South, around the Gulf of Guinea and at a few other places that are in a later stage. These areas largely coincide with the countries called ‘vanguard countries’ by Eloundou-Enyegue and Hirschl (2017), i.e. those countries that started the earliest with the DWO as measured by having a Total Fertility Rate (TFR) of less than 3.5. Fig. 2 Demographic Window Phases across the African continent. Source: Global Data Lab Fig. 2 Demographic Window Phases across the African continent. Source: Global Data Lab 1 3 181 The Demographic Window of Opportunity and Economic Growth at… To have an even more refined view of the demographics in SSA, Table 1 displays the SSA countries that, at the national level, are not in the traditional phase anymore. The tourist destinations Mauritius (Phase 5) and Cape Verde (Phase 3) turn out to be the most developed in this respect, while Gabon and the five most southern countries are in phase 2. Thus, in SSA, only eight countries are not in the traditional phase when countries as a whole are considered. However, demographic developments are generally not homogeneously spread within countries. As was already clear from Fig. 1, in many countries there is substantial sub- national variation in terms of DW phases. Given that fertility reduction tends to start earlier in urban than in rural areas (Easterlin, 1971), we would expect the first signs of an emerg- ing window to be found in the cities (Williamson, 2013). 4.1 Descriptive Analyses For the SSA region, variation in fertility has already been observed for countries like Nigeria and the Democratic Republic of Congo, where fertility is substantially lower in more urban and capital areas than in rural and remote areas of the countries (Jimenez & Pate, 2017; Shapiro et al., 2017). To see whether more of such signs can be discerned in the SSA region, Table 1 also displays the DW phases of the SSA countries with an urban DW phase above phase one (excluding Mauritius and Cape Verde for which no sub-national DW data were available). In 24 coun- tries, the urban areas are in phase two, and in two countries, Lesotho, and South Africa, already in phase three. To look even more in depth, Table 2 displays the urban areas of sub-national regions that have a DW phase of three or over (i.e., the urban areas that have surpassed the pre- window phase and have thus actually entered the window). It shows that the DW is open- ing in more places than one would expect based on Table 1. For instance, while Botswana as a whole is only in the second phase of the DWO and its urban areas as well, the urban area of one of its regions—South-East—is already in the fourth phase, i.e., the mid-win- dow phase. Lastly, while Ethiopia as a whole is in phase one and its urban areas on average in phase two, the country’s capital Addis Ababa is already in phase four. le 1 Demographic window phases of SSA countries with national or urban areas that are not in the tra- onal demographic window phase Table 1 Demographic window phases of SSA countries with national or urban areas that are not in the tra- ditional demographic window phase Country National Urban Country National Urban Mauritius 5 – Eritrea 1 2 Cape Verde 3 – Ethiopia 1 2 Botswana 2 2 Ghana 1 2 Eswatini 2 2 Guinea Bissau 1 2 Gabon 2 2 Kenya 1 2 Lesotho 2 3 Madagascar 1 2 Namibia 2 2 Mauritania 1 2 South Africa 2 3 Malawi 1 2 Burundi 1 2 Rwanda 1 2 Burkina Faso 1 2 Senegal 1 2 Cote D’Ivoire 1 2 Sierra Leone 1 2 Cameroon 1 2 Togo 1 2 Comoros 1 2 Tanzania 1 2 Congo Brazzaville 1 2 Zimbabwe 1 2 1 L. Crombach, J. 4.1 Descriptive Analyses Smits 182 Table 2 Demographic window phases of urban areas of subnational regions in SSA that have entered the DWO (phase 3 or higher) Country Urban Regions Phase Country Urban Regions Phase Ethiopia Addis Ababa 4 Lesotho Leribe 3 Botswana South-East 4 Lesotho Berea 3 South Africa Western Cape 3 Lesotho Maseru 3 South Africa KwaZulu Natal 3 Lesotho Mafeteng 3 South Africa Eastern Cape 3 Lesotho Quthing 3 South Africa North West 3 Namibia Erongo 3 South Africa Gauteng 3 Namibia Khomas 3 South Africa Mpumalanga 3 Namibia Ohangwena 3 South Africa Northern Province 3 Eswatini Hhohho 3 Ethiopia Oromiya 3 Eswatini Lubombo 3 Botswana North-East 3 Table 2 Demographic window phases of urban areas of subnational regions in SSA that have entered DWO (phase 3 or higher) The different countries and the time periods used in the analyses are shown in the online Supplementary Information (SI). In terms of the descriptive statistics, Table 3 shows there is substantial variation in terms of all variables. T1 IWI, i.e., the value for IWI at the start of the analyzed period, ranges from 1.82 to 94.99. Thus, there are regions where the average household in the first year owned almost none of the assets that are included in the index, while there are also regions in which households owned almost all assets in the first year. In terms of economic growth, there is also substantial variation, as the lowest average yearly change between T1 and T2 in IWI was −3.96 while the largest average yearly change was 5.72. In terms of male education, average mean years of schooling ranged from 0.23 years to 13.40 years. Regarding gender ine- quality in education, there are regions where men, on average, went to school 5.77 years longer than women, while there are also regions where women, on average, went to school 2.08 years longer than men. 1 3 Table 3 Sample Descriptive Statistics Variable Mean St. 4.1 Descriptive Analyses Deviation Minimum Maximum ∆ IWI 0.994 1.103 −3.964 5.723 T1 IWI 49.91 24.27 1.82 94.99 T1 Dependency Ratio (%) 77.90 25.37 15.30 204.60 Growth Dependency Ratio (%) −0.20 2.45 −7.96 17.95 T1 Male Schooling (Years) 6.80 2.80 0.23 13.40 ∆ Male Schooling (Years) 0.10 0.15 −0.82 0.89 T1 Male–Female Schooling (Years) 1.29 1.17 −2.08 5.77 ∆ Male–Female Schooling (Years) −0.009 0.106 −0.448 0.818 Governance −0.638 0.499 −2.220 0.707 Financial Development 0.208 0.129 0.026 0.647 Migration 0.888 0.049 0.668 1.100 The Demographic Window of Opportunity and Economic Growth at… 183 4.2 Multivariate Analyses Tables 4 and 5 show the results of our multivariate analyses. Model 1 in Table 4 includes all sub-national determinants of the change in IWI. The demographic window effect is clearly present, as the results show that both a lower T1 dependency ratio and a lower growth rate of the dependency ratio are associated with a significant increase in IWI growth.f Regarding the other factors in the model, we observe the expected negative effect of IWI at T1 on the growth in IWI. IWI growth is also higher in urban regions, in regions with higher levels of male schooling, in regions with a higher growth in male schooling, in regions with a smaller gender difference in schooling and in regions where the gender difference in schooling is decreasing.i f Model 2 in Table 4 introduces all significant interaction terms between the independ- ent variables and the dependency ratios and between the subnational control factors themselves. Regarding the main effects of the independent variable, we observe little Table 4 Coefficients of fixed effects regression models of selected independent variables on economic growth in subnational regions of LMICs t statistics in parentheses. 4.2 Multivariate Analyses p < 0.05,p < 0.01, p < 0.001 Model 1 Model 2 Dependency ratios Beta (β) T-value Beta (β) T-value T1 Dependency Ratio (log) −3.107* (−9.61) −2.572* (−7.97) Growth Dependency Ratio −0.176* (−7.79) −0.144* (−6.17) Control factors T1 IWI −0.0503* (−21.71) −0.0539*** (−23.69) T1 IWI * T1 IWI −0.000299* (−4.88) Urban 0.145 (2.90) 0.213* (4.31) T1 Male Education 0.0861* (4.61) 0.0898*** (4.88) T1 Male–Female Education −0.0702* (−2.54) −0.153* (−5.24) ∆ Male Education 4.098* (26.49) 4.300* (27.40) ∆ Male–Female Education −2.868* (−12.09) −3.294*** (−13.81) Interactions with dependency ratios T1 Dependency Ratio (log) * T1 Male Education −0.461*** (−5.11) T1 Dependency Ratio (log) * Urban 1.429*** (4.00) Growth Dependency Ratio * Urban 0.0845* (2.27) Growth Dependency Ratio * T1 IWI −0.00244* (−2.33) Growth Dependency Ratio * T1 Governance −0.135*** (−3.44) Growth Dependency Ratio * T1 Financial Development 0.514* (2.29) Interactions among sub-national control factors T1 IWI * T1 Male Education −0.00213** (−3.12) T1 IWI * ∆ Male Education −0.0187** (−3.03) Urban * T1 Male–Female Education −0.109*** (−4.02) T1 Male Education * ∆ Male–Female Education 0.264*** (4.04) T1 Male–Female Education * ∆ Male Education −0.374*** (−4.68) # Observations 1,921 1,921 # Countries 91 91 le 4 Coefficients of fixed effects regression models of selected independent variables on economic wth in subnational regions of LMICs 1 3 L. Crombach, J. Smits 184 Table 5 Fixed effects regression models of effects of the demographic window phases on economic growth in subnational regions of LMICs t statistics in parentheses. p < 0.05, p < 0.01, p < 0.001 (1) Model 1 Beta (β) T-value Demographic window phases (2) Pre-window phase 0.271* (5.09) (3) Early-window phase 0.267** (3.21) (4) Mid-window phase 0.252* (2.09) (5) Late-window phase 0.0476 (0.28) (6) Post-window phase −0.0422 (−0.22) Control factors T1 IWI −0.0454*** (−19.80) Urban 0.131* (2.56) T1 Male Education 0.149* (8.41) T1 Male–Female Education −0.101* (−3.59) ∆ Male Education 4.686* (33.66) ∆ Male–Female Education −3.239* (−13.68) # Observations 1,921 # Countries 91 Table 5 Fixed effects regression models of effects of the demographic window phases on economic growth in subnational regions of LMICs t statistics in parentheses. p < 0.05, p < 0.01, **p < 0.001 change. 4.2 Multivariate Analyses The coefficients of the T1 dependency ratio and of the growth of the depend- ency ratio remain highly significant and negative, thus again confirming that both a lower dependency ratio and a decreasing dependency ratio are associated with signifi- cantly more economic growth.fif The interaction coefficients show that the effect of the dependency ratio is condi- tional on the level of urbanization of the region. Both the T1 dependency ratio and the growth rate of the dependency ratio have a significantly weaker negative effect in urban regions. Hence the DWO effect is, on average, stronger in rural areas. Regarding educa- tion, we observe that declines in the T1 dependency ratio are more effective in increas- ing growth when the region has high levels of initial male education (human capital stock). In terms of the growth rate of the dependency ratio, we find that its effect is larger in countries with a stronger institutional environment. In unreported analyses, we find that the governance effect predominantly occurs due to variation in the control of corruption component of the Worldwide Governance Indicators and that no interaction of the other components is significant if control of corruption is included. The interac- tion coefficient of control of corruption is −0.165 and has a t-value of −4.02. Somewhat surprisingly, we find that the DWO is more effective in regions with less financial devel- opment. This suggests that the DWO might be able to create growth even in regions where factors usually associated with job creation (inflation reduction, financial market development, economic openness) are not (yet) well developed.f Our effects are quantitatively important. For instance, a one standard deviation decrease in the T1 dependency ratio or the growth in the dependency ratio, leads to a 0.36 or 0.15 standard deviation increase in IWI growth for an average region, respec- tively. The effect of the T1 dependency ratio is 50% stronger than the average effect in regions with one standard deviation of T1 male education above the average, and 28% 1 3 1 3 The Demographic Window of Opportunity and Economic Growth at… 185 weaker than the average effect in urban regions. Further, the effect of growth in the dependency ratio is 47% stronger in regions with one standard deviation of governance above the average. weaker than the average effect in urban regions. 4.2 Multivariate Analyses Further, the effect of growth in the dependency ratio is 47% stronger in regions with one standard deviation of governance above the average. The negative effect of IWI at T1 remains in the interaction model, but it is now non- linear, and conditional on the level and change in (male) schooling. The larger the level of or increase in schooling, the stronger the T1 IWI effect. Conversely, one could argue that (faster) increases in the level of schooling have a stronger effect in regions with an initially lower level of development. To get a more detailed picture of the relationship between the DWO and economic growth, in Table 5 the dependency ratios are replaced by the T1 DW phases. With regard to the other independent variables, the model is equal to Model 1 of Table 4. The table shows that regions in the second, third and fourth DW phase have significant higher growth rates than the traditional DW phase. This effect is still positive but not significant in the late window phase. In the post- window phase, it has disappeared completely. As such, the results are in line with the idea that the DWO is a temporary period of higher growth. However, given that the effect is already positive in the pre-window phase and not significant anymore in the late window phase, it seems that the positive effects of a decrease of the dependency ratio can already be felt earlier than has been assumed before (e.g., UN, 2004). 5 Discussion and Conclusion In this study, we aim to contribute to the existing literature on the effects of the DWO on economic growth, by studying this relationship at the level of sub-national regions within LMICs. Our data reveal that there is substantial variation in terms of the DWO at the sub-national level, not only in middle-income countries, but also in low-income countries. As such, significant informative value can be gained from analyzing the effect of population age-structures on economic growth at a sub-national level in LMICs.i To achieve this, we created a dataset of 1921 regions in 91 LMICs and we used a fixed effects country dummy model to explain the variation in growth (measured by the Interna- tional Wealth Index) among these regions. The results indicate that the DWO is indeed a statistical reality. Economic growth was largest in regions with a lower dependency ratio, as well as in regions where the dependency ratio decreased during the study period. A more detailed analysis in which economic growth was compared between regions in different phases of the DWO revealed that economic growth was largest in regions that are in the second to fourth phase of the DWO, called here the pre, early and mid-window phases.f Our interaction analysis showed that the effects of the DWO on economic growth are not everywhere the same. The positive associations between a lower dependency ratio and economic growth, as well as of a reduction of the dependency ratio and economic growth, are stronger in rural areas. This indicates that these areas can profit more of the DWO than more urban areas. Also, investments in education have the expected effect with a more effective DWO in areas with a higher educational level. Together, these finding suggest that rural areas, which have invested in reasonable educational facilities, have the highest propensity to reap the demographic dividend.f The positive effect of a reduction in the dependency ratio on economic growth is strengthened in regions with higher levels of development, in regions with good govern- ance and in regions with lower levels of financial development. The first finding makes clear that for very poor regions it is more difficult to make use of the window, which sets sub-Saharan Africa at a disadvantage compared to LMICs in other regions. 4.3 Robustness Tests The models presented in Table S1 in the Supplementary Materials use the same strategy as those in Table 4, but with different data, estimation techniques or variables. The first column shows the coefficients of the model that controls for migration flows within and between regions. Although the coefficient of the migration variable has the expected positive sign, it is not significant at the conventional confidence level of 95%. All other effects are robust to the inclusion of the proxy for migratory flows. In the T0–T1 column, we use the same strategy as before, but we go back one period in time for all countries where another period of at least four years is available. Thus, instead of explaining the variation in changes in the period between times T1 and T2, we now analyze the variation in changes in the period between times T0 and T1. This is possible for 1254 regions in 55 countries. The effects of the main variables—dependency ratio and change in dependency ratio—remain significant. We also note that the main effects of the DWO increase in size, while only the interaction between the change in the dependency ratio and T1 govern- ance remains significant. Of the other variables, we observe that the T1 difference between male and female schooling, and some interactions, lose their significance. The third column shows the results we would have obtained if we only had data at the national level, but with a split between urban/rural regions. This analysis is based on data for 91 countries and 182 observations in total. In this setup, the T1 dependency ratio and the change in the dependency ratio are no longer sig- nificant. In addition, many of the control factors and all interactions lose their significance. This highlights the added value of our sub-national approach.if Overall, we conclude that our main findings regarding DWO effectiveness are robust to the inclusion of migratory flows and the use of older survey data. Nevertheless, T1 Governance is the only significant determinants of DWO effectiveness in the T0-T1 setup. Further, we highlight that sub-national data is better able to capture context-specific effects than national data. 1 3 L. Crombach, J. Smits 186 1 3 5 Discussion and Conclusion Regarding governance, corruption seems the decisive factor, with areas within countries with higher levels of corruption being less able to turn a decreasing dependency ratio into growth.i p g g p y g A low level of financial development, on the other hand, does not seem problematic for reaping the DWO. This might have to do with the way growth is measured: on the basis of household wealth instead of GDP. Whereas GDP growth might depend on business activi- ties that require a well-developed financial infrastructure, this is less the case for increases in household wealth (as measured by the IWI). Our data show that in countries with weak financial institutions households are even better able to translate the extra income due to reduction of the number (and hence costs) of children into asset ownership and improved housing quality. This may have to do with a greater importance of informal social networks in these countries, including local savings and credit associations (Anderson & Baland, 2002; Besley et al., 1993). There are indications that informal institutions may become less effective under stricter formal regulation (Williamson, 2009).i f Important is also our finding that already in the second phase of the DWO, called pre- window phase, a significant increase in economic growth is observed. Hence, regions in an early phase of fertility reduction might already experience benefits in terms of economic growth. On the other hand, regions that are in the late window phase (with less than 20% under 15 and less than 15% over 65) seem to experience less advantages of their demo- graphic situation than countries in an earlier phase. This might mean that in those countries 1 3 The Demographic Window of Opportunity and Economic Growth at… 187 growth chances already start to be affected by the growing number of elderly, and that the effect of the old-age dependency ratio on growth is relatively strong. growth chances already start to be affected by the growing number of elderly, and that the effect of the old-age dependency ratio on growth is relatively strong. f In terms of policy implications, we urge policymakers to invest in education and particu- larly so in rural areas. Control of corruption should be another important policy concern, as the positive effects of good governance that are observed are completely due to improve- ments made in its corruption dimension. 5 Discussion and Conclusion Moreover, we find that investing in education during the DWO leads to a much lower demographic dividend than investing in education early on.f In conclusion, our paper contributes to the existing literature by analyzing the effect of the DWO at subnational level for a large sample of LMICs. Population age-structures matter for economic growth, not only at the national level but also for specific areas within countries. Additionally, our interaction analysis confirms that the relationship between demography and economic growth is not a simple one but depends to a substantial extent on characteristics of the context in which the changes are taking place. Consequently, the implementation of policies such as family planning, labor market policies and education policies should take the unique characteristics of a specific area into account. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s11205-021-02802-8. Acknowledgements We gratefully acknowledge the comments and suggestions made by participants of the 8th African Population Conference held in Entebbe, Uganda in November 2019. Funding Open Access funding provided by ETH Zurich. There was no project-specific funding. Data Availability All data will be made available in case of acceptance. Declarations Conflict of interest The authors declare that they have no conflict of interest. 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 Com- mons 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/. Baerlocher, D., Parente, S., & L., and Rios-Neto, E. . (2019). Economic effects of demographic dividend Brazilian regions. The Journal of the Economics of Ageing. https://doi.org/10.1016/j.jeoa.2019.1001 Anderson, S., & Baland, J. M. (2002). The economics of roscas and intrahousehold resource allocation. T Quarterly Journal of Economics, 117(3), 963–995. https://doi.org/10.1162/003355302760193931.f Allison, P. D. (2002). Missing data. Sage Publications. Anderson, S., & Baland, J. M. (2002). The economics of roscas and intrahousehold resource allocation. The Quarterly Journal of Economics, 117(3), 963–995. https://doi.org/10.1162/003355302760193931. Baerlocher, D., Parente, S., & L., and Rios-Neto, E. . (2019). Economic effects of demographic dividend in Brazilian regions. The Journal of the Economics of Ageing. https://doi.org/10.1016/j.jeoa.2019.100198 Barro, R. J. (1991). Economic growth in a cross section of countries. The Quarterly Journal of Economics, 106(2), 407–443. https://doi.org/10.2307/2937943 , ( ) g g Anderson, S., & Baland, J. M. (2002). The economics of roscas and intrahousehold resource allocation. The Quarterly Journal of Economics, 117(3), 963–995. https://doi.org/10.1162/003355302760193931. Baerlocher, D., Parente, S., & L., and Rios-Neto, E. . (2019). Economic effects of demographic dividend in B ili i Th J l f th E i f A i htt //d i /10 1016/j j 2019 100198 References Aaronson, D., Dehejia, R., Jordan, A., Pop-Eleches, C., Samii, C., & Schulze, K. (2021). The effect of fer- tility on mothers’ labor supply over the last two centuries. The Economic Journal, 131(633), 1–32. https://doi.org/10.1093/ej/ueaa100 Alli P D (2002) d S P bli i Aaronson, D., Dehejia, R., Jordan, A., Pop-Eleches, C., Samii, C., & Schulze, K. (2021). The effect of fer- tility on mothers’ labor supply over the last two centuries. The Economic Journal, 131(633), 1–32. https://doi.org/10.1093/ej/ueaa100 Allison, P. D. (2002). Missing data. Sage Publications. Allison, P. D. (2002). Missing data. Sage Publicatio Baerlocher, D., Parente, S., & L., and Rios-Neto, E. . (2019). Economic effects of demographic dividend in Brazilian regions. The Journal of the Economics of Ageing. https://doi.org/10.1016/j.jeoa.2019.100198 Barro, R. J. (1991). Economic growth in a cross section of countries. The Quarterly Journal of Economics, 106(2), 407–443. https://doi.org/10.2307/2937943 g f f g g p g j j Barro, R. J. (1991). Economic growth in a cross section of countries. The Quarterly Journal of Economics, 106(2), 407–443. https://doi.org/10.2307/2937943 1 3 L. Crombach, J. Smits 188 Besley, T., Coate, S., & Loury, G. (1993). The economics of rotating savings and credit associations. The American Economic Review, 792–810. https://www.jstor.org/stable/2117579.i Bloom, D., & Canning, D. (2008). Global demographic change: Dimensions and economic significance. Population and Development Review, 34, 17–51. Bloom, D., Canning, D., Fink, G., & Finlay, J. E. (2009). Fertility, female labor force participation, and the demographic dividend. Journal of Economic Growth, 14(2), 79–101. https://doi.org/10.1007/ s10887-009-9039-9 Bloom, D., Canning, D., & Sevilla, J. (2003). The demographic dividend: A new perspective on the eco- nomic consequences of population change. RAND Corporation. https://doi.org/10.7249/MR1274 Bloom, D., Kuhn, M., & Prettner, K. (2017). Africa’s prospects for enjoying a demographic dividend. Jour- nal of Demographic Economics, 83(1), 63–76. https://doi.org/10.1017/dem.2016.19 Bloom, D., & Williamson, J. G. (1998). Demographic transitions and economic miracles in emerging Asia. The World Bank Economic Review, 12(3), 419–455. https://doi.org/10.1093/wber/12.3.419 Canning, D., Raja, S., and Yazbeck, A. (Eds.). (2015). Africa’s demographic transition: Dividend or disas- ter? The World Bank.f Collier, P., & Dollar, D. (2001). Can the world cut poverty in half? How policy reform and effective aid can meet international development goals. World Development, 29(11), 1787–1802. https://doi.org/10. 1016/S0305-750X(01)00076-6 ( ) Crespo Cuaresma, J., Lutz, W., & Sanderson, W. (2014). Is the demographic dividend an education divi- dend? Demography, 51(1), 299–315. https://doi.org/10.1007/s13524-013-0245-xfi Cristia, J. P. (2008). References The effect of a first child on female labor supply: Evidence from women seeking fertil- ity services. Journal of Human Resources, 43(3), 487–510. https://doi.org/10.3368/jhr.43.3.487f Dreher, A. (2006). Does globalization affect growth? Evidence from a new index of globalization. Applied Economics, 38(10), 1091–1110. https://doi.org/10.1080/00036840500392078 her, A., Gaston, N., and Martens, P. (2008). Measuring Globalization: Gauging its Consequences. Springer. p g Easterlin, R. A. (1971). Does human fertility adjust to the environment? The American Economic Review, 61(2), 399–407. Eloundou-Enyegue, P. M., Giroux, S., & Tenikue, M. (2017). African transitions and fertility inequality: A demographic Kuznets hypothesis. Population and Development Review, 43, 59–83. https://doi.org/10. 1111/padr.12034 Eloundou-Enyegue, P. M., and Hirschl, N. (2017). Fertility transitions and schooling dividends in Sub-Saha- ran Africa: The experience of Vanguard Countries. In H. Groth and J. F. May (Eds.), Africa’s Popula- tion: In Search of a Demographic Dividend (pp. 101–111). Springer International Publishing. https:// doi.org/10.1007/978-3-319-46889-1_7 Groth, H., and May, J. F. (Eds.). (2017). Africa’s Population: In Search of a Demographic Dividend (1st ed. 2017). Springer International Publishing : Imprint: Springer. https://doi.org/10.1007/ 978-3-319-46889-1 Gygli, S., Haelg, F., Potrafke, N., & Sturm, J.-E. (2019). The KOF globalisation index—revisited. The Review of International Organizations, 14, 543–574. https://doi.org/10.1007/s11558-019-09357-xf Huisman, J., & Smits, J. (2009). Effects of household-and district-level factors on primary school enroll- ment in 30 developing countries. World Development, 37(1), 179–193. https://doi.org/10.1016/j.world dev.2008.01.007. Jimenez, E., and Pate, M. A. (2017). Reaping a demographic dividend in Africa’s largest Country: Nigeria. In H. Groth and J. F. May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 33–51). Springer International Publishing. https://doi.org/10.1007/978-3-319-46889-1_3 Kaufmann, D., Kraay, A., & Mastruzzi, M. (2011). The worldwide governance indicators: Methodology and analytical issues. Hague Journal on the Rule of Law, 3(2), 220–246. https://doi.org/10.1017/S1876 404511200046 Kelley, A. C., & Schmidt, R. M. (2005). Evolution of recent economic-demographic modeling: A synthesis. Journal of Population Economics, 18, 275–300. https://doi.org/10.1007/s00148-005-0222-9i p g Kumar, U. (2013). India’s demographic transition: Boon or bane? Asia & the Pacific Policy Studies, 1(1), 186– 203. https://doi.org/10.1002/app5.9 p g pp Lee, E. S. (1966). A theory of migration. Demography, 3(1), 47. https://doi.org/10.2307/2060063 Loayza, N. V., Rancière, R., Servén, L., & Ventura, J. (2007). Macroeconomic volatility and welfare in devel- oping countries: An introduction. The World Bank Economic Review, 21(3), 343–357. https://doi.org/10. 1093/wber/lhm017 Preston, S. H., Himes, C., & Eggers, M. (1989). Demographic conditions responsible for population aging. Demography, 26(4), 691. References https://doi.org/10.2307/2061266 1 3 The Demographic Window of Opportunity and Economic Growth at… 189 Radelet, S., Sachs, J., & Jong-Wha, L. (2001). The determinants and prospects of economic growth in Asia. International Economic Journal, 15(3), 1–29. https://doi.org/10.1080/10168730100000041 Sander, N., and Charles-Edwards, E. (2017). Internal and International Migration. In H. Groth and J. F. May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 333–349). Springer International Publishing. https://doi.org/10.1007/978-3-319-46889-1_21 Shapiro, D., Tambashe, B. O., and Romaniuk, A. (2017). The Third Biggest African Country: The Democratic Republic of the Congo. In H. Groth and J. F. May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 71–86). Springer International Publishing. https://doi.org/10.1007/978-3-319-46889-1_5 p , , , , , ( ) gg y Republic of the Congo. In H. Groth and J. F. May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 71–86). Springer International Publishing. https://doi.org/10.1007/978-3-319-46889-1_5 Smits, J. (2016). GDL Area Database. Sub-national development indicators for research and policy-making. GDL Working Paper, 16–101. Smits, J., & Steendijk, R. (2015). The international wealth index (IWI). Social Indicators Research, 122, 65–85. https://doi.org/10.1007/s11205-014-0683-x rydzenka, K. (2016). Introducing a New Broad-Based Index of Financial Development. IMF Working Paper No. 16/5, 1–44. Turbat, V. (2017). Economic Growth and Public and Private Investments. In H. Groth and J. F. May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 353–365). Springer International Publish- Turbat, V. (2017). Economic Growth and Public and Private Investments. In H. Groth and J. F. May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 353–365). Springer International Publish- ing. https://doi.org/10.1007/978-3-319-46889-1_22 ing. https://doi.org/10.1007/978-3-319-46889-1_22 United Nations. (2004). World Population to 2300. United Nations Population Division. United Nations. (2018). World Urbanization Prospects: The 2018 Revision. Department of Economic and Social Affairs, Population Division. f Wei, Z., & Hao, R. (2010). Demographic structure and economic growth: Evidence from China. Journal of Comparative Economics, 38(4), 472–491. https://doi.org/10.1016/j.jce.2010.08.002 Williamson, C. R. (2009). Informal institutions rule: Institutional arrangements and economic performan Public Choice, 139(3–4), 371–387. https://doi.org/10.1007/s11127-009-9399-x Williamson, J. G. (2013). Demographic dividends revisited. Asian Development Review, 30(2), 1–25. Wooldridge, J. M. (2013). Introductory econometrics: A modern approach (5th ed). South-Western Cengage Learning.l World Bank. (2021). World Development Indicators. Inflation, consumer prices (annual %). https://databank. worldbank.org/home.aspx ber, A., Blickenstorfer, C., and Groth, H. (2017). Governance, Transparency, and the Rule of Law. In H. Groth and J. F. References May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 367–384). Zuber, A., Blickenstorfer, C., and Groth, H. (2017). Governance, Transparency, and the Rule of Law. In H. Groth and J. F. May (Eds.), Africa’s Population: In Search of a Demographic Dividend (pp. 367–384). Springer International Publishing. https://doi.org/10.1007/978-3-319-46889-1_23 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. 1 3 1 3
W2967146865.txt	https://www.frontiersin.org/articles/10.3389/fevo.2019.00317/pdf	en		Dynamic-Parameter Movement Models Reveal Drivers of Migratory Pace in a Soaring Bird	Frontiers in ecology and evolution	2,019	cc-by	12,046	ORIGINAL RESEARCH published: 27 August 2019 doi: 10.3389/fevo.2019.00317 Dynamic-Parameter Movement Models Reveal Drivers of Migratory Pace in a Soaring Bird Joseph M. Eisaguirre 1,2, Marie Auger-Méthé 3,4 , Christopher P. Barger 5 , Stephen B. Lewis 6 , Travis L. Booms 5 and Greg A. Breed 1,7 1 Department of Biology and Wildlife, University of Alaska Fairbanks, Fairbanks, AK, United States, 2 Department of Mathematics and Statistics, University of Alaska Fairbanks, Fairbanks, AK, United States, 3 Department of Statistics, University of British Columbia, Vancouver, BC, Canada, 4 Institute for the Oceans and Fisheries, University of British Columbia, Vancouver, BC, Canada, 5 Alaska Department of Fish and Game, Fairbanks, AK, United States, 6 United States Fish and Wildlife Service, Juneau, AK, United States, 7 Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK, United States Edited by: Frants Havmand Jensen, Woods Hole Oceanographic Institution, United States Reviewed by: Anne K. Scharf, Max Planck Institute of Ornithology, Germany Peter E. Smouse, Rutgers University, The State University of New Jersey, United States Correspondence: Joseph M. Eisaguirre jmeisaguirre@alaska.edu Specialty section: This article was submitted to Behavioral and Evolutionary Ecology, a section of the journal Frontiers in Ecology and Evolution Received: 11 March 2019 Accepted: 07 August 2019 Published: 27 August 2019 Citation: Eisaguirre JM, Auger-Méthé M, Barger CP, Lewis SB, Booms TL and Breed GA (2019) Dynamic-Parameter Movement Models Reveal Drivers of Migratory Pace in a Soaring Bird. Front. Ecol. Evol. 7:317. doi: 10.3389/fevo.2019.00317 Long distance migration can increase lifetime fitness, but can be costly, incurring increased energetic expenses and higher mortality risks. Stopover and other en route behaviors allow animals to rest and replenish energy stores and avoid or mitigate other hazards during migration. Some animals, such as soaring birds, can subsidize the energetic costs of migration by extracting energy from flowing air. However, it is unclear how these energy sources affect or interact with behavioral processes and stopover in long-distance soaring migrants. To understand these behaviors and the effects of processes that might enhance use of flight subsidies, we developed a flexible mechanistic model to predict how flight subsidies drive migrant behavior and movement processes. The novel modeling framework incorporated time-varying parameters informed by environmental covariates to characterize a continuous range of behaviors during migration. This model framework was fit to GPS satellite telemetry data collected from a large soaring and opportunist foraging bird, the golden eagle (Aquila chrysaetos), during migration in western North America. Fitted dynamic model parameters revealed a clear circadian rhythm in eagle movement and behavior, which was directly related to thermal uplift. Behavioral budgets were complex, however, with evidence for a joint migrating/foraging behavior, resembling a slower paced fly-andforage migration, which could facilitate efficient refueling while still ensuring migration progress. In previous work, ecological and foraging conditions are usually considered to be the key aspects of stopover location quality, but taxa, such as the golden eagle, that can tap energy sources from moving fluids to drive migratory locomotion may pace migration based on both foraging opportunities and available flight subsidies. Keywords: Bayesian, correlated random walk, golden eagle, movement ecology, soaring flight Frontiers in Ecology and Evolution \| www.frontiersin.org 1 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace INTRODUCTION the earth’s surface to generate thermal uplift. Other forms arise from turbulent eddies over small landscape features and ocean waves modifying the air. The dynamic nature of atmosphericallydriven flight subsidies requires detailed movement data as well as carefully designed analytical techniques to investigate certain mechanisms hidden in those data. Our understanding of migratory processes has advanced enormously in the past 30 years, as animal tracking technology developed from a novelty of coarse observation to a core method for observing animal behavior and movement in incredible detail (Luschi et al., 1998; Sawyer et al., 2005; Bridge et al., 2011; Katzner et al., 2015; Hooten et al., 2017). Global Positioning System (GPS) telemetry, in particular, allows remote observation of animal relocations across a broad spatiotemporal scale. GPS transmitters are now light and reliable enough to study the complete migrations of many large soaring migrants, including golden eagles Aquila chrysaetos, which often rely on flight subsidies during migration (Katzner et al., 2015). Golden eagles and other large soaring birds have been used as model systems for phenomenologically evaluating questions about migratory flight performance and migration strategies (sensu Duerr et al., 2012; Lanzone et al., 2012; Katzner et al., 2015; Vansteelant et al., 2015; Miller et al., 2016; Shamoun-Baranes et al., 2016; Rus et al., 2017). For example, Lanzone et al. (2012) and Katzner et al. (2015) found that golden eagles use both thermal and orographic uplift to subsidize migratory flight, although thermal soaring was often more efficient in long distance, directed flight (Duerr et al., 2012). While these studies have contributed to our understanding of soaring migration and have laid a foundation for more detailed approaches, they relate meteorology to derived movement metrics, rather than incorporate them into process-based models that mechanistically predict movement, and ignore the temporal dependence between serially observed locations (i.e., autocorrelation). Not accounting for such autocorrelation imparts bias on certain estimated parameters (e.g., variances) thereby affecting inference through, for example, underestimating uncertainty. Consequently, the links between resources distributed over the landscape, such as flight subsidies, and behavioral budgets, including stopover behavior, during migrations of soaring birds remain unclear. Unlike previous approaches, process-based, mechanistic movement models allow explicit inference of the underlying mechanisms driving movement (e.g., changes in behavior) that may not be available from conventional phenomenological analytical approaches (Turchin, 1998; Nathan et al., 2008; Hooten et al., 2017). While it is impossible to understand fully the intricacies in animal movement, we can pose mathematical models (e.g., correlated random walks) to approximate the movement process (Kareiva and Shigesada, 1983; Turchin, 1998). We can then fit these models statistically to observed data to estimate parameters describing behavior and its relationship with dynamic environmental features that moving animals experience (Blackwell, 1997, 2003; Morales et al., 2004; Breed et al., 2017; Hooten et al., 2017). Many of the recently developed mechanistic movement models are built in a discrete stateswitching framework, where animals switch between discrete behavioral states (see Hooten et al., 2017, and references Long-distance migration can relax competition and permit use of seasonally available resources, helping many animals maximize lifetime fitness (Newton, 2008; Avgar et al., 2014). Those benefits, however, come at substantial costs, including greater vulnerability to predators, uncertain conditions, mechanical wear, elevated energy expenditure, and time (Alerstam and Hedenström, 1998; Clark and Butler, 1999; Hedenström, 2008; Newton, 2008; Avgar et al., 2014). As many migrant species cannot store sufficient energy for nonstop, long-distance migration, stopover evolved as a behavior for strategically resting and refueling en route (Gill, 2007). Migrant species are adapted for utilizing either soaring or flapping flight, and the different flight modes translate into stopover strategy (Hedenström, 1993; Gill, 2007). Generally, soaring flight is favorable for larger birds and flapping flight for smaller birds, though the partitioning of time for each flight mode during migration is dependent on the tradeoff between time and energy (Hedenström, 1993; Duerr et al., 2015; Katzner et al., 2015; Miller et al., 2016). In theory, a timeminimizing migrator would be expected to fly with greater directional persistence and stronger directional bias than would an energy-expenditure minimizer. Such net energy maximizers would be expected to take advantage of en route foraging opportunities and may divert or delay to replenish energy reserves. (Note that “energy minimization” has been used to describe this strategy e.g., Alerstam, 2011; Miller et al., 2016, but we use “net energy maximization” for clarity.) If time is less important, a net energy maximizer is less restricted and can spend additional time seeking an energetically superior path; the emergent path would then be more tortuous with less directional bias toward the final destination at any given point along the route. Time minimization and net energy maximization strategies are not mutually exclusive, however, and the emergent strategy and behaviors in any given migrating individual lies along a continuum (Alerstam, 2011; Miller et al., 2016). Obligate soaring migrants must also consider routes based on their energy landscape (Shamoun-Baranes et al., 2010), the energetic constraints of movement over space (Shepard et al., 2013), which also contributes to a migrant’s location along the behavioral continuum. While soaring migrants can stopover, their energy landscape is more complex. Meteorological conditions are at least as important as foraging resources for soaring migrants, which can be extremely dynamic and subsidize the energetic cost of flight directly via uplift (Pennycuick, 1971; Alerstam, 1979; Spaar and Bruderer, 1997; Gill, 2007; Duerr et al., 2012; Murgatroyd et al., 2018). The flight performance of soaring migrants relative to subsidies provided by meteorological conditions has been well documented (Pennycuick, 1971; Alerstam, 1979; Spaar and Bruderer, 1997), establishing a clear link between diurnal migrant behavior and development of the atmospheric boundary layer. Two primary forms of uplift arise by (1) wind interacting with topography to form upslope wind or mountain waves (air currents forming standing waves established on the lee side of mountains; hereafter orographic uplift) and (2) solar heating of Frontiers in Ecology and Evolution \| www.frontiersin.org 2 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace Data Collection cited therein). Choosing both the biologically relevant and quantitatively supported number of states, as well as interpreting the identified states in a biological context, remains challenging (Patterson et al., 2017; Pohle et al., 2017). Often, this challenge leads researchers to artificially limit the number of states and/or collapse two or more states into one biologically interpretable state. For example Pirotta et al. (2018), presented a model with five discrete kinds of avian flight, but the complexity of the model made interpreting those states difficult and poorly matched classifications manually identified by an expert. In many cases, a more natural approach to modeling an animal’s movement process is along a dynamic continuum, rather than as switching between discrete behavioral states (Breed et al., 2012; Auger-Méthé et al., 2017; Jonsen et al., 2019). Modeling along a continuum may be an especially useful approach for understanding movement behavior in soaring birds, considering the dynamic nature of atmospheric processes that influence movements. Here, we developed and applied a flexible mechanistic movement model based on a correlated random walk with time-varying parameters. This novel model was fit to movement data collected via GPS telemetry to understand how individuals in a population of long-distance soaring migrants use flight subsidies and budget stopover and migration behavior. Specifically, we were interested in identifying which flight subsidies influence stopover and migratory behavior and how the effect of key subsides and behaviors varied between spring and fall migrations. Our approach resembled continuous-time correlated random walks (Johnson et al., 2008; Blackwell et al., 2015; Gurarie et al., 2017; Michelot and Blackwell, 2019), but was easily implemented and yielded a relatively small number of dynamic parameters that could be directly interpreted biologically. A set of candidate models could be ranked, with model selection approaches, providing inference on how behavioral budgets and meteorological variables interacted to give rise to the observed migration paths. Modeling the effects of dynamic wind and uplift variables as time-varying movement behaviors of migratory golden eagles further allowed new details to emerge without imposing artificially discrete states. We captured golden eagles with a remote-fired net launcher, placed over carrion bait near Gunsight Mountain, Alaska (61.67◦ N 147.35◦ W). Captures occurred between mid-March and mid-April 2014-2016. Fifty-three adult and sub-adult eagles were equipped with 45-g back pack solar-powered Argos/GPS platform transmitter terminals (PTTs; Microwave Telemetry, Inc., Columbia, MD, USA). Eagles were sexed molecularly and aged by plumage. PTTs were programmed to record GPS locations on duty cycles, ranging from 8 to 14 fixes per day during migration, depending on year of deployment. PTTs deployed in 2014 were set to record 13 locations at 1-h intervals centered around solar noon plus a location at midnight local time. PTTs deployed in 2015 were programmed to record 8 locations with 1-h intervals centered around solar noon, and PTTs deployed in 2016 took eight fixes daily at regular 3-h time intervals. Note that the PTTs deployed in 2015 did not record locations overnight. Poor battery voltage from September to March often resulted in PTTs failing to take all programmed fixes, so the resulting GPS tracks had missing observations during these periods. Tags lasted multiple seasons, and in fact many are still deployed and transmitting at this writing. We chose to limit this analysis to the migrations that occurred in 2016. The spring and fall migratory pathways of the 2016 migration from 26 tags were available and suitable for analysis in that year: 11 deployed in 2014, 7 deployed 2015, and 8 deployed 2016. Tracks were suitable for analysis based on having few missing data, with no more than a few days of consecutive missing locations. Movement data were managed in the online repository Movebank (https://www.movebank.org/), and we used the Track Annotation Service (Dodge et al., 2013) to extract flight subsidy (wind and uplift) data, specific to each PTT location and time of recording that location, along eagle tracks. The Track Annotation Service derives uplift variables from elevation models and weather and atmospheric reanalyses (Bohrer et al., 2012). We followed the Movebank recommendations for interpolation methods; details are below. Movement Model METHODS We developed a correlated random walk (CRW) movement model to reveal how changes in behavior give rise to the movement paths of migrating eagles. We chose to use a dynamic, time-varying correlation parameter, which represents behavior as a continuum rather than discrete categories, to capture complex behavioral patterns that could occur on multiple temporal and spatial scales (Breed et al., 2012; Auger-Méthé et al., 2017; Jonsen et al., 2019). We believe this approach can offer substantial flexibility, as a continuous range of behaviors is more realistic and, as we show, more naturally allows modeling behavior as a function of covariates. The basic form of the model was a first-difference CRW presented by Auger-Méthé et al. (2017), which can take the form: Model System The golden eagle is a large, soaring raptor, distributed across the Holarctic (Watson, 2010). Golden eagles are predatory and opportunistic, utilizing many taxa for food resources, ranging from small mammals and birds to ungulates, often scavenging carrion (Kochert et al., 2002; Watson, 2010). While many populations are classified as partial migrants, most individuals that summer and breed above approximately 55◦ N in North America are considered true long-distance migrants (Kochert et al., 2002; Watson, 2010). The population we observed in this study migrates over the mountainous regions of western North America between a breeding range primarily in southcentral Alaska, USA and a broad overwintering range in western North America that ranges from the southwestern US to central British Columbia and Alberta, Canada (Bedrosian et al., 2018). Frontiers in Ecology and Evolution \| www.frontiersin.org 1ti (xi−1 − xi−2 ), 6 i , xi \|xi−1 , xi−2 ∼ N2 xi−1 + γi 1ti−1 3 (1) August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace where 6i = 1ti2 σx2 0 0 , σx , σy > 0. 1ti2 σy2 as a linear combination of continuously-distributed random variables (Jonsen et al., 2019). These variables were different meteorological conditions affecting flight subsidies. Now, (2) Here, 1ti = ti − ti−1 represents the time interval between Cartesian coordinate vectors xi and xi−1 for the observed locations of the animal at times ti and ti−1 . Incorporating autocorrelation in behavior, γi constitutes a random walk, such that γi \|γi−1 ∼ N γi−1 , 1ti2 σν2 , σν > 0. (3) γi′ = log γi 1 − γi , (4) ′ + ZTi β + ǫi , γi′ = γi−1 ǫi ∼ N 0, 1ti2 σν2 , (5) where γi correlates displacements (or “steps”) and can be interpreted to understand the type of movement, and thus behavior, of migrating individuals: estimates of γi closer to one indicate directionally-persistent, larger-scale migratory movement, while estimates of γi closer to zero indicate more-tortuous, smallerscale stopover movement (Breed et al., 2012; Auger-Méthé et al., i and the variance components by 1ti2 2017). Scaling γi by 1t1ti−1 allows us to accommodate unequal time intervals (Auger-Méthé et al., 2017), which can arise from a PTT’s pre-programmed duty cycles and/or missed location attempts. This assumes that over longer time intervals an animal is likely to move greater distances and that the previous step will have less influence on the current step. Notably, in introducing 1ti , this CRW essentially becomes a correlated velocity model presented in terms of displacement vectors (xi−1 −xi−2 ) (Johnson et al., 2008; Blackwell et al., 2015; Gurarie et al., 2017), most closely resembling the autocorrelated velocity model presented by Gurarie et al. (2017). Because location error of GPS data is negligible compared to the movement of most large vertebrates (Hooten et al., 2017), we did not incorporate an observation equation to handle location error. While a covariance parameter could be added to the model, we chose to fix covariance to zero (equation 2), which assumes that movement in the x and y dimensions are independent. This assumption has been suggested to be potentially problematic (Dunn and Gipson, 1977; Blackwell, 1997); however, it is common and has been shown to draw reasonable inference from real data, as well as recover known parameters from simulated data (Breed et al., 2012, 2017; Auger-Méthé et al., 2017; Jonsen et al., 2019). To support this, we compared results from the model assuming zero covariance to one fit assuming equal variance in x and y—like estimating covariance, this ensures invariance under linear transformation of the coordinate system—to illustrate that inference remains unaffected by this assumption (Appendix 1). Extending this CRW to introduce environmental covariates, we first made the assumption that an individual’s behavior can be adequately explained by the previous behavior plus some effects of environmental conditions and random noise. This modeling approach and philosophy aligns with the movement ecology paradigm presented by Nathan et al. (2008): An animal’s movement path is influenced by its internal state and the environmental conditions it experiences. We modified the behavioral (or internal state) process—previously described above as a pure random walk in one dimension (Equation 3)—similar to a linear model with a logit link function. The logit link constrains γi ∈ [0, 1] and allowed us to model it Frontiers in Ecology and Evolution \| www.frontiersin.org (6) and ZTi is the row vector of environmental covariates associated with xi . Each element of the vector β is an estimated parameter representing the magnitude and direction of the effect of its respective covariate on the correlation parameter γi in addition to the effect of γi−1 . Note that including γi−1 here preserves explicit serial correlation in the behavioral process so that any additional environmental effect is not overestimated. γi′ is only used to estimate γi ; any behavioral interpretations are made in terms of γi . Model Fitting Subsetting Tracks Of the 26 eagles producing suitable data in 2016, we fit the model to 15 spring and 16 fall adult golden eagle migration tracks recorded by 18 adult males and 8 adult females in 2016. This included both spring and fall migrations for five individuals. In reporting the results, we assumed any individual random effects of including both migrations for these few individuals to be negligible, which seems reasonable given fitted parameters presented in Table S1 in Appendix. The model was fit only to the migratory periods, plus two fixes prior to departure to ensure valid parameter estimates at the onset of migration. Data were constrained to migratory periods under the following rules: The first migration step was identified as the first directed movement away from what was judged to be an individual’s summer (or winter) range with no subsequent return to that range, and the final migration step was defined as the step terminating in the apparent winter (or summer) range. This assignment was usually straightforward; however, in some cases there were apparent pre-migration staging areas. These were not considered part of migration and excluded from the analysis here; movement data from these individuals collected during the breeding and overwintering periods are neither presented nor analyzed here. Environmental Covariates Golden eagles can switch between using thermal and orographic uplift as flight subsidies (Lanzone et al., 2012; Katzner et al., 2015), so we included both variables as covariates affecting the correlation parameter in the behavioral process of the CRW (equation 5). Thermal uplift ztu and orographic uplift zou are measured in m/s with ztu , zou ∈ [0, ∞). Thermal uplift was bilinearly interpolated from European Center for Medium-Range Weather Forecasts (ECMWF) reanalyses, and orographic uplift from the nearest neighbor (grid cell) by pairing National Center for Environmental Predictions (NCEP) North 4 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace American Regional Reanalysis (NARR) data with the Advanced Spaceborne Thermal Emission Reflection Radiometer (ASTER) Global Digital Elevation Model (GDEM; Brandes and Ombalski, 2004; Bohrer et al., 2012). We also introduced wind as a covariate in the behavioral process, as it can influence eagle flight as well as the flight and energy landscape of many birds during migration (Shamoun-Baranes et al., 2017). Wind data were bilinearly interpolated from the NCEP NARR u (easterly/zonal) and v (northerly/meridional) components of wind predicted 30 m above ground in m/s, from which we calculated the wind support ztw , such that ztw ∈ (−∞, ∞) (Safi et al., 2013), where positive values correspond to tailwind and negative values headwind. The bearings used to calculate each ztw,i were the compass bearings required to arrive at xi+1 from xi . We included a time of day interaction in the model because of clear diurnal effects. This also helped reduce zero inflation, particularly for thermal uplift, which often decays to zero after sunset due to heat flux and atmospheric boundary layer dynamics. To introduce the interaction, we used a dummy variable z0 , such that z0,i = 0 when ti fell after sunset but before sunrise and z0,i = 1 when ti fell after sunrise but before sunset. This assumed behavior was not dependent on the covariates at night—the combination of covariates becomes zero when z0,i = 0—which is sensible given observed diurnal behavioral cycles. Sunrise and sunset times local to each GPS point were calculated in R with the “sunriset” function in the package “maptools” (Bivand and Lewin-Koh, 2016; R Core Team, 2016). Writing out the matrix operation in Equation (5), the final overall formulation of the behavioral process for the full model was: ′ γi′ = γi−1 + β0 + βou (zou,i × z0,i ) + βtu (ztu,i × z0,i ) + βtw (ztw,i × z0,i ) + ǫi , We implemented HMC with R and Stan through the package “rstan” (R Core Team, 2016; Stan Development Team, 2016). Working R and Stan code, including details on prior choice, and example data are provided as Supplementary Material, as well as supplementary tables and figures (Appendices 1–3). The model was fit to each track independently with five chains of 300,000 HMC iterations, including a 200,000 iteration warm-up phase, and retaining every tenth sample. Convergence to the posterior distribution was checked with trace plots, effective sample sizes, posterior plots of parameters, and Gelman diagnostics (R̂) for each model fit. We compared candidate models with leave-one-out crossvalidation approximated by Pareto-smoothed importance sampling (PSIS-LOO) in R with the package “loo” (Vehtari et al., 2016, 2017). The candidate models included possible combinations of environmental covariates plus a null CRW model without covariates. To limit model complexity and because we were interested in competing hypotheses about key predictors of behavior, we chose not to include interactions beyond time-of-day. We ranked the models by the expected log pointwise predictive density (elpd; i.e., out-of-sample predictive accuracy) transformed onto the deviance scale (looic; Vehtari et al., 2017), which created a measure on the same scale as common information criterion (e.g., AIC) and allowed applying the rules of more traditional information-theoretic model selection (e.g., Burnham and Anderson, 2004). The model with the lowest looic was considered the best fit to the data, but if other models were within two looic of the top model, each, including the top model, were considered equally supported by the data. To understand how the predictive ability of the full model varied among tracks, we also computed a pointwise average of the elpd for each track (Vehtari et al., 2017). Normalizing by the sample size allowed comparing the out of sample predictive ability of the full model across individual migration tracks (Table S1 in Appendix). The elpd (and looic), being sums, are otherwise dependent on the sample size for each model fit. (7) Prior to fitting the model, we followed Gelman et al. (2008) and log-transformed the uplift covariates and standardized variance to 0.25. We used a shifted log-transformation (Fox and Weisberg, 2019); adding one to the covariates prior to the logtransformation preserved zeros (i.e., zeros mapped to zero under the transformation). The distribution of raw wind support data appeared Gaussian, so it was only centered and standardized. RESULTS Model Performance and Diagnostics We fit eight candidate formulations of our CRW model to 31 migration tracks, equating to 248 total model fits. Chain mixing, Gelman diagnostics (R̂) close to one, and large effective sample sizes for all parameters indicated convergence to the posterior for most model fits. Posteriors of parameters appeared symmetric, also indicating the model was well behaved (Figure S1 in Appendix). Across all migration tracks, the full model showed strong evidence of convergence, but for five tracks, we did not consider the null model converged to the posterior (e.g., R̂ > 1.01). The five migrations for which the null model did not converge were not included from formal model selection. Parameter Estimation and Model Selection We fit our correlated random walk (CRW) in a Bayesian framework. Because the model has explicit serially correlated parameters, we used Hamiltonian Monte Carlo (HMC) over more conventional Markov-chain Monte Carlo (MCMC; e.g., Metropolis steps) to sample efficiently from a posterior with such correlation. Gelman et al. (2008) suggested Cauchy priors for logistic regression parameters; however, Ghosh et al. (2015) found that sampling from the posterior can be inefficient due to the fat tails of the Cauchy distribution. We thus chose Studentt priors centered on zero (µ = 0 and σ = 2.5) with five degrees of freedom as weakly informative priors for the covariate parameters. Weakly informative normal priors were placed on the variance parameters of the model. Frontiers in Ecology and Evolution \| www.frontiersin.org Behavior During Migration Median (interquartile range) departure and arrival dates were 5 March (4.5 d) and 27 March (6.4 d) in the spring and 29 September (11.7 d) and 16 November (15.5 d) in the fall. On 5 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace more highly-correlated displacements, or migratory movements. Despite that, there were some migration bouts not associated with great thermal uplift (Figures 1, 2). Coefficients close to zero for orographic uplift and wind support indicate that, in general, they were not strong drivers of directionallypersistent movements. Based on the model selection, the best-fitting formulation of the environmental drivers of the behavioral process was variable across individuals. However, in almost all cases, some form of flight subsidy was used and there was little difference between the spring and fall seasons in the pattern of subsidy use (Table 2). The high variability across individuals (Table S1 in Appendix) was likely due to differing weather patterns and thus subsidy sources encountered and/or used by each eagle as migrations were not synchronous (in time or space) across individuals. In addition, inter-individual variation was much larger than any difference attributable to demographic variables; we found no evidence that difference in sex or age explained patterns of flight subsidy use during migration. Note, though, that all eagles included in this analysis were in adult plumage, so strong age effects would not necessarily be expected. Comparing the pointwise elpd across tracks revealed that the out of sample predictive ability of the full model varied among individuals (Table S1 in Appendix). It also showed that predictive ability was greater for fall migrations than spring. TABLE 1 \| Summary statistics of flight subsidies encountered by migrating golden eagles that summer in Alaska. Season Orographic uplift Thermal uplift Wind support meana (s.d.) mean (s.d.) mean (s.d.) Spring 0.41 (0.71) 0.61 (0.48) 2.09 (3.06) Fall 0.43 (0.71) 0.39 (0.34) 1.37 (3.45) Variables were interpolated in space and time from weather reanalyses to eagle locations recorded by GPS telemetry. Units for all variables are m/s. a grand mean across discrete GPS locations with individual migration tracks pooled. average, eagles encountered similar orographic uplift in spring and fall but more intense thermal uplift and tailwind in the spring (Table 1). The model revealed that eagles changed their behavior on multiple scales. First, there were very strong daily rhythms in behavior during migration, with birds migrating or moving more slowly and tortuously during the day and stopping at night (Figures 1, 2). Explicitly including a time-of-day interaction could cause a daily rhythm to emerge as an artifact of model specification. However, accounting for serial correlation in behavior (Equation 5) limited that possibility. Additionally, prolonged periods of movement without an apparent daily rhythm suggest that, where daily rhythms are observed they are not a product of model specification (Figure 2). Second, there was some evidence of stopover-like behavior, but with individuals continually moving along the migration route while exhibiting less directional persistence in movement (Figure 3). The continuation along the migration route while in a stopoverlike state is highlighted by track segments extended over space associated with low and intermediate estimates of γi (blue/purple in Figure 3). There was also a clear effect of season on movement patterns and behavior. Spring was characterized by straighter, more direct trajectories and punctuated by slower, more tortuous, stopoverlike movement; whereas, fall movements were much more tortuous overall and regular patterns in changes in movement rate and/or tortuosity less clear (Figures 1–3). The distributions of estimated γ values also clearly indicate that daytime movements were most frequently directed migratory moves in the spring; whereas, in the fall, the bimodal distribution indicates more equivalent partitioning between directed migratory moves and slower stopover type movement, with significant time spent exhibiting behaviors associated with intermediate tortuosity and movement rate (Figure 3). DISCUSSION Here, we develop and demonstrate how dynamic parameter CRW models fit to GPS data reveal the effects of variable flight subsides available along migration routes. Use of these subsidies gives rise to diverse patterns in the movement of a longdistance soaring migrant. Behavioral changes occur continuously as available subsidies shift over time and space. These key driving mechanisms underlie emergent movement paths, yet such processes are often hidden in the discrete satellite observations available. Our mechanistic modeling approach allowed linking of dynamic meteorology to changes in behavior, and those changes in behavior to the observed movement paths, revealing time series of behaviors more complex than individuals simply apportioning time between migration and stopover. Model Performance Incorporating time-varying parameters into movement models has been a relatively infrequently utilized approach (Breed et al., 2012; Auger-Méthé et al., 2017; Jonsen et al., 2019). Here we provide a case study for its utility and developed the approach for achieving practical biological inference about movement processes. Modeling the serial correlation in movement as a function of environmental covariates (equation 4), allowed simultaneous inference of behavior and the effect of environmental covariates on behavior from animal trajectories with regular and irregular duty cycles and containing missing observations. While other methods exist to handle missing data, the behavioral patterns we found would be more difficult to reveal with a state-switching movement model (e.g., hidden Markov models (HMMs); Michelot et al., 2016) because each Environmental Covariates While there were differences in some environmental covariates between spring and fall (Table 1), parameter estimates from the full model (all covariates) indicate that there was little to no difference in effect of flight subsidies (i.e., wind and uplift) on behavior between spring and fall (Figure 4, Table S1 in Appendix). Including environmental covariates in the behavioral process, though, improved model fit for almost all fitted migrations (Table 2). Positive coefficients on the thermal uplift covariate indicate that increasing thermal uplift resulted in Frontiers in Ecology and Evolution \| www.frontiersin.org 6 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace FIGURE 1 \| Time series of behavior parameter γ from correlated random walk model with full behavioral process (orographic uplift, thermal uplift, and wind support as predictors) for two golden eagles during spring migration with PTTs reporting on different duty cycles. Upper panel is 13 hourly centered on solar noon plus one at midnight, and the lower panel is 8 hourly centered on solar noon. γ close to one reflect movements associated with migratory behavior, and γ close to zero stopover behavior. Points are times of observations, and lines are linear interpolations between points. Hue indicates intensity of thermal uplift, with yellow indicating greater and blue lower. Note the daily rhythm in behavior associated with intense thermal uplift, stopover periods of one or more days, and the intermediate periods suggesting fly-and-forage. FIGURE 2 \| Time series of behavior parameter γ from correlated random walk model with full behavioral process (orographic uplift, thermal uplift, and wind support as predictors) for three golden eagles during fall migration with PTTs reporting on different duty cycles. Upper panel is 13 hourly centered on solar noon plus one at midnight, middle panel is 8 hourly centered on solar noon, and lower panel is fixed 3-h interval. γ close to one reflect movements associated with migratory behavior, and γ close to zero stopover behavior. Points are times of observations, and lines are linear interpolations between points. Hue indicates intensity of thermal uplift, with yellow indicating greater thermal uplift and blue lower. Note the daily rhythm in behavior and extended stopovers as well as periods intermediate values suggesting fly-and-forage. states (e.g., McClintock et al., 2012), these states can require ancillary data streams (e.g., accelerometry) to discriminate and remain extremely difficult to employ and interpret in practice step would be forced into a discrete behavioral state from a set of usually 2–3 discrete states. Moreover, although hidden-state models have been introduced that have more than five discrete Frontiers in Ecology and Evolution \| www.frontiersin.org 7 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace FIGURE 3 \| Golden eagle migration trajectories (N = 15 spring and N = 16 fall). Hue indicates value of behavioral parameter γ estimated with the correlated random walk model with full behavioral process, including orographic uplift, thermal uplift, and wind support as predictors. Insets show the relative frequencies of estimates of γ assigned to the displacements between observed daytime GPS locations. γ close to one reflect movements associated with migratory behavior, and γ close to zero stopover behavior. Daily rhythms, revealed in Figures 1, 2, are not apparent here because the birds moved so little at night. TABLE 2 \| Number of golden eagle migration tracks recorded by GPS transmitters that each candidate formulation of the behavioral process in the correlated random walk model fit the best, according to approximate leave-one-out cross-validation (Table S1). looic tallya Model Spring Fall Total Fullb 4 3 7 Therm + twind 2 4 6 Oro + therm 3 3 6 Oro 3 3 6 Therm 2 3 5 Oro + twind 2 2 4 Twind 0 3 3 Null 1 1 2 “therm” corresponds to thermal uplift, “oro” to orographic uplift, and “twind” to wind support. a tally given to model with lowest information criterion (looic; Vehtari et al., 2016); if one or more models were within two looic of the top model, each was given a tally. b oro + therm + twind. are neither biologically meaningful nor sensible (Pohle et al., 2017). Implementing models with dynamic parameters that can be interpreted along a behavioral continuum seems a more natural approach for many animal movement questions. FIGURE 4 \| Point estimates of environmental covariate effect parameters (βou , βtu , βtw ) on golden eagle behavior and movements during migration (N = 15 spring and N = 16 fall). Estimates are from the correlated random walk model with full behavioral process, including orographic uplift, thermal uplift, and wind support as predictors. Effects of Tag Programming While our CRW model revealed the same trends across duty cycles and was generally robust to the different duty cycles (Figures 1, 2), the most detail in daily behavioral rhythms was revealed in tracks with a fixed 3-hr time interval (lower panel in Figure 2), as it provided data throughout the 24-h day at regular intervals. The other duty cycles were initially chosen to minimize (Patterson et al., 2017; Pohle et al., 2017). Finally, as HMMs include greater numbers of potential states, they tend to fit better than models with fewer states as judged by classical model selection approaches, such as AIC, even when additional states Frontiers in Ecology and Evolution \| www.frontiersin.org 8 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace the risk of battery depletion overnight. Although generally robust, duty cycles did affect model fitting. HMC permitted Bayesian inference rather efficiently for our model, considering elevated correlation in the posterior of parameters due to the model formulation. Model fits typically took no more than a few hours, though tracks with much more than several hundred locations sometimes took longer. Preliminary fitting of our model with Stan and Template Model Builder (TMB; following Auger-Méthé et al., 2017) suggested that Maximum Likelihood estimation (when fit with TMB) tends to fail frequently when tag programming results in uneven temporal coverage of each day (e.g., our 2015 duty cycle), while Bayesian inference still provided sensible parameter estimates in most cases. Although the model presented herein and the model presented by AugerMéthé et al. (2017) can make up for irregular time intervals between observations, they do have limitations. Breed et al. (2011) offer an in-depth discussion of tag programming and its effects on model fitting and inference. However, model selection for models including those variables did indicate they explained some variance in eagle movement, which we discuss further below. Despite meteorological conditions along migration paths that differed between spring and fall and a stark difference between behavioral budgets, our results showed no clear difference in the use of flight subsidies between the spring and fall seasons (Figure 4). This finding contrasts with season-specific effects of flight subsidies on golden eagle migration shown phenomenologically in eastern North America, where thermal uplift was shown to be the key subsidy in migratory performance during spring, while wind with some additional support from thermal uplift is most important in the fall (Duerr et al., 2015; Rus et al., 2017). Although our results indicate that eagles use similar flight subsidizing strategies in both seasons, consistent with the differences from the eastern population, the actual behaviors performed during spring and fall migrations differed considerably. In spring, eagles used subsidies to drive a migration that allows timely arrival on the breeding grounds, consistent with a time minimization strategy. In the fall, flight was subsidized to minimize net energy use, which emerged as a much more diverse behavioral repertoire during a slower fall migration (Figure 3; Miller et al., 2016). The more rapid and direct flight punctuated by bouts of tortuous, stopover-like movement in the spring (Figure 3), suggest eagles pause, refuel, and/or perhaps wait for better migration conditions. This suggests eagles may employ, at least in part, a net energy maximization strategy (Hedenström, 1993; Miller et al., 2016), despite the need for timely arrival on the breeding grounds to avoid fitness costs (Both and Visser, 2001). The behavioral time series of spring migrations showed some evidence of individuals responding less to thermal uplift as latitude increased (Figure 1). This likely corresponded to a general decay in thermal uplift as individuals migrated northward (Supplementary Material, Figure S2 in Appendix). Reduced thermal uplift availability would be expected at higher latitudes due to the larger amounts of remaining spring snowpack and lower solar angles. Thus, golden eagles, and likely other soaring birds, migrating to high latitudes may need to budget behaviors carefully between time minimization and net energy maximization during spring migration to best take advantage of the reduced flight subsidy from thermal uplift and mitigate the greater energy demands of flight at higher latitudes. While our results show that thermal uplift is the most important flight subsidy for the majority of migrating eagles sampled, the model selection indicated orographic uplift and wind support improved out of sample predictive accuracy and explained some variance in eagle movement. Additionally, variability in top models across individuals (Table 2, Table S1 in Appendix) suggests among-individual variance in flightsubsidizing strategy. Although some of this variability can be attributed to real individual differences in behavioral strategy, it is at least as likely that individuals encountered different subsidies en route and used the subsidies they had available, as migrations across our sample were not synchronous. Given that orographic uplift and wind support parameter estimates were negative or close to zero for many individuals (Figure 4, Table S1 Flight Subsidies as Drivers Migration of Behavior Thermal uplift is a flight subsidy dependent on daily atmospheric boundary layer dynamics, and it was clearly an important driver of the daily rhythm in eagle movement (Figure 4, Table S1 in Appendix). Intense thermal uplift was often associated with the peaks in daily migration bouts (Figure 1). The larger magnitude of the thermal uplift effect, relative to orographic uplift, was somewhat surprising, as many individuals in our sample followed the Rocky Mountains, a large potential source of orographic uplift. Golden eagles are known to use orographic uplift as a flight subsidy while migrating through the Appalachian Mountains in eastern North America (Katzner et al., 2015). Much of the Appalachians, however, is characterized by long, unbroken, linearly-oriented ridges. Wind blowing over these ridges produces long stretches of predictable orographic uplift (Rus et al., 2017). The Rocky Mountains, by contrast, are far more rugged and nonuniform, and conditions that might produce suitable upslope winds and mountain waves, as well as strong tailwinds, likely also generate violent turbulence and could impede efficient migratory flight. Soaring raptors have been shown to use small-scale turbulence to achieve subsidized flight (Allen et al., 1996; Mallon et al., 2016); however, unpredictable, nonstationary violent turbulence, which can occur in large, highelevation mountain ranges (Ralph et al., 1997), could produce unfavorable migratory conditions. The large effect of thermal uplift, thus, could indicate that the Rocky Mountains, a spine that spans almost the entire migration corridor for this population, as well as some areas further west (Bedrosian et al., 2018), serves as a network of thermal streets for migrating eagles (Pennycuick, 1998). More explicitly, intense sun on south facing slopes would be expected to generate linear series of thermals that birds could glide between during both spring and fall migration. It is important to keep in mind that the migrants could capitalize on fine-scale, localized features of certain flight subsidies, like orographic uplift and tailwind, that may not have been captured by the interpolated meteorological data used in our analyses. Frontiers in Ecology and Evolution \| www.frontiersin.org 9 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace paradigm (Nathan et al., 2008); it used the observed data— GPS locations, rather than a derived metric—and a theoretical movement process to infer behavior from movement patterns along tracks on a spectrum ranging from stopped to rapid, directionally-persistent movement. Our analyses, however, showed that eagles still tended to continue along their migration route during periods of movement most resembling stopover, but with reduced movement rate and directional persistence (Figures 1–3). This pattern suggests a joint migration/opportunistic foraging behavior that resembles fly-and-forage migration (Strandberg and Alerstam, 2007; Åkesson et al., 2012; Klaassen et al., 2017), which is consistent with observations of en route hunting behavior of golden eagles by Dekker (1985). Such behavior could be used to maintain balance between time expenses and energy intake, as it allows simultaneous migration progress and foraging. This pattern does not fall very well within the “stopover” paradigm (Gill, 2007; Newton, 2008), however, as true stops during the migrations we observed were rare, except for expected nightly stops. Rather, migrants seemed to change their pace—either by slowing down, moving more tortuously, or both—but still generally moved toward their migratory destination (Figures 1–3). Thus, instead of a discrete behavioral framework, whereby migrants switch between two migratory phases (migration and stopover) with very different movement and behavioral properties, we propose that, for certain taxa, a continuous alternative framework “migratory pacing” may be more appropriate and a natural way to interpret en route migratory behavior and movement dynamics. Such taxa would include some and perhaps many soaring migrants, as well some migrating species in other fluid environments such as fishes and marine mammals. Soaring birds, even when energy reserves are relatively depleted, likely can still make steady progress toward a migratory goal when flight subsidies are available. Flapping migrants, on the other hand, would not be able to achieve this as readily, due in part to the greater energy demands for sustained flight, and would require more regular refueling stopovers where migration progress is temporarily completely arrested. Both opportunism in foraging and use of energetic subsidies are likely key characters of fly-and-forage behavior and the ability to change pace of migration without actually stopping, as they relax the need for individuals to stopover in specific, food-rich habitats, which are required by most migrants with less flexibility in food and that lack the morphological specialization to maximally exploit the energetic subsidies available in moving fluids (Gill, 2007; Piersma, 2007). Our model results revealed seasonal variability in migratory pacing by golden eagles. The tendency for eagles to exhibit movements matching fly-and-forage behavior, and pace their migrations more slowly was most apparent during fall migration. In contrast, spring migration was usually composed of much more punctuated events of slower-paced movements but these were still extended over space (Figure 3), indicating the eagles pace their migration and employ a mixed behavioral strategy to some extent in spring as well. During spring, hibernating mammalian prey would be minimally available, leaving carrion, along with a few non-migratory and -hibernating species (e.g., in Appendix), those covariates likely predicted the periods of slower, more tortuous movements (i.e., γi closer to zero). Wind support occurred in top models for more individuals in the fall (Table 2, Table S1 in Appendix), which is consistent with findings from others (McIntyre et al., 2008; Rus et al., 2017) and suggests it may be important during southbound migrations. Additionally, although there was variance among the types and combinations of subsidies used, the null model (without flight subsidies) was the best fitting model for very few tracks (Table 2, Table S1 in Appendix), evidence that weather and flight subsidies are of importance to migrating golden eagles, and likely also to the migrations of similar soaring species. Lastly, we found that the full model had, on average, better predictive ability in fall than spring (Table S1 in Appendix), suggesting that the weather variables explained more of the variance within movement paths in fall compared to spring; during spring migration, other internal state variables associated with greater time limitation that were not explicitly accounted for in the models were likely responsible for this seasonal difference. Daily Rhythm and Migratory Pace The full movement model revealed two clear, nested behavioral patterns in the long-distance migrations of golden eagles. First, there was a daily rhythm where inferred directed migratory movements (i.e., γi close to one) occurred most frequently around midday or early afternoon (Figures 1, 2). Mechanistic models of animal movement have revealed diel behavioral rhythms in other taxa (Jonsen et al., 2006). The basic aspects of daily rhythms in vertebrate behavior have hormone controls (Cassone, 1990), but the benefits can include balancing migration progress and foraging bouts (Newton, 2008). Soaring migrants also benefit by synchronizing diel movement patterns with diel atmospheric cycles. That is, consistent with our results, diurnal soaring migrants express a general circadian behavioral rhythm, where flight performance and behavior is strongly tied to thermal development of the planetary boundary layer to take best advantage of atmospherically generated flight subsides (Kerlinger et al., 1985; Leshem and Yom-Tov, 1989; Spaar and Bruderer, 1996, 1997; Mateos-Rodríguez and Liechti, 2012). The second behavioral pattern revealed was a general stopover pattern, whereby eagles changed behavior for one to several days while en route (Figures 1–3). These changes were consistent with searching movements (i.e., γ intermediate or close to zero), possibly representing foraging behavior. In terms of soaring raptors, however, very few reports of movement patterns and behavior during stopovers have been published. Stopover segments have been previously identified by speed or some other metric calculated from tracks, then excluded from subsequent analyses (e.g., Katzner et al., 2015; Vansteelant et al., 2015); occasionally, authors noted apparent enhanced tortuosity but explored it no further (e.g., Vansteelant et al., 2017). On occasions where stopover behavior was considered, classifications based on stay duration and travel distance or speed with hard, often arbitrarily chosen cutoffs between migrating and stopover segments were used (Chevallier et al., 2011; Katzner et al., 2012; Duerr et al., 2015; Miller et al., 2016). In contrast, our modeling framework aligned with the movement ecology Frontiers in Ecology and Evolution \| www.frontiersin.org 10 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace behavioral patterns during migration of long-distance soaring migrants. While time-varying, dynamic parameters have been infrequently employed in movement modeling (Breed et al., 2012; Jonsen et al., 2013; Auger-Méthé et al., 2017), we have shown it is a promising approach that can overcome certain limitations in discrete state-switching models and help provide novel insight into animal behavior. This approach also has potential for further development and for revealing additional new patterns in soaring bird movement; it has already been shown to help provide new insight for other taxa as well (Jonsen et al., 2019). Although we demonstrated the approach for several individual eagles, applying our methods across a larger sample and across more years will increase the inferential strength of our results. For example, previous work found effects of wind support and orographic uplift (e.g., Katzner et al., 2015; Vansteelant et al., 2015), where we, in accounting for an eagles’ underlying movement process and the inherent autocorrelation in that process, found that those meteorological variables may be of less importance, at least compared to thermal uplift. It remains unclear though, whether these are systemspecific findings or a more general result. Additionally, the model we present has potential to help assess effects of habitat on the movement decisions of soaring birds and other species. One potential avenue for such would be incorporating the model into a resource selection framework (e.g., step selection function). Furthermore, given the movement process is parameterized in terms of coordinate vectors, the position likelihood could be straightforwardly extended to include the z axis to investigate questions regarding flight height of soaring birds or dive depth of marine species, assuming data of acceptable temporal resolution and location error are, or become, available. ptarmigan Lagopus spp. and hare Lepus spp.), as major food sources, which could help explain the more punctuated bouts of slower-pacing. Alternatively, individuals could have been slowed by poor weather conditions (Rus et al., 2017). Scavenging large ungulate carcasses would be extremely rewarding in terms of energy accumulation. Much of the carrion we used successfully to capture eagles was large ungulate (e.g., moose Alces alces), strongly suggesting that the population we sampled uses carcasses during migration. The bimodal distribution for the behavioral parameter γ in fall shows that eagles tended to budget daytime behaviors approximately equally between rapid, directed and slower-paced movements (Figure 3); the high frequency and range of intermediate values are, again, evidence for the more complex fly-and-forage and pacing dynamic, rather than eagles simply switching between stopover and migration. This behavioral complexity might be biologically important, allowing eagles to arrive on winter home ranges in better condition compared to migration strategies that do not incorporate en route foraging opportunity. In contrast to fall, daytime movements in the spring were typically faster-paced (i.e., larger-scale and directionally-persistent; Figure 3), consistent with a time minimization strategy, where eagles need to partition time more in favor of migration progress to ensure timely arrival on breeding grounds (Hedenström, 1993; Alerstam, 2011; Miller et al., 2016). We thus see in eagles, and propose more generally, that such pacing varies between and within seasons along the continuum between time minimization and net energy maximization strategies (Alerstam, 2011; Miller et al., 2016). A migrant’s pace would be expected to depend upon their energetic demands, energetic subsidies available from the environment, and the importance of arriving at the migration terminus in a timely fashion (Nathan et al., 2008). DATA AVAILABILITY Implications and Conclusions We developed and applied a movement model with timevarying parameters to help reveal the mechanisms underlying the migration of a long-distance soaring migrant that relies on incredibly dynamic flight subsidies. We found that variation in flight subsidies gives rise to changes in migrant behavior with thermal uplift seemingly most important. While these findings might be expected given previous phenomenological analyses (e.g., Duerr et al., 2012; Lanzone et al., 2012; Katzner et al., 2015; Vansteelant et al., 2015; Miller et al., 2016; ShamounBaranes et al., 2016; Rus et al., 2017), we were able to show how meteorology is a mechanism influencing changes in movement patterns and thus behavior. In the behavioral budgets of migrating golden eagles, we identified an expected daily rhythm, as well as evidence for behavioral dynamics that would allow nearly simultaneous foraging and migration, which is greater complexity than the traditional stopover paradigm allows. Migratory pacing, facilitated by fly-and-forage behavior, expands the traditional notion of stopover, whereby a bird migrates until resting and refueling is required, at which point it stops for a brief period in specific habitat suitable for efficient foraging (Gill, 2007; Newton, 2008). This advance was enabled by incorporating time-varying parameters into the movement model, which revealed new Frontiers in Ecology and Evolution \| www.frontiersin.org All movement data used for this manuscript are managed in the online repository Movebank (https://www.movebank.org/; IDs 17680093 and 19389828). The data contain information considered sensitive by the State of Alaska, but they could be made available at the discretion of the Alaska Department of Fish & Game and U.S. Fish & Wildlife Service. Code to fit the movement model to data and example data are provided as Supplementary Material. ETHICS STATEMENT Field procedures were conducted following the ADF&G Animal Care & Use Committee protocol #2013-036 and University of Alaska Fairbanks Institutional Animal Care & Use Committee protocol #859448. AUTHOR CONTRIBUTIONS JE and GB conceived the ideas of the research presented herein. JE, MA-M, and GB designed analytical methods. TB, CB, and SL designed the field methodology. TB, CB, SL, and JE collected the data. JE analyzed the data, and led the manuscript. All authors 11 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace and data. JE was supported by the Calvin J. Lensink Fund during part of the project. MA-M acknowledges the support of the Natural Sciences and Engineering Research Council of Canada. The findings and conclusions of this paper are those of the authors and do not necessarily represent the views of the USFWS. This manuscript has been released as a pre-print at Eisaguirre et al. (2019). contributed to drafts and editing of the manuscript and provided final approval for publication. ACKNOWLEDGMENTS We thank M. Kohan, B. Robinson, T. & D. Hawkins and many others for support in the field and J. Liguori and N. Paprocki for help aging eagles. We also thank T. Avgar, P. Doak, T. Katzner, K. Kielland, C. McIntyre, A. Scharf, and P. Smouse for helpful comments on drafts of the manuscript. Funding was provided by the Alaska Department of Fish & Game (ADF&G) through the federal State Wildlife Grant Program, and the U.S. Fish & Wildlife Service (USFWS) provided PTTs SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fevo. 2019.00317/full#supplementary-material REFERENCES behavioral dynamics from animal tracks. Ecol. Model. 235–236, 49–58. doi: 10.1016/j.ecolmodel.2012.03.021 Breed, G. A., Golson, E. A., and Tinker, M. T. (2017). Predicting animal homerange structure and transitions using a multistate Ornstein-Uhlenbeck biased random walk. Ecology 98, 32–47. doi: 10.1002/ecy.1615 Bridge, E. S., Thorup, K., Bowlin, M. S., Chilson, P. B., Diehl, R. H., Fléron, R. W., et al. (2011). Technology on the Move: recent and Forthcoming Innovations for Tracking Migratory Birds. BioScience 61, 689–698. doi: 10.1525/bio.2011.61.9.7 Burnham, K. P., and Anderson, R. (2004). Multimodel inference: understanding AIC and BIC in model selection. Sociol. Methods Res. 33, 261–304. doi: 10.1177/0049124104268644 Cassone, V. M. (1990). Effects of melatonin on vertebrate circadian systems. Trends Neurosci. 13, 457–464. doi: 10.1016/0166-2236(90)90099-V Chevallier, D., Le Maho, Y., Brossault, P., Baillon, F., and Massemin, S. (2011). The use of stopover sites by Black Storks (Ciconia nigra) migrating between West Europe and West Africa as revealed by satellite telemetry. J. Ornithol. 152, 1–13. doi: 10.1007/s10336-010-0536-6 Clark, C. W., and Butler, R. W. (1999). Fitness components of avian migration: a dynamic model of Western Sandpiper migration. Evol. Ecol. Res. 1, 443–453. Dekker, D. (1985). Hunting behavior of golden eagles, Aquila chrysaetos, migrating in southwestern Alberta. Can. Field-Natur. 99, 383–385. Dodge, S., Bohrer, G., Weinzierl, R., Davidson, S. C., Kays, R., Douglas, D., et al. (2013). The environmental-data automated track annotation (Env-DATA) system: linking animal tracks with environmental data. Movem. Ecol. 1:3. doi: 10.1186/2051-3933-1-3 Duerr, A. E., Miller, T. A., Lanzone, M., Brandes, D., Cooper, J., O’Malley, K., et al. (2012). Testing an emerging paradigm in migration ecology shows surprising differences in efficiency between flight modes. PLoS ONE 7:e35548. doi: 10.1371/journal.pone.0035548 Duerr, A. E., Miller, T. A., Lanzone, M., Brandes, D., Cooper, J., O’Malley, K., et al. (2015). Flight response of slope-soaring birds to seasonal variation in thermal generation. Funct. Ecol. 29, 779–790. doi: 10.1111/1365-2435.12381 Dunn, J. E., and Gipson, P. S. (1977). Analysis of radio telemetry data in studies of home range. Biometrics 33, 85–101. doi: 10.2307/2529305 Eisaguirre, J. M., Auger-Méthé, M., Barger, C. P., Lewis, S. B., Booms, T. L., and Breed, G. A. (2019). Environmentally-driven dynamic parameters in mechanistic movement models reveal complex migratory pacing in a soaring bird. bioRxiv [Preprint]. doi: 10.1101/465427 Fox, J., and Weisberg, S. (2019). An R Companion to Applied Regression, 3rd Edn. Thousand Oaks, CA: SAGE Publications Inc. Gelman, A., Jakulin, A., Pittau, M. G., and Su, Y. S. (2008). A weakly informative default prior distribution for logistic and other regression models. Ann. Appl. Stat. 2, 1360–1383. doi: 10.1214/08-AOAS191 Ghosh, J., Li, Y., and Mitra, R. (2015). On the use of Cauchy prior distributions for Bayesian logistic regression. ArXiv 1507.07170. Gill, F. B. (2007). Ornithology, 3rd Edn. New York, NY: W. H. Freeman and Company. Gurarie, E., Fleming, C. H., Fagan, W. F., Laidre, K. L., Hernández-Pliego, J., and Ovaskainen, O. (2017). Correlated velocity models as a fundamental Åkesson, S., Klaassen, R., Holmgren, J., Fox, J. W., and Hedenström, A. (2012). Migration routes and strategies in a highly aerial migrant, the common swift Apus apus, revealed by light-level geolocators. PLoS ONE 7:e41195. doi: 10.1371/journal.pone.0041195 Alerstam, T. (1979). Optimal use of wind by migrating birds: combined drift and overcompensation. J. Theor. Biol. 79, 341–353. doi: 10.1016/0022-5193(79)90351-5 Alerstam, T. (2011). Optimal bird migration revisited. J. Ornithol. 152, 5–23. doi: 10.1007/s10336-011-0694-1 Alerstam, T., and Hedenström, A. (1998). The development of bird migration theory. J. Avian Biol. 29, 343–369. doi: 10.2307/3677155 Allen, P. E., Goodrich, L. J., and Bildstein, K. L. (1996). Within- and among-year effects of cold fronts on migrating raptors at Hawk Mountain, Pennsylvania, 1934-1991. Auk 113, 329–338. doi: 10.2307/4088899 Auger-Méthé, M., Albertsen, C. M., Jonsen, I. D., Derocher, A. E., Lidgard, D. C., Studholme, K. R., et al. (2017). Spatiotemporal modelling of marine movement data using Template Model Builder (TMB). Mar. Ecol. Prog. Ser. 565, 237–249. doi: 10.3354/meps12019 Avgar, T., Street, G., and Fryxell, J. M. (2014). On the adaptive benefits of mammal migration. Can. J. Zool. 92, 481–490. doi: 10.1139/cjz-2013-0076 Bedrosian, B. E., Domenech, R., Shreading, A., Hayes, M. M., Booms, T. L., and Barger, C. R. (2018). Migration corridors of adult Golden Eagles originating in northwestern North America. PLoS ONE 13:e0205204. doi: 10.1371/journal.pone.0205204 Bivand, R., and Lewin-Koh, N. (2016). maptools: Tools for Reading and Handling Spatial Objects, Version 0.8-39. Blackwell, P. G. (1997). Random diffusion models for animal movement. Ecol. Model. 100, 87–102. doi: 10.1016/S0304-3800(97)00153-1 Blackwell, P. G. (2003). Bayesian inference for Markov processes with diffusion and discrete components. Biometrika 90, 613–627. doi: 10.1093/biomet/90.3.613 Blackwell, P. G., Niu, M., Lambert, M. S., and Lapoint, S. D. (2015). Exact Bayesian inference for animal movement in continuous time. Methods Ecol. Evol. 7, 184–195. doi: 10.1111/2041-210X.12460 Bohrer, G., Brandes, D., Mandel, J. T., Bildstein, K. L., Miller, T. A., Lanzone, M., et al. (2012). Estimating updraft velocity components over large spatial scales: contrasting migration strategies of golden eagles and turkey vultures. Ecol. Lett. 15, 96–103. doi: 10.1111/j.1461-0248.2011.01713.x Both, C., and Visser, M. E. (2001). Adjustment to climate change is constrained by arrival date in a long-distance migrant bird. Nature 411, 296–298. doi: 10.1038/35077063 Brandes, D., and Ombalski, D. W. (2004). Modeling raptor migration pathways using a fluid-flow analogy. J. Rapt. Res. 38, 195–207. Breed, G. A., Costa, D. P., Goebel, M. E., and Robinson, P. W. (2011). Electronic tracking tag programming is critical to data collection for behavioral time-series analysis. Ecosphere 2:10. doi: 10.1890/ES10-00021.1 Breed, G. A., Costa, D. P., Jonsen, I. D., Robinson, P. W., and MillsFlemming, J. (2012). State-space methods for more completely capturing Frontiers in Ecology and Evolution \| www.frontiersin.org 12 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace Michelot, T., and Blackwell, P. G. (2019). State-switching continioustime correlated random walks. Methods Ecol. Evol. 10, 637–649. doi: 10.1111/2041-210X.13154 Michelot, T., Langrock, R., and Patterson, T. A. (2016). moveHMM: an R package for the statistical modelling of animal movement data using hidden Markov models. Methods Ecol. Evol. 7, 1308–1315. doi: 10.1111/2041-210X.12578 Miller, T. A., Brooks, R. P., Lanzone, M. J., Brandes, D., Cooper, J., Tremblay, J. A., et al. (2016). Limitations and mechanisms influencing the migratory performance of soaring birds. Ibis 158, 116–134. doi: 10.1111/ibi.12331 Morales, J. M., Haydon, D. T., Frair, J., Holsinger, K. E., and Fryxell, J. M. (2004). Extracting more out of relocation data: building movement models as mixtures of random walks. Ecology 85, 2436–2445. doi: 10.1890/03-0269 Murgatroyd, M., Photopoulou, T., Underhill, L. G., Bouten, W., and Amar, A. (2018). Where eagles soar: fine-resolution tracking reveals the spatiotemporal use of differential soaring modes in a large raptor. Ecol. Evol. 8, 6788–6799. doi: 10.1002/ece3.4189 Nathan, R., Getz, W. M., Revilla, E., Holyoak, M., Kadmon, R., Saltz, D., et al. (2008). A movement ecology paradigm for unifying organismal movement research. Proc. Natl. Acad. Sci. U.S.A. 105, 19052–19059. doi: 10.1073/pnas.0800375105 Newton, I. (2008). The Migration Ecology of Birds. London: Academic Press. Patterson, T. A., Parton, A., Langrock, R., Blackwell, P. G., Thomas, L., and King, R. (2017). Statistical modelling of individual animal movement: an overview of key methods and a discussion of practical challenges. AStA Adv. Stat. Anal. 101, 399–438. doi: 10.1007/s10182-017-0302-7 Pennycuick, C. (1998). Field observations of thermals and thermal streets, and the theory of cross-country soaring flight. J. Avian Biol. 29, 33–43. doi: 10.2307/3677338 Pennycuick, C. J. (1971). Soaring behavior and performance of some East African birds, observed from a motor-glider. Ibis 114, 178–218. doi: 10.1111/j.1474-919X.1972.tb02603.x Piersma, T. (2007). Using the power of comparison to explain habitat use and migration strategies of shorebirds worldwide. J. Ornithol. 148(Suppl. 1):S45– S59. doi: 10.1007/s10336-007-0240-3 Pirotta, E., Katzner, T., Miller, T. A., Duerr, A. E., Braham, M. A., and New, L. (2018). State-space modelling of the flight behaviour of a soaring bird provides new insights to migratory strategies. Funct. Ecol. 32, 2205–2215. doi: 10.1111/1365-2435.13180 Pohle, J., Langrock, R., van Beest, F. M., and Schmidt, N. M. (2017). Selecting the number of states in hidden Markov models: pragmatic solutions illustrated using animal movement. J. Agricult. Biol. Environ. Stat. 22, 270–293. doi: 10.1007/s13253-017-0283-8 R Core Team (2016). R: A Language and Environment for Statistical Computing. R Core Team. Ralph, F. M., Neiman, P. J., and Levinson, D. (1997). Lidar observations of a breaking mountain wave associated with extreme turbulence. Geophys. Res. Lett. 24, 663–666. doi: 10.1029/97GL00349 Rus, A. I., Duerr, A. E., Miller, T. A., Belthoff, J. R., and Katzner, T. E. (2017). Counterintuitive roles of experience and weather on migratory performance. Auk 134, 485–497. doi: 10.1642/AUK-16-147.1 Safi, K., Kranstauber, B., Weinzierl, R., Griffin, L., Rees, E. C., Cabot, D., et al. (2013). Flying with the wind: scale dependency of speed and direction measurements in modelling wind support in avian flight. Movem. Ecol. 1:4. doi: 10.1186/2051-3933-1-4 Sawyer, H., Lindzey, F., and McWhirter, D. (2005). Mule deer and pronghorn migration in western Wyoming. Wildl. Soc. Bull. 33, 1266–1273. doi: 10.2193/ 0091-7648(2005)33[1266:MDAPMI]2.0.CO;2 Shamoun-Baranes, J., Bouten, W., Loon, E. E. V., Meijer, C., Camphuysen, C. J., and Shamoun-baranes, J. (2016). Flap or soar? How a flight generalist responds to its aerial environment. Philos. Trans. R. Soc. B 371:20150395. doi: 10.1098/rstb.2015.0395 Shamoun-Baranes, J., Bouten, W., and Van Loon, E. E. (2010). Integrating meteorology into research on migration. Integr. Compar. Biol. 50, 280–292. doi: 10.1093/icb/icq011 Shamoun-Baranes, J., Liechti, F., and Vansteelant, W. M. (2017). Atmospheric conditions create freeways, detours and tailbacks for migrating birds. J. Compar. Physiol. A 203, 509–529. doi: 10.1007/s00359-017-1181-9 unit of animal movement: synthesis and applications. Movem. Ecol. 5, 1–18. doi: 10.1186/s40462-017-0103-3 Hedenström, A. (1993). Migration by soaring or flapping flight in birds: the relative importance of energy cost and speed. Philos. Trans. R. Soc. B Biol. Sci. 342, 353–361. doi: 10.1098/rstb.1993.0164 Hedenström, A. (2008). Adaptations to migration in birds: behavioural strategies, morphology and scaling effects. Philos. Trans. R. Soc. B 363, 287–299. doi: 10.1098/rstb.2007.2140 Hooten, M. B., Johnson, D. S., McClintock, B. T., and Morales, J. M. (2017). Animal Movement: Statistical Models for Telemetry Data. New York, NY: CRC Press. Johnson, D. S., London, J. M., Lea, M.-A., and Durban, J. W. (2008). Continuoustime correlated random walk model for animal telemetry data. Ecology 89, 1208–1215. doi: 10.1890/07-1032.1 Jonsen, I. D., Basson, M., Bestley, S., Bravington, M. V., Patterson, T. A., Pedersen, M. W., et al. (2013). State-space models for bio-loggers: a methodological road map. Deep-Sea Res. II Top. Stud. Oceanogr. 88–89, 34–46. doi: 10.1016/j.dsr2.2012.07.008 Jonsen, I. D., McMahon, C. R., Patterson, T. A., Auger-Méthé, M., Harcourt, R., Hindell, M. A., et al. (2019). Movement responses to environment: fast inference of variation among southern elephant seals with a mixed effects model. Ecology 100, 1–8. doi: 10.1002/ecy.2566 Jonsen, I. D., Myers, R. A., and James, M. C. (2006). Robust hierarchical state-space models reveal diel variation in travel rates of migrating leatherback turtles. J. Anim. Ecol. 75, 1046–1057. doi: 10.1111/j.1365-2656.2006.01129.x Kareiva, P. M., and Shigesada, N. (1983). Analyzing insect movement as a correlated random walk. Oecologia 56, 234–238. doi: 10.1007/BF00379695 Katzner, T. E., Brandes, D., Miller, T., Lanzone, M., Maisonneuve, C., Tremblay, J. A., et al. (2012). Topography drives migratory flight altitude of golden eagles: implications for on-shore wind energy development. J. Appl. Ecol. 49, 1178–1186. doi: 10.1111/j.1365-2664.2012.02185.x Katzner, T. E., Turk, P. J., Duerr, A. E., Miller, T. A., Lanzone, M. J., Cooper, J. L., et al. (2015). Use of multiple modes of flight subsidy by a soaring terrestrial bird, the golden eagle Aquila chrysaetos, when on migration. J. R. Soc. Interface 12:20150530. doi: 10.1098/rsif.2015.0530 Kerlinger, P., Bingman, V., and Able, K. P. (1985). Comparative flight behavior of migrating hawks studied with tracking radar during autumn in Central New York. Can. J. Zool. 63, 755–761. doi: 10.1139/z85-110 Klaassen, R. H., Schlaich, A. E., Bouten, W., and Koks, B. J. (2017). Migrating Montagu’s harriers frequently interrupt daily flights in both Europe and Africa. J. Avian Biol. 48, 180–190. doi: 10.1111/jav.01362 Kochert, M. N., Steenhof, K., McIntyre, C. L., and Craig, E. H. (2002). “Golden Eagle (Aquila chrysaetos),” in The Birds of North America, ed A. Poole (Ithaca, NY: Cornell Lab of Ornithology). Available online at: https://birdsna.org/ Species-Account/bna/species/684/articles/introduction Lanzone, M. J., Miller, T. A., Turk, P., Brandes, D., Halverson, C., Maisonneuve, C., et al. (2012). Flight responses by a migratory soaring raptor to changing meteorological conditions. Biol. Lett. 8, 710–713. doi: 10.1098/rsbl. 2012.0359 Leshem, Y., and Yom-Tov, Y. (1989). The use of thermals by soaring migrants. Ibis 183, 667–674. doi: 10.1111/j.1474-919X.1996.tb04768.x Luschi, P., Hays, G. C., Del Seppia, C., Marsh, R., and Papi, F. (1998). The navigational feats of green sea turtles migrating from Ascension Island investigated by satellite telemetry. Proc. R. Soc. Lond. Ser. B Biol. Sci. 265, 2279–2284. doi: 10.1098/rspb.1998.0571 Mallon, J. M., Bildstein, K. L., and Katzner, T. E. (2016). In-flight turbulence benefits soaring birds. Auk 133, 79–85. doi: 10.1642/AUK-15-114.1 Mateos-Rodríguez, M., and Liechti, F. (2012). How do diurnal long-distance migrants select flight altitude in relation to wind? Behav. Ecol. 23, 403–409. doi: 10.1093/beheco/arr204 McClintock, B. T., King, R., Thomas, L., Matthiopoulos, J., McConnell, B. J., and Morales, J. M. (2012). A general discrete-time modeling framework for animal movement using multistate random walks. Ecol. Monogr. 82, 335–349. doi: 10.1890/11-0326.1 McIntyre, C. L., Douglas, D. C., and Collopy, M. W. (2008). Movements of Golden Eagles (Aquila chrysaetos) from interior Alaska during their first year of independence. Auk 125, 214–224. doi: 10.1525/auk.2008.125. 1.214 Frontiers in Ecology and Evolution \| www.frontiersin.org 13 August 2019 \| Volume 7 \| Article 317 Eisaguirre et al. Dynamic-Parameter Models and Migratory Pace Vansteelant, W. M. G., Shamoun-Baranes, J., McLaren, J., van Diermen, J., and Bouten, W. (2017). Soaring across continents: decision-making of a soaring migrant under changing atmospheric conditions along an entire flyway. J. Avian Biol. 48, 887–896. doi: 10.1111/jav.01298 Vehtari, A., Gelman, A., and Gabry, J. (2016). loo: efficient leave-one-out crossvalidation and WAIC for Bayesian models. R package version 1.0.0. Vehtari, A., Gelman, A., and Gabry, J. (2017). Practical Bayesian model evaluation using leave-one-out cross-validation and WAIC. Stat. Comput. 27, 1413–1432. doi: 10.1007/s11222-016-9696-4 Watson, J. (2010). The Golden Eagle, 2nd Edn. New Haven, CT: Yale University Press. Shepard, E. L. C., Wilson, R. P., Rees, W. G., Grundy, E., Lambertucci, S. A., and Vosper, S. B. (2013). Energy landscapes shape animal movement ecology. Am. Nat. 182, 298–312. doi: 10.1086/671257 Spaar, R., and Bruderer, B. (1996). Soaring migration of Steppe Eagles Aquila nipalensis in Southern Israel: flight behaviour under various wind and thermal conditions. J. Avian Biol. 27, 289–301. doi: 10.2307/36 77260 Spaar, R., and Bruderer, B. (1997). Optimal flight behavior of soaring migrants: a case study of migrating steppe buzzards, Buteo buteo vulpinus. Behav. Ecol. 8, 288–297. doi: 10.1093/beheco/8.3.288 Stan Development Team (2016). RStan: The R Interface to Stan, Version 2.10.1. Stan Development Team. Strandberg, R., and Alerstam, T. (2007). The strategy of fly-and-forage migration, illustrated for the osprey (Pandion haliaetus). Behav. Ecol. Sociobiol. 61, 1865– 1875. doi: 10.1007/s00265-007-0426-y Turchin, P. (1998). Quantitative Analysis of Movement: Measuring and Modeling Population Redistribution in Animals and Plants. Sunderland, MA: Sinauer Associates. Vansteelant, W. M. G., Bouten, W., Klaassen, R. H. G., Koks, B. J., Schlaich, A. E., Diermen, J. V., et al. (2015). Regional and seasonal flight speeds of soaring migrants and the role of weather conditions at hourly and daily scales. J. Avian Biol. 46, 25–39. doi: 10.1111/jav. 00457 Frontiers in Ecology and Evolution \| www.frontiersin.org 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. Copyright © 2019 Eisaguirre, Auger-Méthé, Barger, Lewis, Booms and Breed. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 14 August 2019 \| Volume 7 \| Article 317
https://openalex.org/W3107552890	https://epjquantumtechnology.springeropen.com/track/pdf/10.1140/epjqt/s40507-021-00119-6	English	null	Deep reinforcement learning for universal quantum state preparation via dynamic pulse control	EPJ quantum technology	2,021	cc-by	10,227	© The Author(s) 2021. 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/. Abstract Accurate and eﬃcient preparation of quantum state is a core issue in building a quantum computer. In this paper, we investigate how to prepare a certain single- or two-qubit target state from arbitrary initial states in semiconductor double quantum dots with only a few discrete control pulses by leveraging the deep reinforcement learning. Our method is based on the training of the network over numerous preparing tasks. The results show that once the network is well trained, it works for any initial states in the continuous Hilbert space. Thus repeated training for new preparation tasks is avoided. Our scheme outperforms the traditional optimization approaches based on gradient with both the higher eﬃciency and the preparation quality in discrete control space. Moreover, we ﬁnd that the control trajectories designed by our scheme are robust against stochastic ﬂuctuations within certain thresholds, such as the charge and nuclear noises. Keywords: Quantum control; Quantum state preparation; Semiconductor double quantum dots; Deep reinforcement learning Keywords: Quantum control; Quantum state preparation; Semiconductor double quantum dots; Deep reinforcement learning Deep reinforcement learning for universal quantum state preparation via dynamic pulse control He1 , Rui Wang1, Shen-Shuang Nie1, Jing Wu1, Jia-Hui Zhang1 and Zhao-Ming Wang1* Run-Hong He1 , Rui Wang1, Shen-Shuang Nie1, Jing Wu1, Jia-Hui Zhang1 and Zhao-Ming Wang1* Run-Hong He1 , Rui Wang1, Shen-Shuang Nie1, Jing Wu1, Jia-Hui Zhang1 and Zhao-Min Correspondence: mingmoon78@126.com 1College of Physics and Optoelectronic Engineering, Ocean University of China, Qingdao, China He et al. EPJ Quantum Technology ( 2021) 8:29 https://doi.org/10.1140/epjqt/s40507-021-00119-6 He et al. EPJ Quantum Technology ( 2021) 8:29 https://doi.org/10.1140/epjqt/s40507-021-00119-6 He et al. EPJ Quantum Technology ( 2021) 8:29 https://doi.org/10.1140/epjqt/s40507-021-00119-6 Open Access Correspondence: mingmoon78@126.com 1College of Physics and Optoelectronic Engineering, Ocean University of China, Qingdao, China 1 Introduction Future quantum computers promise exponential speed-ups over their classical counter- parts in solving certain problems like search and simulation [1]. A wide variety of promis- ing modalities emerges in the race to realize the quantum computer, such as trapped ions [2, 3], photonic system [4–7], nitrogen-vacancy centers [8], nuclear magnetic resonance [9], superconducting circuits [10, 11] and semiconductor quantum dots [12–18]. Among these the semiconductor quantum dots is a powerful competitor for potential scalability, integrability with existing classical electronics and well-established fabrication technol- ogy. Spins of electrons trapped in quantum dots structure based on Coulomb eﬀect can serve as spin-qubits for quantum information [19]. Spin qubits can be encoded in many ways, such as spin-1/2, singlet-triplet (S-T0) and hybrid systems [20]. In particular, the spin S-T0 qubit in double quantum dots (DQDs) attracts much attention for the merit that it can be manipulated solely with electrical pulses [21–23]. It has been proved that several arbitrary single-qubit gates plus an entangling two-qubit gate are the prototypes of all other logic gates in quantum algorithm implemented on a ( 2021) 8:29 Page 2 of 16 He et al. EPJ Quantum Technology circuit-model quantum computer [1, 24]. In an authentic sense, the implementation of any single- and two-qubit gates can be reduced to the state preparation problems. Arbitrary manipulations of a single-qubit can be achieved by successive rotations around the x- and z-axes on the Bloch sphere. In the context of S-T0 single-qubit in semiconductor QDQs, the only tunable parameter J is the rotation rate around the z-axis, while the rotation rate h around the x-axis is diﬃcult to be changed [25]. Various schemes have been proposed to add proper pulses on J to control the qubits [26–28]. It is typically required to iteratively solve a set of nonlinear equations [29, 30] for analytically tailoring the control trajectory, so it is a computationally exorbitant and time- consuming task in practice. There are also several traditional optimal methods based on gradient that can be used to design the control trajectory, such as stochastic gradient de- scent (SGD) [31], chopped random-basis optimization (CRAB) [32, 33] and gradient as- cent pulse engineering (GRAPE) [34, 35]. However, the intensities of their pulses are nearly continuous, which may leave challenges to the experimental implementation. While the requirement of discrete pulses will inevitably reduce their performance [36]. 1 Introduction In addition, their eﬃciency is limited by their iterative nature, which makes the task of designing pulses a big burden especially when there exist a large number of states waiting to be processed. Except for these traditional routes, recently the deep reinforcement learning (RL) [37] shows a wide applicability in quantum control problems [38–51]. For example, how to drive a qubit from a ﬁxed initial state to another ﬁxed target state with discrete pulses by leveraging the deep RL [52] has been investigated [36]. Recently, the generation of arbitrary states from a speciﬁc state [53] in nitrogen-vacancy center has been realized with the aid of the deep RL. Then it is intriguing to check if the deep RL can be used to realize a contrary problem: preparing a certain target state from arbitrary initial states, i.e., universal state preparation (USP). In practical quantum computation, it is often required to reset an arbitrary state to a speciﬁc target state [54–56]. For example, the initial state of the system always needs to be set to the ground state when transferring a quantum state through a spin chain [54, 55]. In the realization of quantum Toﬀoli or Fredkin gate, the ancilla state must be preprepared to the standard state \|0⟩ in certain cases [57–59]. Generation of two-qubit entangled state is also required [1] in completing quantum information processing tasks, such as the teleportation [60, 61]. Note that the network typically requires being trained again once the preparing task changes [36, 46]. Thus, the designing task of control pulses could be an exhausting work when there are lots of diﬀerent states waiting to be prepared to a certain target state. In this paper, we investigate this USP problem with the deep RL in such a constrained driving parameters system. Beneﬁted from a more suﬃcient learning on numerous preparing tasks, we ﬁnd that the USP can be achieved with a single training of the network. Evaluation results show that our scheme outperforms the alternative optimization approaches both in terms of the eﬃciency of pulses designing and preparation quality in discrete control space. In addition, we ﬁnd that the average step of control trajectories designed by our USP algorithm is obviously less than that of alternatives. Moreover, we discuss the robustness of the control trajectories designed by our USP algorithm against various noises and explore the major source of errors in control accuracy. 1 Introduction We point out that by combining our scheme with Ref. [53], the driving between arbitrary states can be realized. ( 2021) 8:29 Page 3 of 16 He et al. EPJ Quantum Technology 2.1 Voltage-controlled single-qubit in semiconductor DQDs The eﬀective control Hamiltonian of a single-qubit encoded by S-T0 states in semicon- ductor DQDs can be written as [62–65], H(t) = J(t)σz + hσx. (1) (1) H(t) = J(t)σz + hσx. It is written under the computational basis states: the spin singlet state \|0⟩= \|S⟩= (\| ↑↓⟩– \| ↓↑⟩)/ √ 2 and the spin triplet state \|1⟩= \|T0⟩= (\| ↑↓⟩+ \| ↓↑⟩)/ √ 2. Here the ar- rows indicate the spin projections of the electron in the left and right dots, respectively. σz and σx are the Pauli matrices. h accounts for the Zeeman energy spacing of two spins. Considering h is diﬃcult to be changed experimentally [20], here we assume it is a con- stant h = 1 and set it to be the unit of pulse intensity. We also take the reduced Planck constant ℏ= 1 and the 1/ℏas the time-scale throughout. Physically the exchange coupling J(t) is tunable and non-negative [20]. In addition, if the J(t) is limited to a ﬁnite range, so that not to destroy the charge conﬁguration of the DQDs, the leakage of population to the non-computational space will be suppressed and we can study the evolution of the system safely within the Hilbert space spanned by the two bases [29, 30, 38]. Arbitrary single-qubit states can be written as Arbitrary single-qubit states can be written as \|s⟩= cos θ 2\|0⟩+ eiϕ sin θ 2\|1⟩, \|s⟩= cos θ 2\|0⟩+ eiϕ sin θ 2\|1⟩, (2) (2) where θ and ϕ are real numbers that deﬁne points on the Bloch sphere. For an initial state \|sini⟩on the Bloch sphere, any target state \|star⟩can be achieved by successive rotations around the x- and z-axes of the Bloch sphere. In the context of semiconductor DQDs, h and J(t) cause rotations around the x-axis and z-axis of the Bloch sphere, respectively. 2 Models and methods At ﬁrst, we present the models of electrically controlled S-T0 single- and two-qubit in semiconductor DQDs in Sects. 2.1 and 2.2, respectively. Then we present our USP algo- rithm in Sect. 2.3. 2.3 Universal state preparation via deep reinforcement learning Our target is to drive arbitrary initial states to a certain target state with discrete pulses. The control trajectory is discretized as a piece-wise constant function, i.e., the pulses have rectangular shapes [34]. While, the conclusion still holds if one take into account the ﬁnite rise time of the pulses that can be available with an arbitrary wave generator [23, 28, 63, 66, 68] in actual experiments: we need just alter the parameters of the pulses generated by our algorithm slightly as demonstrated in [26] and [29]. So, it is a reasonable simpliﬁcation to perform the optimization with ideal, zero rise time pulses. The strategy used here is to generate this control trajectory with the deep Q network algorithm (DQN) [69, 70], which is an important member of deep RL. The details of the DQN are described in Appendix. Here we just refer it as a neural network, i.e., the Main Net θ. Our scheme of obtaining a competent Main Net goes as follows: Firstly, a database com- prised of numerous potential initial quantum states is divided randomly into the training set, the validation set and the test set. The states in the training set will be used to train the Main Net in turn. The validation set will be utilized to estimate the generalization error of the Main Net during the training process. The test set will be employed to evaluate the Main Net’s ﬁnal performance after training. Secondly, the random-initialized Main Net is initially fed with a sampled initial state s from the training set at step k = 1 and then out- puts the predicted “best action” ak ( i.e., the pulse intensity J(t)). According to the current state s and the action ak, calculate the next state \|s′⟩= exp(–iH(ak)dt)\|s⟩and the corre- sponding ﬁdelity F = \|⟨star\|s′⟩\|2. The ﬁdelity F indicates how close the next state is to the target state. Then the next state s′ is fed to the Main Net as the new current state with the step k ←k +1. The reward will envelopes the ﬁdelity r = r(F) and be used to train the Main Net. Then repeat the above operations until the episode terminates when k reaches the maximum step or the ﬁdelity excesses a certain satisfactory threshold. Correspondingly, the control trajectory is constructed by these predicted actions orderly. 2.3 Universal state preparation via deep reinforcement learning After more than enough episodes of training over diﬀerent preparing tasks, the Main Net learns to assign an action-value (also named Q-value) to each state and action pair gradually according to the correspondence between them and the target state. With accurate Q-values, it is easily to determinate which action should be chosen in a given state. So that the Main Net can match every potential state with a reasonable action towards the target state. Finally, the well-trained Main Net can be used to tailor the appropriate control trajectories for these initial states databased in the test set and even all other states in the continuous Hilbert space. The overview of this training and pulses designing process is pictured in Fig. 1. And a full description of the training process is given in Algorithm 1. 2.2 Capacitively coupled S-T0 qubits in semiconductor DQDs Operations on two entangled qubits are often required in quantum information process- ing. In semiconductor DQDs, interqubit operations can be performed on two adjacent and capacitively coupled S-T0 qubits. In the basis of {\|SS⟩,\|ST0⟩,\|T0S⟩,\|T0T0⟩}, the Hamilto- nian can be written as [21, 23, 28, 63, 66, 67], H2–qubit = ℏ 2 ⎛ ⎜⎜⎜⎝ J1 + J2 h2 h1 0 h2 J1 – J2 0 h1 h1 0 J2 – J1 h2 0 h1 h2 –J1 – J2 + 2J12 ⎞ ⎟⎟⎟⎠, (3) (3) where hi and Ji are the Zeeman energy spacing and exchange coupling of the ith qubit respectively. J12 ∝J1J2 refers to the strength of Coulomb coupling between two qubits. Ji > 0 is required to maintain the interqubit coupling all the time. For simplicity, we take h1 = h2 = 1 and J12 = J1J2/2 here. where hi and Ji are the Zeeman energy spacing and exchange coupling of the ith qubit respectively. J12 ∝J1J2 refers to the strength of Coulomb coupling between two qubits. Ji > 0 is required to maintain the interqubit coupling all the time. For simplicity, we take h1 = h2 = 1 and J12 = J1J2/2 here. ( 2021) 8:29 Page 4 of 16 He et al. EPJ Quantum Technology Algorithm 1 The pseudocode for training the USP algorithm Algorithm 1 The pseudocode for training the USP algorithm Initialize the Experience Memory D to empty. Randomly initialize the Main-network θ. Initialize the Target-network θ– by: θ– ←θ. Set the ϵ = 0. for episode = 0, episodemax do Initialize the state s = sini according to the training point selected randomly from the training set. while True do With probability 1 – ϵ select a random action ai, otherwise ai = argmaxa Q(s,a;θ). Set the ϵ = ϵ + δϵ, except ϵ = ϵmax. Execute ai and observe the reward r, and the next state s′. Store experience unit = (s,ai,r,s′) in D. Select batch size Nbs of experiences units randomly from D. Update θ by minimizing the Loss function. Every C steps, set θ– ←θ. break if F > Fthreshold or step ≥T/dt. end while end for Algorithm 1 The pseudocode for training the USP algorithm Initialize the Experience Memory D to empty. Randomly initialize the Main-network θ. Initialize the Target-network θ– by: θ– ←θ. Set the ϵ = 0. for episode = 0, episodemax do Initialize the state s = sini according to the training point selected randomly from the training set. while True do With probability 1 – ϵ select a random action ai, otherwise ai = argmaxa Q(s,a;θ). Set the ϵ = ϵ + δϵ, except ϵ = ϵmax. Execute ai and observe the reward r, and the next state s′. Store experience unit = (s,ai,r,s′) in D. Select batch size Nbs of experiences units randomly from D. Update θ by minimizing the Loss function. Every C steps, set θ– ←θ. break if F > Fthreshold or step ≥T/dt. end while end for 3 Results In this section, we compare and contrast the performance of our scheme with two so- phisticated optimization approaches based on gradient for the USP problem. As demon- stration, we consider the preparation of a single-qubit state \|0⟩and a two-qubit Bell state (\|00⟩+ \|11⟩)/ √ 2. We stress that our USP scheme is applicable to any other target states as long as it is trained speciﬁcally. ( 2021) 8:29 Page 5 of 16 He et al. EPJ Quantum Technology Figure 1 Overview of the USP algorithm to learn control trajectory designing. The details of this algorithm is described in the main text of Sect. 2.3 and in the pseudocode Algorithm 1 Figure 1 Overview of the USP algorithm to learn control trajectory designing. The details of this algorithm is described in the main text of Sect. 2.3 and in the pseudocode Algorithm 1 Figure 1 Overview of the USP algorithm to learn control trajectory designing. The details of this algorithm is described in the main text of Sect. 2.3 and in the pseudocode Algorithm 1 Figure 1 Overview of the USP algorithm to learn control trajectory designing. The details of this algorithm is described in the main text of Sect. 2.3 and in the pseudocode Algorithm 1 3.1 Universal single-qubit state preparation Now we consider the preparation of the single-qubit state \|0⟩by using our USP algorithm. Considering the challenges to implement pulses with continuous intensity, our scheme takes only several discrete allowed actions on J(t): 0, 1, 2 or 3 with duration dt = π/10. We stress that these settings are made experimentally and can be further tailored as required. The maximum total operation time is limited to be 2π, which is uniformly discretized into 20 slices. The Main Net consists of two hidden layers with 32 neurons each. The reward function should be set to allow a growth in itself as the ﬁdelity increases, thus the Main Net can be inspirited to pursue a higher ﬁdelity. In practice, we ﬁnd that the function r = F works well. For training the Main Net and evaluating the performance of our algorithm, we sample 128 points on the Bloch sphere uniformly with respective to the θ and the ϕ as the initial states. Both the training and validation sets contain 32 points, while the test set ( 2021) 8:29 Page 6 of 16 He et al. EPJ Quantum Technology Table 1 List of hyperparameters for USP Parameters\Target state \|0⟩ Bell state Allowed actions a (J(t)) 0, 1, 2, 3 a Size of the training set 32 256 Size of the validation set 32 256 Size of the test set 64 6400 Batch size Nbs 32 32 Memory size M 20,000 40,000 Learning rate α 0.01 0.0001 Replace period C 200 200 Reward discount factor γ 0.9 0.9 Number of hidden layers 2 3 Neurons per hidden layer 32/32 256/256/128 Activation function Relu Relu ϵ-greedy increment δϵ 0.001 0.0001 Maximal ϵ in training ϵmax 0.95 0.95 ϵ in validation and testing 1 1 Fthreshold per episode 0.999 0.999 episodemax for training 33 731 Total time T 2π 20π Action duration dt π/10 π/2 Maximum steps per episode 20 40 a The allowed actions of two-qubit operations satisfy {(J1,J2)\|J1,J2 ∈{1,2,3,4,5}}. a The allowed actions of two-qubit operations satisfy {(J1,J2)\|J1,J2 ∈{1,2,3,4,5}}. consists of the remaining 64 points. The details of all hyperparameters for this algorithm has been listed in Table 1. As shown in Fig. 2(a), after about 33 episodes of training, the average ﬁdelity and total reward over the validation set have no obvious increase as the episode grows up, which implies the Main Net converges and can be used to implement the USP task. 3.1 Universal single-qubit state preparation To evaluate the performance of our algorithm, we compare it with two alternatives: the GRAPE and the CRAB. Considering that the eﬃciency of an algorithm is also an important metric when facing a large number of diﬀerent preparation tasks, we plot their preparation ﬁdelities of state \|0⟩versus the corresponding runtime of designing the control trajecto- ries in Fig. 2(b). The average ﬁdelities ¯F = 0.9968, 0.9721, 0.9655 and the average pulses designing time ¯t = 0.0120, 0.0268, 0.7504 with USP, GRAPE and CRAB, respectively. The ﬁdelities of the three algorithms are the maximums that can be achieved within the maxi- mum step. To satisfy the limitation of discrete pulses, for the GRAPE and the CRAB, their continuous control strengths are discretized into the nearest allowed actions when the designing process is completed [36]. Although the two traditional algorithms can achieve high average ﬁdelities after convergence with continuous control pulses, ¯F = 0.9997 for GRAPE and ¯F = 0.9995 for CRAB, they do not perform well in discrete control space. Figure 2(b) shows that our USP algorithm outperforms the alternative optimization ap- proaches both in terms of preparation quality and pulses designing eﬃciency in discrete control space. Clearly, CRAB algorithm performs the worst, and GRAPE algorithm is in the middle. The average steps to achieve the maximum ﬁdelities are 12.297, 14.109, 13.375 with USP, GRAPE and CRAB, respectively. A trajectory with fewer steps required for a given state preparation task corresponds to a faster control scheme in experiment. Over- all, the control trajectories generated by our USP algorithm are better than that of the alternatives. To show the control trajectory designed by our USP algorithm visually, as an example we plot one in Fig. 3(a), where the position of the initial state on the Bloch sphere is θ = 2π/7, ( 2021) 8:29 Page 7 of 16 He et al. EPJ Quantum Technology Figure 2 (a) The average ﬁdelity and total reward over the validation set as functions of the number of episodes in the training process for the single-qubit \|0⟩USP. (b) The distribution of state preparation ﬁdelities F versus pulses designing time for preparing single-qubit \|0⟩over 128 sampled tasks in the test set with diﬀerent optimization algorithms. 3.1 Universal single-qubit state preparation The average ﬁdelities F = 0.9968, 0.9721, 0.9655 and the average pulses designing time t = 0.0120, 0.0268, 0.7504 with USP, GRAPE and CRAB, respectively Figure 2 (a) The average ﬁdelity and total reward over the validation set as functions of the number of episodes in the training process for the single-qubit \|0⟩USP. (b) The distribution of state preparation ﬁdelities F versus pulses designing time for preparing single-qubit \|0⟩over 128 sampled tasks in the test set with diﬀerent optimization algorithms. The average ﬁdelities F = 0.9968, 0.9721, 0.9655 and the average pulses designing time t = 0.0120, 0.0268, 0.7504 with USP, GRAPE and CRAB, respectively ϕ = 3π/7 and the target state is \|0⟩. It shows that the USP algorithm takes only 6 steps to complete this task. The reason is that the DQN algorithm favors the policy with fewer steps due to the discounted reward (See the details of the DQN described in Appendix). In Fig. 3(b), we plot the corresponding motion trail of the quantum state on the Bloch sphere during operations. It shows that the ﬁnal quantum state reaches a position that is very close to the target state \|0⟩on the Bloch sphere and the ﬁnal ﬁdelity F = 0.9999. 3.2 Universal two coupled S-T0 qubits state preparation Now we consider the preparation of the Bell state (\|00⟩+ \|11⟩)/ √ 2 [1] from arbitrary initial states. The allowed pulse strengths on each qubit are deﬁned as {(J1,J2)\|J1,J2 ∈ {1,2,3,4,5}}. The reward function is set to be r = F. The architecture of the Main Net employed in this task is diﬀerent from the one used for the manipulation of single-qubit and the detailed hyper-parameters are captured in Table 1. The database used to train and to test the Main Net contains 6912 points that are deﬁned as {[a1,a2,a3,a4]T}. aj = bcj refers to the probability amplitude corresponding to the jth basis state. b ∈{1,i,–1,–i}. cjs ( 2021) 8:29 Page 8 of 16 He et al. EPJ Quantum Technology Figure 3 (a) Control trajectory designed by our USP algorithm. The task is to reset the point θ = 2/7π, ϕ = 3/7π on the Bloch sphere to the target state \|0⟩. The pulses only take discrete values 0, 2 and 3. This reset task is completed at the sixth step. (b) The corresponding motion trail for the reset task on the Bloch sphere with the ﬁnal ﬁdelity F = 0.9999 Figure 3 (a) Control trajectory designed by our USP algorithm. The task is to reset the point θ = 2/7π, ϕ = 3/7π on the Bloch sphere to the target state \|0⟩. The pulses only take discrete values 0, 2 and 3. This reset task is completed at the sixth step. (b) The corresponding motion trail for the reset task on the Bloch sphere with the ﬁnal ﬁdelity F = 0.9999 together deﬁne points on a four-dimensional unit hypersphere, together deﬁne points on a four-dimensional unit hypersphere, ⎧ ⎪⎪⎪⎪⎪⎨ ⎪⎪⎪⎪⎪⎩ c1 = cosθ1, c2 = sinθ1 cosθ2, c3 = sinθ1 sinθ2 cosθ3, c4 = sinθ1 sinθ2 sinθ3, (4) (4) where θi ∈{π/8,π/4,3π/8}. The normalization condition is satisﬁed for each quantum state represented by these points. The database is divided randomly into the training set, the validation set and the test set with 256, 256 and 6400 points, respectively. As depicted in Fig. 4(a), the Main Net converges after about 700 episodes of training. With 731 episodes of training, the average ﬁdelity of the Bell state preparation over all the test points ¯F = 0.9695. The maximum total operation time is taken as 20π and be discretized into 40 slices with pulse duration dt = π/2. In Fig. 4(b), we plot the distribution of the ﬁdelities of the test points under control trajectories designed by our USP scheme in this two-qubit preparing ( 2021) 8:29 Page 9 of 16 He et al. EPJ Quantum Technology Figure 4 (a) The average ﬁdelity and total reward over the validation set as functions of the number of training episodes for two-qubit Bell state USP. (b) The ﬁdelities distribution of 6400 sampled preparation tasks in the test set for the two-qubit Bell state USP with ¯F = 0.9695 Figure 4 (a) The average ﬁdelity and total reward over the validation set as functions of the number of training episodes for two-qubit Bell state USP. (b) The ﬁdelities distribution of 6400 sampled preparation task in the test set for the two-qubit Bell state USP with ¯F = 0.9695 task. The average pulses designing time ¯t = 0.0477 and the average step to complete the preparation tasks is 24.014. It shows that although the ﬁdelities are distributed unevenly between the interval [0.91,1], the overall performance is good. 3.3 USP in noisy environments In the preceding section, we have studied the USP problem without considering the sur- rounding environment. However the qubits will suﬀer from a variety of ﬂuctuations in a practical experiment, which prevents the accessibility of high precision control over the system. There exist works studied the corrected gate operations that employ the additional pulses to counteract the impact of various noises, such as the SUPCODE [26, 29]. However they treat diﬀerent noises equally resulting the designed control trajectories are too long to implement in actual experiment (about 300π of rotation for a single quantum gate). Thus it is worth exploring which noise will lead to the most serious threat to the control accuracy. Then designing the compensating pulses to shorten the total control trajectory using the SUPCODE as well as to improve the physical platform accordingly. Next we will study the performance of the control trajectories designed by our USP al- gorithm under two main noises leading to stochastic errors in the system Hamiltonian: the charge noise and nuclear noise. Considering that they vary on a typical time scale He et al. EPJ Quantum Technology ( 2021) 8:29 ( 2021) 8:29 Page 10 of 16 He et al. EPJ Quantum Technology (∼100 μs) much longer than a gate duration (∼10 ns), we take them constants during the preparation task. We point out that these noises are integrated into the system’s evolution after the control trajectories have been designed by our Main Net, which is trained on a clean model. This is a reasonable assumption since normally the environment is unpre- dictable. The charge noise stems from the imperfection of the external voltage ﬁeld, while the nuclear noise comes from the uncontrolled hyperﬁne coupling with spinful nuclei of the host material [63, 71, 72]. They can be represented by an additional term δσz (or δσx) in the Hamiltonian (1) for the single-qubit case or by additional terms δiσz (or δiσx) in the Hamiltonian (3) for the two-qubit case. Where i ∈{1,2} indicate the corresponding qubit and δ (δi) are the amplitudes of the noises. In addition, for the two-qubit Bell state preparation, we assume that the amplitudes of the noises on the two qubits are identical. Average ﬁdelities of the target states \|0⟩and Bell state preparation with control trajecto- ries generated by our USP over all test points versus amplitudes of two noises are plotted in Fig. 5(a) and (b), respectively. 3.3 USP in noisy environments It can be seen from Fig. 5, the average ﬁdelities do not change signiﬁcantly and the control trajectories exhibit robustness against considered imperfec- tions within certain thresholds. We also ﬁnd that in the analyzed parameter windows the F in nuclear noise are always higher than that in charge noise with the same amplitudes for Figure 5 Average ﬁdelities of the target state (a) \|0⟩and (b) Bell state preparation with USP over all test points versus amplitudes of charge and nuclear noises Figure 5 Average ﬁdelities of the target state (a) \|0⟩and (b) Bell state preparation with USP over all test points versus amplitudes of charge and nuclear noises ( 2021) 8:29 Page 11 of 16 He et al. EPJ Quantum Technology both single- and two-qubit cases. It reveals that the charge noise leaves the most impact to the preparation tasks. A meaningful point worth stating is that the best average ﬁdelity can even be obtained in non-zero nuclear noise from Fig. 5(b). That is to say, certain noises can be helpful to boost the ﬁdelity due to subtle adjustments on parameters. A possible explanation may be the limitation of the discrete value in our calculation. We believe that there is still a room for the achievement of better performance by employing more allowed actions and more deliberate Zeeman energy spacing, just as what these noises do. Of course, more suﬃcient training on Main Net is also helpful for the enhancement of the ﬁdelity. Given the limitations of quantum computing hardwares presently accessible, we simu- late quantum computing on a classical computer and generate data to train the network. Our algorithm is implemented with PYTHON 3.7.9, TensorFlow 2.6.0 and QuTip 4.6.2 and have been run on an 4-core 1.80 GHz CPU with 8 GB memory. Details of the run- ning environment of the algorithm can be found in Availability of data and materials. The runtime for the training process of USP algorithms are about tens of seconds in the single-qubit case and about an hour in the two-qubit case. 4 Conclusions Precise and eﬃcient quantum state preparation is crucial for quantum information pro- cessing. In this paper, we proposed an eﬃcient scheme to generate appropriate control trajectories to reset arbitrary single- or two-qubit states to a certain target state with the aid of deep RL. Our scheme has the advantage that once the network is well trained, it works for arbitrary initial states and does not require training again. Taking the control of spin S-T0 qubits in semiconductor DQDs as an example, the evaluation results show that our scheme outperforms traditional optimization approaches with both preparation quality and pulses designing eﬃciency. In addition, the average step required to complete the preparation tasks of our USP algorithm is obviously less than that of the alternatives, which implies faster control schemes in experiment. Moreover, we found that the control trajectories designed by our scheme exhibit robustness against two main noises within certain thresholds and discovered the charge noise leaves the most impact to the con- trol precision. Although we only considered the single and two-qubit state preparation in semiconductor DQDs, this scheme can be extended to a wide variety of quantum control problems. A.1 Deep reinforcement learning and deep Q network A.1 Deep reinforcement learning and deep Q network In this section, we will introduce the deep RL and DQN algorithm, which underlie our USP scheme. In this section, we will introduce the deep RL and DQN algorithm, which underlie our USP scheme. The deep RL combines the deep learning algorithm that is good at nonlinear ﬁtting and the RL algorithm that is expert in dynamic programming problems [37, 52, 73]. In RL, an Agent is generally used to represent an object with decision-making and action ca- pability, such as a robot. We consider a Markov decision process in which the next state depends only on the current state as well as the action performed by the Agent and has no relation with the past states [52]. In the interaction between the Agent and the Envi- ronment, the current state s of the Environment will be changed to another next state s′, ( 2021) 8:29 Page 12 of 16 He et al. EPJ Quantum Technology after the Agent selecting and performing an action ai chose from the set of allowed actions a = {a1,a2,...,an} at time t. In return, the Environment also gives a feedback, or immedi- ate reward r to the Agent. A Policy π represents which action the Agent will be chose in a given state, i.e., ai = π(s). The process is deﬁned as an episode in which the Agent starts from an initial state until it completes the task or terminates in halfway. The total discounted reward R gained in an N-steps episode can be written as [52]: R = r1 + γ r2 + γ 2r3 ··· + γ N–1rN = N t=1 γ t–1rt, (5) (5) where γ is a discount factor within the interval [0,1], which indicates that the immedi- ate reward r discounts with the steps increasing. The goal of the Agent is to maximize R, because a greater R implies a better performance of the Agent. Because the discounted r, the Agent tends naturally to get a bigger reward as quickly as possible to ensure a consid- erable R. To determine which action should to be chose in a given state, we introduce the action-value function, which is also named Q-value [74]: Qπ(s,ai) = E[rt + γ rt+1 + ···\|s,ai] = E rt + γ Qπ s′,a′ \|s,ai . A.1 Deep reinforcement learning and deep Q network (6) (6) The Q-value indicates the expectation of R, which the Agent will get after it executing an action ai in a given state s under the policy π, and this value can be obtained iteratively according to the Q-values of the next state. Because there are multiple allowed actions can be chosen in each state, and diﬀerent actions will lead to diﬀerent next states, it is a time- consuming task to calculate Q-values in a multi-step process. To reduce the overhead, there are various algorithms used to calculate approximations of that expectation, such as Q-learning [74] and SARSA [52]. In Q-learning, the current Q(s,ai) value is obtained by the Q-value of the next state’s “best action” [74]: (7) (7) Q(s,ai) ←Q(s,ai) + α[rt + γ max a′ Q(s′,a′) – Q(s,ai)], (7) where α is the learning rate, which aﬀects the convergence of this function. The part of Qtarget(s′,a′) = rt + γ maxa′ Q(s′,a′) is called the Qtarget value. All the Q-values of diﬀerent states and actions can be recorded in a so-called Q-Table. With a precise Q-Table, it is easily to identify which action should be chose in a given state. However, on the one hand, we need the best action to calculate iteratively the Q-value; on the other hand, we must know all the Q-values to determine which action is the best. To solve this dilemma of “ex- ploitation” and “exploration”, we adopt the ϵ-greedy strategy in choosing action to execute, i.e., choose the action corresponding to the current maximum Q-value with a probability of ϵ to calculate Q-value eﬃciently, or choose an action randomly with a probability of 1 – ϵ to expand the range of consideration. At the beginning, since it is not known that which action is the best one in a certain state, the ϵ is set to be 0 to explore as many states and actions as possible. When suﬃcient states and actions are explored, that parameter gradually increases with the amplitude of δϵ until to ϵmax, which is slightly smaller than 1, to calculate the Q-values eﬃciently. where α is the learning rate, which aﬀects the convergence of this function. The part of Qtarget(s′,a′) = rt + γ maxa′ Q(s′,a′) is called the Qtarget value. All the Q-values of diﬀerent states and actions can be recorded in a so-called Q-Table. A.1 Deep reinforcement learning and deep Q network With a precise Q-Table, it is easily to identify which action should be chose in a given state. However, on the one hand, we need the best action to calculate iteratively the Q-value; on the other hand, we must know all the Q-values to determine which action is the best. To solve this dilemma of “ex- ploitation” and “exploration”, we adopt the ϵ-greedy strategy in choosing action to execute, i.e., choose the action corresponding to the current maximum Q-value with a probability of ϵ to calculate Q-value eﬃciently, or choose an action randomly with a probability of 1 – ϵ to expand the range of consideration. At the beginning, since it is not known that which action is the best one in a certain state, the ϵ is set to be 0 to explore as many states and actions as possible. When suﬃcient states and actions are explored, that parameter gradually increases with the amplitude of δϵ until to ϵmax, which is slightly smaller than 1, to calculate the Q-values eﬃciently. Page 13 of 16 ( 2021) 8:29 He et al. EPJ Quantum Technology For an Environment with a large number or even an inﬁnite number of states, the Q- Table would be prohibitively large. To solve this “dimensional disaster”, we can substitute this table with a multi-layer neural network. After learning, the network will be capable to match a suited Q-value to each action after be fed with a certain state. The deep Q net- work algorithm (DQN) [69, 70] are based on the Equation (7). A network, the Main Net θ, is used to predict the term Q(s,ai), and another network, the Target Net θ– is used to predict the term maxa′ Q(s′,a′) in Equation (7) respectively. In order to ensure the abil- ity of generalization, the data used to train the Main Net must meet the assumption of independent and identically distributed, i.e. each sample of the dataset is independent of another while the training and test set are identically distributed. So we adopt the ex- perience memory replay strategy [70]: the Agent could get an experience unit (s,a,r,s′) at each step. After numerous steps, the Agent will collect a lot of such units that can be stored in an Experience Memory D with capacity of Memory size M. Authors’ contributions d d Z-MW and R-HH conceived the project, R-HH carried out the numerical simulations and prepared the ﬁrst version of the manuscript. All authors participated in the discussions and approved the ﬁnal manuscript. Abbreviations DQD, double quantum dot; SGD, stochastic gradient descent; GRAPE, gradient ascent pulse engineering; CRAB, chopped random-basis optimization; RL, reinforcement learning; USP, universal state preparation; SUPCODE, soft uniaxial positive control for orthogonal drift error; DQN, deep Q network; SARSA, State action reward state action; MBGD, mini-batch grade descent. Availability of data and materials The code, running environment of algorithm and all data supporting the conclusions of this article are available from the corresponding author on reasonable request or on Gitee repository under MIT License (https://gitee.com/herunhong/DL for USP). Competing interests Th h d l h p g The authors declare that they have no competing interests. Funding Funding This work was supported by the Shandong Provincial Natural Science Foundation (Grant. Nos. ZR2021LLZ004 and ZR2014AM023) and the Natural Science Foundation of China (Grant No. 11475160). A.1 Deep reinforcement learning and deep Q network In the process of training, the Agent randomly samples batch size Nbs of experience units from the Experi- ence Memory to train the Main Net at each time step. Notice that to ensure the stability of the algorithm only the Main Net is trained in every time step by minimizing the Loss function: Loss = 1 Nbs Nbs i=1 r + γ max a′ Q s′,a′ i – Q(s,a)i 2 , (8) (8) where Nbs is the sample batch size through mini-batch gradient descent (MBGD) algo- rithm [37, 69, 70]. While the Target Net θ– is not updated in real time, instead, it copies the parameters from the Main Net θ every C steps. A schematic of this DQN algorithm is shown in Fig. 6. Figure 6 Schematic for the DQN algorithm. See the main text of the Appendix for details of the algorithm Figure 6 Schematic for the DQN algorithm. See the main text of the Appendix for details of the algorithm Page 14 of 16 ( 2021) 8:29 He et al. EPJ Quantum Technology Acknowledgements Acknowledgements We would like to thank Xin-Hong Han, Jing-Hao Sun and Chen Chen for useful discussions. Received: 27 May 2021 Accepted: 13 December 2021 Received: 27 May 2021 Accepted: 13 December 2021 Received: 27 May 2021 Accepted: 13 December 2021 References Zajac DM, Sigillito AJ, Russ M, Borjans F, Taylor JM, Burkard G, Petta JR. Resonantly driven cnot gate for electron spins. Science. 2018;359(6374):439–42. 13. Huang W, Yang C, Chan K, Tanttu T, Hensen B, Leon R, Fogarty M, Hwang J, Hudson F, Itoh KM et al. Fidelity benchmarks for two-qubit gates in silicon. Nature. 2019;569(7757):532–6. 13. Huang W, Yang C, Chan K, Tanttu T, Hensen B, Leon R, Fogarty M, Hwang J, Hudson F, Itoh KM et al. Fidelity benchmarks for two qubit gates in silicon Nature 2019 569(7757) 532 6 13. Huang W, Yang C, Chan K, Tanttu T, Hensen B, Leon R, Fogarty M, Hwang J, Hudson F benchmarks for two-qubit gates in silicon. Nature. 2019;569(7757):532–6. 14. Watson T, Philips S, Kawakami E, Ward D, Scarlino P, Veldhorst M, Savage D, Lagally M, Friesen M, Coppersmith S et al. A programmable two qubit quantum processor in silicon Nature 2018;555(7698):633 7 14. Watson T, Philips S, Kawakami E, Ward D, Scarlino P, Veldhorst M, Savage D, Lagally M, Friesen M, Coppersmith S et al A programmable two-qubit quantum processor in silicon. Nature. 2018;555(7698):633–7. , p , , , , , g , g y , , pp A programmable two-qubit quantum processor in silicon. Nature. 2018;555(7698):633–7. 15. Jang W, Cho M-K, Kim J, Chung H, Umansky V, Kim D. Three individual two-axis control of singlet-triplet qubits in a i i d d 2020 Xi i 2009 13182 15. Jang W, Cho M-K, Kim J, Chung H, Umansky V, Kim D. Three individual two-axis c micromagnet integrated quantum dot array. 2020. arXiv preprint. 2009.13182. 15. Jang W, Cho M-K, Kim J, Chung H, Umansky V, Kim D. Three individual two-axis control of singlet-triplet qubits in a micromagnet integrated quantum dot array. 2020. arXiv preprint. 2009.13182. 16. Hanson R, Kouwenhoven LP, Petta JR, Tarucha S, Vandersypen LM. Spins in few-electron quantum dots. Rev Mod Phys. 2007;79(4):1217. 17. Eriksson MA, Friesen M, Coppersmith SN, Joynt R, Klein LJ, Slinker K, Tahan C, Mooney P, Chu J, Koester S. Spin-based quantum dot quantum computing in silicon. Quantum Inf Process. 2004;3(1–5):133–46. 18. Zwanenburg FA, Dzurak AS, Morello A, Simmons MY, Hollenberg LC, Klimeck G, Rogge S, Coppersmith SN, Eriksson MA. Silicon quantum electronics. Rev Mod Phys. 2013;85(3):961. Zwanenburg FA, Dzurak AS, Morello A, Simmons MY, Hollenberg L 18. Zwanenburg FA, Dzurak AS, Morello A, Simmons MY, Hollenberg MA. Silicon quantum electronics. Rev Mod Phys. References 1. Nielsen MA, Chuang I. Quantum computation and quantum information. American Association of Physics Teachers. 2002. 1. Nielsen MA, Chuang I. Quantum computation and quantum information. American Association of Physics Teachers. 2002. 1. Nielsen MA, Chuang I. Quantum computation and quantum information. American Association of Physics Teach 2002. 2. Richerme P, Gong Z-X, Lee A, Senko C, Smith J, Foss-Feig M, Michalakis S, Gorshkov AV, Monroe C. Non-local 2. Richerme P, Gong Z-X, Lee A, Senko C, Smith J, Foss-Feig M, Michalakis S, Gorshkov AV, Monroe C. Non-local propagation of correlations in quantum systems with long-range interactions. Nature. 2014;511(7508):198–201. 2. Richerme P, Gong Z-X, Lee A, Senko C, Smith J, Foss-Feig M, Michalakis S, Gorshkov AV, Monroe C. Non-local propagation of correlations in quantum systems with long-range interactions. Nature. 2014;511(7508):198–2 g g propagation of correlations in quantum systems with long-range interactions. Nature. 2014;511(7508):198–201. 3. Casanova J, Mezzacapo A, McClean JR, Lamata L, Aspuru-Guzik A, Solano E, et al. From transistor to trapped-ion computers for quantum chemistry. Sci Rep. 2014. 4. Bellec M, Nikolopoulos GM, Tzortzakis S. Faithful communication Hamiltonian in photonic lattices. Opt Lett. 2012;37(21):4504–6. 5. Perez-Leija A, Keil R, Moya-Cessa H, Szameit A, Christodoulides DN. Perfect transfer of path-entangled photons in j x photonic lattices. Phys Rev A. 2013;87(2):022303. 5. Perez-Leija A, Keil R, Moya-Cessa H, Szameit A, Christodoulides DN. Perfect transfer of path-entangled photons in j x photonic lattices. Phys Rev A. 2013;87(2):022303. y 6. Peruzzo A, McClean J, Shadbolt P, Yung M-H, Zhou X-Q, Love PJ, Aspuru-Guzik A, O’brien JL. A variational eigenvalue solver on a photonic quantum processor. Nat Commun. 2014;5:4213. 6. Peruzzo A, McClean J, Shadbolt P, Yung M-H, Zhou X-Q, Love PJ, Aspuru-Guzik A, O’brien JL. A variational eigenvalue solver on a photonic quantum processor. Nat Commun. 2014;5:4213. 7. Chapman RJ, Santandrea M, Huang Z, Corrielli G, Crespi A, Yung M-H, Osellame R, Peruzzo A. Experimental perfect state transfer of an entangled photonic qubit. Nat Commun. 2016;7:11339. 8. Childress L, Hanson R. Diamond nv centers for quantum computing and quantum networks. Mater Res Soc Bull. 2013;38(2):134–8. 9. Vandersypen LM, Chuang IL. Nmr techniques for quantum control and computation. Rev Mod Phys. 2005;76(4) 10. Devoret MH, Schoelkopf RJ. Superconducting circuits for quantum information: an outlook. Science. 2013;339(6124):1169–74. 11. Wendin G. Quantum information processing with superconducting circuits: a review. Rep Prog Phys. 2017;80(10):106001. 12. Publisher’s Note Publisher s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. References 2013;85(3):961. MA. Silicon quantum electronics. Rev Mod Phys. 2013;85(3):961. 19. Loss D, DiVincenzo DP. Quantum computation with quantum dots. Phys Rev A. 1998;57(1):120. 19. Loss D, DiVincenzo DP. Quantum computation with quantum dots. Phys Rev A. 1998;57(1):120. 9. Loss D, DiVincenzo DP. Quantum computation with quantum do Page 15 of 16 ( 2021) 8:29 He et al. EPJ Quantum Technology 20. Zhang X, Li H-O, Cao G, Xiao M, Guo G-C, Guo G-P. Semiconductor quantum computation. Nat Sci Rev. 2019;6(1):32–54. 21. Taylor J, Engel H-A, Dür W, Yacoby A, Marcus C, Zoller P, Lukin M. Fault-tolerant architecture for quantum computation using electrically controlled semiconductor spins Nat Phys 2005;1(3):177–83 21. Taylor J, Engel H-A, Dür W, Yacoby A, Marcus C, Zoller P, Lukin M. Fault-tolerant architecture for quantum using electrically controlled semiconductor spins. Nat Phys. 2005;1(3):177–83. g y p y 22. Wu X, Ward DR, Prance J, Kim D, Gamble JK, Mohr R, Shi Z, Savage D, Lagally M, Friesen M et al. Two-axis control of a Wu X, Ward DR, Prance J, Kim D, Gamble JK, Mohr R, Shi Z, Savage D, 22. Wu X, Ward DR, Prance J, Kim D, Gamble JK, Mohr R, Shi Z, Savage D, Lagally M, Friesen M et al. Two-axis con singlet–triplet qubit with an integrated micromagnet. Proc Natl Acad Sci. 2014;111(33):11938–42. singlet–triplet qubit with an integrated micromagnet. Proc Natl Acad Sci. 2014;111(33):11938–42. g g g 23. Nichol JM, Orona LA, Harvey SP, Fallahi S, Gardner GC, Manfra MJ, Yacoby A. High double-quantum-dot spin qubits. npj Quantum Inf. 2017;3(1):1–5. 23. Nichol JM, Orona LA, Harvey SP, Fallahi S, Gardner GC, Manfra MJ, Yacoby A. High-ﬁdelity entangling gate for double quantum dot spin qubits npj Quantum Inf 2017;3(1):1 5 23. Nichol JM, Orona LA, Harvey SP, Fallahi S, Gardner GC, Manfra MJ, Yacoby A. High-ﬁdelity entangling gate fo double-quantum-dot spin qubits. npj Quantum Inf. 2017;3(1 24. Bishnoi B. Quantum-computation and applications. 2020. 25. Throckmorton RE, Zhang C, Yang X-C, Wang X, Barnes E, Sarma SD. Fast pulse sequences for dynamically correc gates in singlet triplet qubits Phys Rev B 2017;96(19):195424 25. Throckmorton RE, Zhang C, Yang X-C, Wang X, Barnes E, Sarma SD. Fast pulse sequenc gates in singlet-triplet qubits. Phys Rev B. 2017;96(19):195424. 25. Throckmorton RE, Zhang C, Yang X-C, Wang X, Barnes E, Sar gates in singlet-triplet qubits. Phys Rev B. 2017;96(19):195424. 26. References Palmieri AM, Kovlakov E, Bianchi F, Yudin D, Straupe S, Biamonte JD, Kulik S. Experimental neural network enhanced quantum tomography. npj Quantum Inf. 2020;6(1):1–5. 44. Palmieri AM, Kovlakov E, Bianchi F, Yudin D, Straupe S, Bia quantum tomography. npj Quantum Inf. 2020;6(1):1–5. y 45. Wang ZT, Ashida Y, Ueda M. Deep reinforcement learning control of quantum cartpoles. Phys Rev Lett. 2020;125(10). 46 Zheng A Zhou DL Deep reinforcement learning for quantum gate control Europhys Lett 2019;126(6):60002 y 45. Wang ZT, Ashida Y, Ueda M. Deep reinforcement learning control of quantum cartpoles. Phys Rev Lett. 2020;125(10). 46. Zheng A, Zhou DL. Deep reinforcement learning for quantum gate control. Europhys Lett. 2019;126(6):60002. 45. Wang ZT, Ashida Y, Ueda M. Deep reinforcement learning control of quantum cartpoles. Phys Rev Lett. 2020;125( 45. Wang ZT, Ashida Y, Ueda M. Deep reinforcement learning 45. Wang ZT, Ashida Y, Ueda M. Deep reinforcement learning control of quantum cartpoles. Phys Rev Lett. 2020;125(10) 46. Zheng A, Zhou DL. Deep reinforcement learning for quantum gate control. Europhys Lett. 2019;126(6):60002. 46. Zheng A, Zhou DL. Deep reinforcement learning for quantum gate control. Europhys Lett. 2019;126(6):6 47. Niu MY, Boixo S, Smelyanskiy V, Neven H. Universal quantum control through deep reinforcement learning. npj Quantum Inf. 2019;5(33). 48. Gratsea A, Metz F, Busch T. Universal and optimal coin sequences for high entanglement generation in 1d discrete time quantum walks. J Phys A, Math Theor. 2020. q y 49. Lin J, Lai ZY, Li X. Quantum adiabatic algorithm design using reinforcement learning. Phys Rev A. 2020;101:052327. https://doi.org/10.1103/PhysRevA.101.052327. https://doi.org/10.1103/PhysRevA.101.052327. 50. Ma H, Dong D, Ding SX, Chen C. Curriculum-based deep reinforcement learning for quantum control. 2021. 2012.15427. 50. Ma H, Dong D, Ding SX, Chen C. Curriculum-based deep reinforcement learning for quantum control. 2021. 2012.15427. 51. Bukov M, Day AGR, Sels D, Weinberg P, Polkovnikov A, Mehta P. Reinforcement learning in diﬀerent phases of quantum control Phys Rev X 2018;8:031086 https://doi org/10 1103/PhysRevX 8 031086 51. Bukov M, Day AGR, Sels D, Weinberg P, Polkovnikov A, Mehta P. Reinforcement learning in diﬀerent phases of quantum control. Phys Rev X. 2018;8:031086. https://doi.org/10.1103/PhysRevX.8.031086. q y p g y 52. Sutton RS, Barto AG. Reinforcement learning: an introduction. IEEE Trans Neural Netw. 199 52. Sutton RS, Barto AG. Reinforcement learning: an introduction. IEEE Trans Neural Netw. 1998;9(5):1054. 53. Haug T, Mok WK, You JB, Zhang W, Png CE, Kwek LC. References When does reinforcement learning stand out in quantum control? A comparative study on state preparation npj Quantum Inf 2019;5(1):1–7 36. Zhang X-M, Wei Z, Asad R, Yang X-C, Wang X. When does reinforcement learn comparative study on state preparation. npj Quantum Inf. 2019;5(1):1–7. comparative study on state preparation. npj Quantum Inf. 2019;5(1):1–7. 37. Goodfellow I, Bengio Y, Courville A. Deep learning. Cambridge: MIT Press; 2016. 7. Goodfellow I, Bengio Y, Courville A. Deep learning. Cambridge: M 38. Zhang X-M, Cui Z-W, Wang X, Yung M-H. Automatic spin-chain learning to explore the quantum speed limit. Phy Rev A. 2018;97(5):052333. 39. Yang X, Liu R, Li J, Peng X. Optimizing adiabatic quantum pathways via a learning algorithm. Phys Rev A. 2020;102(1):012614. 40. Lin J, Lai ZY, Li X. Quantum adiabatic algorithm design using reinforcement learning. Phys Rev A. 2020;101(5):052327. g g g g y 41. Bukov M. Reinforcement learning for autonomous preparation of Floquet-engineered states: inverting the quantum kapitza oscillator. Phys Rev B. 2018;98(22):224305. 41. Bukov M. Reinforcement learning for autonomous preparation of Floquet-engineered states: inverting the quantum kapitza oscillator. Phys Rev B. 2018;98(22):224305. p y 42. Bukov M, Day AG, Sels D, Weinberg P, Polkovnikov A, Mehta P. Reinforcement learning in diﬀerent phases of quan control. Phys Rev X. 2018;8(3):031086. y 42. Bukov M, Day AG, Sels D, Weinberg P, Polkovnikov A, Mehta P. Reinforcement learning in diﬀerent phases of quantum control. Phys Rev X. 2018;8(3):031086. y 43. Kong X, Zhou L, Li Z, Yang Z, Qiu B, Wu X, Shi F, Du J. Artiﬁcial intelligence enhanced two-dimensional nanoscale nuclear magnetic resonance spectroscopy. npj Quantum Inf. 2020;6(1):1–10. 43. Kong X, Zhou L, Li Z, Yang Z, Qiu B, Wu X, Shi F, Du J. Artiﬁcial intelligence enhanced nuclear magnetic resonance spectroscopy. npj Quantum Inf. 2020;6(1):1–10. 43. Kong X, Zhou L, Li Z, Yang Z, Qiu B, Wu X, Shi F, Du J. Artiﬁcial intelligence enhanced two-dimensional nanoscale l ti t j Q t I f 2020 6(1) 1 10 43. Kong X, Zhou L, Li Z, Yang Z, Qiu B, Wu X, Shi F, Du J. Artiﬁcial intelligence enhanced two nuclear magnetic resonance spectroscopy. npj Quantum Inf. 2020;6(1):1–10. 43. Kong X, Zhou L, Li Z, Yang Z, Qiu B, Wu X, Shi F, Du J. Artiﬁcial intelligence enhanced two-dimensional nanoscale nuclear magnetic resonance spectroscopy. npj Quantum Inf. 2020;6(1):1–10. 44. References Wang X, Bishop LS, Kestner J, Barnes E, Sun K, Sarma SD. Composite pulses for robust universal control of singlet–triplet qubits. Nat Commun. 2012;3(1):1–7. 26. Wang X, Bishop LS, Kestner J, Barnes E, Sun K, Sarma SD. Co singlet–triplet qubits. Nat Commun. 2012;3(1):1–7. 27. Kestner J, Wang X, Bishop LS, Barnes E, Sarma SD. Noise-resistant control for a spin qubit array. Phys Rev Lett. 2013;110(14):140502. 28. Wang X, Barnes E, Sarma SD. Improving the gate ﬁdelity of capacitively coupled spin qubits. npj Quantum Inf. 2015;1(1):1–7. 29. Wang X, Bishop LS, Barnes E, Kestner J, Sarma SD. Robust quantum gates for singlet-triplet spin qubits using composite pulses. Phys Rev A. 2014;89(2):022310. 29. Wang X, Bishop LS, Barnes E, Kestner J, Sarma SD. Robust quantum gates for singlet-triplet spin qubits using i l Ph R A 2014 89(2) 022310 composite pulses. Phys Rev A. 2014;89(2):022310. 30. Yang X-C, Yung M-H, Wang X. Neural-network-designed pulse sequences for robust control of singlet-triplet qu Phys Rev A. 2018;97(4):042324. 30. Yang X-C, Yung M-H, Wang X. Neural-network-designed pulse sequences for robust control of singlet-triplet qubits. Phys Rev A. 2018;97(4):042324. y 31. Ferrie C. Self-guided quantum tomography. Phys Rev Lett. 2014;113(19). 32. Doria P, Calarco T, Montangero S. Optimal control technique for many body quantum systems dynamics. Phys Rev Lett. 2010;106(19):237. 32. Doria P, Calarco T, Montangero S. Optimal control technique for many body quantum systems dynamics. Phys Re Lett. 2010;106(19):237. 33. Caneva T, Calarco T, Montangero S. Chopped random-basis quantum optimization. Phys Rev A. 2011;84(2):17864. 33. Caneva T, Calarco T, Montangero S. Chopped random-basis quantum optimization. Phys Rev A. 2011;84(2):17864 34. Khaneja N, Reiss T, Kehlet C, Schulte-Herbrüggen T, Glaser SJ. Optimal control of coupled spin dynamics: design nmr pulse sequences by gradient ascent algorithms – sciencedirect. J Magn Res. 2005;172(2):296–305. 35. Rowland B, Jones JA. Implementing quantum logic gates with gradient ascent pulse engineering: principles and practicalities. Philos Trans A Math Phys Eng. 2012;370(1976):4636–50. 35. Rowland B, Jones JA. Implementing quantum logic gates with gradient ascent pulse engineering: principles and l h l h h ( ) 5. Rowland B, Jones JA. Implementing quantum logic gates with g practicalities. Philos Trans A Math Phys Eng. 2012;370(1976):4636–5 y g 36. Zhang X-M, Wei Z, Asad R, Yang X-C, Wang X. When does reinforcement learning stand out in quantum control? A 36. Zhang X-M, Wei Z, Asad R, Yang X-C, Wang X. References Classifying global state preparation via deep reinforcement learning. Mach Learn Sci Technol. 2021;2(1):01. g 54. Wang Z-M, Sarandy MS, Wu L-A. Almost exact state transfer in a spin chain via pulse control. Phys Rev A. 2020;102:022601. https://doi.org/10.1103/PhysRevA.102.022601. g 54. Wang Z-M, Sarandy MS, Wu L-A. Almost exact state transfer in a spin chain via pulse control. Phys Rev A. 54. Wang Z-M, Sarandy MS, Wu L-A. Almost exact state transfer in a sp 2020;102:022601. https://doi.org/10.1103/PhysRevA.102.022601. 54. Wang Z-M, Sarandy MS, Wu L-A. Almost exact state transfer in g y 2020;102:022601. https://doi.org/10.1103/PhysRevA.102.02260 p g y 55. Wang Z-M, Ren F-H, Luo D-W, Yan Z-Y, Wu L-A. Almost-exact 55. Wang Z-M, Ren F-H, Luo D-W, Yan Z-Y, Wu L-A. Almost-exact state transfer by leakage-elimination-operator contro M k i i t Ph R A 2020 102 042406 htt //d i /10 1103/Ph R A 102 042406 55. Wang Z-M, Ren F-H, Luo D-W, Yan Z-Y, Wu L-A. Almost-exact state transfer by leakage-elimination-operator control in a non-Markovian environment. Phys Rev A. 2020;102:042406. https://doi.org/10.1103/PhysRevA.102.042406. g , , , , y g p a non-Markovian environment. Phys Rev A. 2020;102:042406. https://doi.org/10.1103/PhysRevA.102.042406. y p g y 56. Ren F-H, Wang Z-M, Wu L-A. Accelerated adiabatic quantum search algorithm via pulse control in a non-Markovian y y 56. Ren F-H, Wang Z-M, Wu L-A. Accelerated adiabatic quantum search algorithm via pulse control in a non-Markovian i Ph R A 2020 102 062603 h //d i /10 1103/Ph R A 102 062603 56. Ren F-H, Wang Z-M, Wu L-A. Accelerated adiabatic quantum search algorithm via pulse control in a non-Markov environment. Phys Rev A. 2020;102:062603. https://doi.org/10.1103/PhysRevA.102.062603. 56. Ren F H, Wang Z M, Wu L A. Accelerated adiabatic quantum search algorithm via pulse control in a non Markovi environment. Phys Rev A. 2020;102:062603. https://doi.org/10.1103/PhysRevA.102.062603. environment. Phys Rev A. 2020;102:062603. https://doi.org/10.1103/PhysRevA.102.062603. o DP. Two-bit gates are universal for quantum computation. Phys Re 58. Feynman RP. Simulating physics with computers. Int J Theor Phys. 1982;21(6/7). 59. Smolin JA, DiVincenzo DP. Five two-bit quantum gates are suﬃcient to implement the quantum Fredkin gate. Phys Rev A. 1996;53(4):2855. 59. Smolin JA, DiVincenzo DP. Five two-bit quantum gates are suﬃcient to implement the quantum Fredkin gate. Ph Rev A. 1996;53(4):2855. 60. Bennett CH, Brassard G, Crépeau C, Jozsa R, Peres A, Wootters WK. Teleporting an unknown quantum state via du classical and Einstein–Podolsky–Rosen channels. Phys Rev Lett. 1993;70(13):1895. References Page 16 of 16 ( 2021) 8:29 He et al. EPJ Quantum Technology 61. Bouwmeester D, Pan J-W, Mattle K, Eibl M, Weinfurter H, Zeilinger A. Experimental quantum teleportation. Nature. 1997;390(6660):575–9. 62. Malinowski FK, Martins F, Nissen PD, Barnes E, Cywi´nski Ł, Rudner MS, Fallahi S, Gardner GC, Manfra MJ, Marcus CM et 62. Malinowski FK, Martins F, Nissen PD, Barnes E, Cywi´nski Ł, Rudner MS, Fallahi S, Gardner GC, Manfra MJ, Marcus C al. Notch ﬁltering the nuclear environment of a spin qubit. Nat Nanotechnol. 2017;12(1):16–20. 62. Malinowski FK, Martins F, Nissen PD, Barnes E, Cywinski Ł, Rudner MS, Fallahi S, Gardner GC, Manfra MJ, Marcu al. Notch ﬁltering the nuclear environment of a spin qubit. Nat Nanotechnol. 2017;12(1):16–20. 63. Petta JR, Johnson AC, Taylor JM, Laird EA, Yacoby A, Lukin MD, Marcus CM, Hanson MP, Gossard AC. Coherent manipulation of coupled electron spins in semiconductor quantum dots. Science. 2005;309(5744):2180–4. 64. Bluhm H, Foletti S, Mahalu D, Umansky V, Yacoby A. Universal quantum control of two electron spin qubits via dynamic nuclear polarization. APS. 2009. 17–008. y 65. Maune BM, Borselli MG, Huang B, Ladd TD, Deelman PW, Holabird KS, Kiselev AA, Alvarado-Rodriguez I, Ross RS, Schmitz AE et al Coherent singlet triplet oscillations in a silicon based double quantum dot Nature 65. Maune BM, Borselli MG, Huang B, Ladd TD, Deelman PW, Holabird KS, Kiselev AA, Alvarado-Rodriguez I, Ross RS, Schmitz AE et al. Coherent singlet-triplet oscillations in a silicon-based double quantum dot. Nature. 2012;481(7381):344–7. 66. Shulman MD, Dial OE, Harvey SP, Bluhm H, Umansky V, Yacoby A. Demonstration of entanglement of electrostatically coupled singlet-triplet qubits. Science. 2012;336(6078):202–5. 67. Van Weperen I, Armstrong B, Laird E, Medford J, Marcus C, Hanson M, Gossard A. Charge-state conditional operation of a spin qubit. Phys Rev Lett. 2011;107(3):030506. 68. Krantz P, Kjaergaard M, Yan F, Orlando TP, Oliver WD. A quantum engineer’s guide to superconducting qubits. Appl Phys Rev. 2019;6(2):021318. y 69. Mnih V, Kavukcuoglu K, Silver D, Graves A, Antonoglou I, Wierstra D, Riedmiller M. Playing atari with deep reinforcement learning. 2013. arXiv preprint. 1312.5602. 70. Mnih V, Kavukcuoglu K, Silver D, Rusu AA, Veness J, Bellemare MG, Graves A, Riedmiller M, Fidjeland AK, Ost al. Human-level control through deep reinforcement learning. Nature. 2015;518(7540):529–33. 70. Mnih V, Kavukcuoglu K, Silver D, Rusu AA, Veness J, Bellemare MG, Graves A, Riedmiller M, Fidjeland AK, Ostrovski G et al. References Human-level control through deep reinforcement learning. Nature. 2015;518(7540):529–33. 71. Barnes E, Cywi´nski Ł, Sarma SD. Nonperturbative master equation solution of central spin dephasing dynamics. Phys Rev Lett. 2012;109(14):140403. 71. Barnes E, Cywi´nski Ł, Sarma SD. Nonperturbative master equation solution of central spin dephasing dynamics. Phy Rev Lett. 2012;109(14):140403. 72. Nguyen NT, Sarma SD. Impurity eﬀects on semiconductor quantum bits in coupled quantum dots. Phys Rev B. 2011;83(23):235322. 72. Nguyen NT, Sarma SD. Impurity eﬀects on semiconductor quantum bits in coupled quantum dots. Phys Rev B. 2011;83(23):235322. 73. Shalev-Shwartz S, Ben-David S. Understanding machine learning: from theory to algorithms. Cambridge: Cambridge University Press; 2014. 73. Shalev-Shwartz S, Ben-David S. Understanding machine learning: from theory to algorithms. Cambridge: Cambridge University Press; 2014. 74. Watkins CJ, Dayan P. Q-learning. Mach Learn. 1992;8(3–4):279–92. 74. Watkins CJ, Dayan P. Q-learning. Mach Learn. 1992;8(3–4):279–92.
https://openalex.org/W3032070729	https://pubs.rsc.org/en/content/articlepdf/2020/ra/d0ra01316a	English	null	A remarkable mixture of germanium with phosphorus and arsenic atoms making stable pentagonal hetero-prisms [M@Ge5E5]+, E = P, As and M = Fe, Ru, Os	RSC advances	2,020	cc-by	7,587	aDepartment of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium bLaboratory of Computational Chemistry and Modelling, Quy Nhon University, Quy Nhon, Vietnam cInstitute for Computational Science and Technology (ICST), Ho Chi Minh City, Vietnam dComputational Chemistry Research Group, Ton Duc Thang University, Ho Chi Minh City, Vietnam. E-mail: nguyenminhtam@tdtu.edu.vn eFaculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City, Vietnam † Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra01316a Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6: This article is licensed under a Creative Commons Attribution 3.0 U Hung Tan Pham, a Cam-Tu Dang Phan, b Minh Tho Nguyen c and Nguyen Minh Tam *de A pentagonal hetero-prismatic structural motif was found for singly transition metal doped M@Ge5E5 + clusters, where the transition metal atom is located at the centre of a (5/5) Ge5E5 prism in which Ge is mixed with either P or As atoms. Structural characterization indicates that each (5/5) Ge5E5 prism is established by joining of two Ge3E2 and Ge2E3 strings in a prismatic fashion rather than two Ge5 and E5 strings. Each string results from a remarkable mixture of Ge and E atoms and contains only one E–E connection due to the fact that Ge–E bonds are much stronger than E–E connections. From the donor– acceptor perspective, the Ge5E5 tube donates electrons to the M center, which behaves as an acceptor. NBO atomic charge and ELI_D analyses demonstrate such electrostatic interactions of the M dopant with a Ge5E5 + tube which likely induce thermodynamic stability for the resulting M@Ge5E5 + cluster. CMO analysis illustrates that the conventional 18 electron count is recovered in the M@Ge5E5 + cations. Received 11th February 2020 Accepted 12th May 2020 DOI: 10.1039/d0ra01316a rsc.li/rsc-advances Some previous theoretical studies found that the three- dimensional star-like structure can be constructed by the ionic interactions of seven satellite alkali cations with a at E5 6- pentagonal ring in which E is one of elements of group 14.22–24 Moreover, by using DFT calculations including van der Waals eﬀects, Li et al. has point out the small Ge6, Ge9, and Ge10 clusters can play as the block units which can be connect together in order to form assembly materials and the van der Waals force impressively strengthens the covalent bond between diﬀerent units, but plays less important role on the bonds in unit.25 Remarkably, it is highly particular that the pentagonal prism shape was experimentally observed for the CoGe10 3 and FeGe10 3 clusters in which either the Co or the Fe atom is centered in a D5h (5/5) Ge10 pentagonal prism.26,27 A large number of systematic investigations were carried out to elucidate the structural evolution of singly metal doped germanium clusters at various charged states.18–20,28–35 Accord- ingly, an interplay between the metal dopant and the Ge-host gives rise to the richness on geometries varying from incom- plete cage through encapsulated tube to Frank–Kasper polyhedron. RSC Advances Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. View Article Online View Journal \| View Issue This journal is © The Royal Society of Chemistry 2020 A remarkable mixture of germanium with phosphorus and arsenic atoms making stable pentagonal hetero-prisms [M@Ge5E5]+, E ¼ P, As and M ¼ Fe, Ru, Os† Cite this: RSC Adv., 2020, 10, 19781 3.1 Geometries For Ge5P5 +, the lowest energy structure P.1 contains four P–P connections, whereas P.2 turns out to contain a P5 cycle connected to a Ge5 counterpart and is only 3 kcal mol1 higher in energy than P.1. The next isomers including P.3, P.4 and P.5 are signicantly less stable. Regarding the Ge5As5 + cations, As.1 contains only one As–As bond but it emerges as the lowest-energy structure. The geometric characteristic of As.1 is completely diﬀerent from that of the isovalent P.1. Remarkably, As.3 possesses an As5 pentagonal string and is 8 kcal mol1 higher. Other higher energy isomers of the Ge5As5 + cation are also shown in Fig. 1. 3.1 Geometries As for a convention, we label the structures considered as A.M.x in which A ¼ P and As stand for Ge5P5 and Ge5As5 hosts, respectively, M ¼ Fe, Ru and Os denotes the TM dopant, and nally, x ¼ 1, 2, ., indicates the isomers with increasing rela- tive energy. For the Ge5E5 + cations, the structures are denoted as A.x. Relative energies given here under are consistent with respect to the corresponding isomer x ¼ 1. p p g To probe the eﬀects of the metal dopant on the geometries of Ge5P5 + and Ge5As5 + cations, we rst present in Fig. 1 the lower- lying isomers of both Ge5P5 + and Ge5As5 + cations obtained at the B3P86/6-311+G(d) level. No special shape is observed for both Ge–P and Ge–As mixed systems (Fig. 1). For Ge5P5 +, the lowest energy structure P.1 contains four P–P connections, whereas P.2 turns out to contain a P5 cycle connected to a Ge5 counterpart and is only 3 kcal mol1 higher in energy than P.1. The next isomers including P.3, P.4 and P.5 are signicantly less stable. Regarding the Ge5As5 + cations, As.1 contains only one As–As bond but it emerges as the lowest-energy structure. The geometric characteristic of As.1 is completely diﬀerent from that of the isovalent P.1. Remarkably, As.3 possesses an As5 pentagonal string and is 8 kcal mol1 higher. Other higher energy isomers of the Ge5As5 + cation are also shown in Fig. 1. Geometry identication for M@Ge5P5 + cations with M ¼ Fe, Ru and Os clearly points out that a metal dopant M stabilizes the Ge5P5 + host into a double ring shape. The lower-lying isomers of M@Ge5P5 + clusters are displayed in Fig. 2, and also in Fig. S1–S3 of the ESI le.† Accordingly, the M@Ge5P5 + cations mainly feature a pentagonal prism, and each metal To probe the eﬀects of the metal dopant on the geometries of Ge5P5 + and Ge5As5 + cations, we rst present in Fig. 1 the lower- lying isomers of both Ge5P5 + and Ge5As5 + cations obtained at the B3P86/6-311+G(d) level. No special shape is observed for both Ge–P and Ge–As mixed systems (Fig. 1). Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. All guessing structures of each series are geometrically optimized by using B3P86 functional in conjunction with small LANL2DZ basis set.49 Subsequently, the obtained structures, which have relative energy in range 50 kcal mol1, will be selected to re-optimize using the same functional but in conjunction with a larger basis set, including the 6-311+G(d) set50 for Ge, P and As atoms, and the aug-cc-pVTZ or aug-cc- pVTZ-PP for Fe, Ru and Os51,52 in which PP stands for pseudo- potential. The current study utilizes the hybrid B3P86 func- tional due to it has previously been tested as suitable for treatment of geometrical and electronic structures of mixed clusters containing transition metals.53 All geometric optimi- zations and electronic structure calculations are performed using the Gaussian 09 suite of program.54 It should be noted that the cationic state is considered in order to probe the closed- shell electron conguration with a low spin state. g pp g g y y Although the MGe10 q prismatic structures have gained so much attentions, their hetero-derivatives with P and As have been not considered yet. Indeed, while the FeGe10 q was identi- ed as a pentagonal prism in nine charge states with q being from 5 to +3, the isovalent RuGe10 q clusters are of polyhedral geometry.34,38 Additionally, the compounds containing a P5 or As5 pentagon were found in the carbon-free as well as mixed M(Cp)E5 sandwich complexes. Within these coordination compounds, each P5 or As ring coordinates to a transition metal rather than forms any mixed-ring.39–43 It is subsequently pre- dicted that M@Ge5P5 + and M@Ge5As5 + could be stable in a sandwich form where the M center is coordinated by both Ge5 and E5 rings. In this context, it is of interest to explore the eﬀects of the P and As hetero-atoms to geometry of Fe@Ge10 q pentagonal prism. With the aim to search for novel clusters possessing a stable tubular structural motif, we set out to carry out a theoretical investigation on geometries and electronic structure of the species Ge5E5 + in mixing the ve germanium atoms with ve P or As counterparts, and then they are singly doped by a transition metal (TM) giving rise to the doped M@Ge5E5 + clusters. The main role of the TM dopant is to stabilize the high symmetry tubular prism motif which is usually not stable in free forms. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For a systematic exploration, we consider the elements of group 8 including Fe, Ru and Os as the dopant M. It turns out that such a mixture between Ge with either P or As leads to a set of remarkably stable pentagonal prisms containing an unprecedented combination of these elements. This journal is © The Royal Society of Chemistry 2020 Paper w Article Online View Article Online View Article Online RSC Advances Paper showed that the V and Nb dopants are located at the central region of the Ge8As4 and Ge8As6 hetero-cages, respectively.36,37 Similarly, with dopant being Cr, Mo and W atoms, high symmetry structures were also observed in which the metal dopant is covered by a D3h Ge8E6 frame with E ¼ P and As. Subsequent theoretical studies pointed out that these M@Ge8E6 hetero-cages share an electron shell of [1S21P61D101F141G182S22P62D10] enclosing 68 electrons. The existence of Ge-cages with mixed P and As elements suggests that a doping of P or As into a germanium host emerges as a good approach to generate high symmetry hetero-structures. subroutine in which each atom exchanges its position with all the others. For each MGe5E5 + system, we generate 1000 initial isomers for geometry optimization. This algorithm has been proven to be highly eﬃcient in the search for the energetically lower-lying isomers of the systems containing various compo- nents.46–48 Additionally, on the basis of the well-known MGe10 q structures that have already been reported in previous studies, we substitute Ge atoms by either P or As atoms, and thereby generate the initial isomers for the mixed M@Ge5E5 + systems. subroutine in which each atom exchanges its position with all the others. For each MGe5E5 + system, we generate 1000 initial isomers for geometry optimization. This algorithm has been proven to be highly eﬃcient in the search for the energetically lower-lying isomers of the systems containing various compo- nents.46–48 Additionally, on the basis of the well-known MGe10 q structures that have already been reported in previous studies, we substitute Ge atoms by either P or As atoms, and thereby generate the initial isomers for the mixed M@Ge5E5 + systems. 1. Introduction 1. Due to a potentially important role of germanium based compounds in semiconductors and optoelectronic indus- tries,1–4 the geometric, electronic, thermodynamic and spec- troscopic properties of small Ge clusters and their doped varieties have carefully been investigated by both theoretical and experimental methods alike.5–15 According to numerous previous studies on doped germanium clusters, singly transi- tion metal doped germanium clusters provide us with a wide range of geometrical features. It is known that the M@Ge16 clusters with M ¼ Ti, Zr, and Hf establish Frank–Kasper poly- hedrons in which each metal dopant is encapsulated by a Td Ge16 cage.16 Similar to silicon clusters, a hexagonal prism shape has been identied for the V@Ge12 , Mo@Ge12 and W@Ge12 clusters.17–20 Of the transition metal doped MGe12 /0 clusters, the gold doped AuGe12 anion presents a high symmetry structure whose Au dopant is encapsulated by an Ih Ge12 host.21 Within a great eﬀort in the search for novel geometrical motifs of germanium-based cluster, multiple doping of P and As hetero-atoms to germanium hosts produced some symmetric hetero-fullerene structures. Following introduction of As atoms the mixed [V@Ge8As4]3 and [Nb@Ge8As6]3 hetero-fullerenes were generated. Structural identications for the experimen- tally prepared [V@Ge8As4]3 and [Nb@Ge8As6]3 clusters This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 19781–19789 \| 19781 Paper View Article Online 2. Computational methods 2. In order to explore the potential energy surface (PES) of each of the M@Ge5E5 + systems considered, its guess geometries are generated by using a stochastic algorithm previously imple- mented by us.44 Our stochastic search method was improved based on the ‘random kick’ procedure reported by Saunders45 for exploring the low-lying isomers of compounds. According to this procedure, each atom of an initial structure is kicked to randomly move within a sphere of radius r, then the structures, generated from that, become the inputs for subsequent geom- etry optimizations using electronic structure calculations, and the “moving radius” r of atoms is the only variable controlled in this procedure. In our modied stochastic searching procedure, three additional variables will be controlled to provide better structures constructed for the following geometry optimiza- tions. We modify this algorithm by adding a permutation Geometry identication for M@Ge5P5 + cations with M ¼ Fe, Ru and Os clearly points out that a metal dopant M stabilizes the Ge5P5 + host into a double ring shape. The lower-lying isomers of M@Ge5P5 + clusters are displayed in Fig. 2, and also in Fig. S1–S3 of the ESI le.† Accordingly, the M@Ge5P5 + cations mainly feature a pentagonal prism, and each metal This journal is © The Royal Society of Chemistry 2020 19782 \| RSC Adv., 2020, 10, 19781–19789 RSC Advances View Article Online Paper Paper Fig. 1 Shapes and relative energies (in kcal mol1) of lower-lying isomers of Ge5P5 + and Ge5As5 + clusters. Geometry optimizations and energy calculations were performed at the B3P86/6-311+G(d) level. Fig. 1 Shapes and relative energies (in kcal mol1) of lower-lying isomers of Ge5P5 + and Ge5As5 + clusters. Geometry optimizations and energy calculations were performed at the B3P86/6-311+G(d) level. dopant, involving Fe, Ru and Os, is found to be located in the central region of a mixed (5/5) Ge5P5 double ring, which actually is formed by connecting Ge4P, Ge3P2, Ge2P3 and GeP4 penta- gons together in a prismatic fashion. Of the latter, a combina- tion of both Ge3P2 and Ge2P3 strings establishes the global energy minimum structure for the M@Ge5P5 + cation. No isomer having a (Ge5)M(E5)+ sandwich complex has been found. The appearance of M@Ge5P5 + double ring prism emphasizes the crucial role of the metal dopant Fe, Ru and Os in stabilizing a Ge5P5 + cation in a high symmetry form. This journal is © The Royal Society of Chemistry 2020 Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. For a further characterization of the electron distribution, the bond length and Wiberg bond index (WBI) of the Ge–E, Ge– Ge and E–E bonds are tabulated in Table 2. For free M–E molecules, a bond length of 2.1 ˚A is found for M–P connec- tions with M ¼ Fe, Ru and Os, but their WBI values vary from 2.6, 3.2 to 3.5, respectively (Table 1). According to the usual meaning of WBI, P atom forms a triple bond with Fe and Ru while a nearly quadruple bond is identied for OsP dimer. A similar result is observed in M–As and M–Ge diatomic mole- cules where Os establishes a nearly quadruple bond with As and Ge, and a triple bond character is found for Fe–As, Fe–Ge, Ru–As and Ru–Ge dimeric species. The strength of M–E and M–Ge dimers tends to increase in going from M ¼ Fe to Os. The WBI values of free Ge2, GeP and GeAs dimers are calculated to be 2.5, 2.7 and 3.1, respectively. As a consequence, they can be formally classied as a triple bond. Particularly, P2 and As2 have bond lengths of 1.9 and 2.2 ˚A, respectively, and the corresponding WBI values amount to 3.6 and 3.5. Regarding the M@Ge5As5 + clusters, a similar behavior is again observed. DFT calculations emphasize that a hetero- prismatic shape is again dominating as displayed in Fig. S4– S6 of the ESI le.† On the structural aspect, each of the Fe, Ru and Os dopants occupies a place of the central region of a prismatic cage formed by the Ge4As, Ge3As2, Ge2As3 and GeAs4 pentagons. Similar to M@Ge5P5 +, disposition of both Ge3As2 and Ge2As3 pentagonal strings in an anti-prism form gives rise to the most stable structure for M@Ge5As5 +, as depicted in Fig. 2. The similarity on geometric characteristic of M@Ge5As5 + clusters and their P homologues (M@Ge5P5 + cations) again emphasizes the crucial stabilizing role of Fe, Ru and Os metals in turning an irregular cage to a tubular structure. In case of FeGe5As5 +, there is a competition for the ground state. Actually, the triplet As.Fe.1 and As.Fe.2 isomers, which are structures containing two and three As–As connections, are only 1 kcal mol1 more stable than the singlet As.Fe.3, an isomer containing only one As–As bond. 2. Computational methods Similar to the Fe@Ge10 q cluster, a mixed Fe@Ge5P5 + cluster is thus stabilized in a pentagonal prism. Moreover, such Fig. 2 Shapes of the global energy minimum structures of M@Ge5E5 + with M ¼ Fe, Ru and Os and E ¼ P and As. Geometry optimizations were performed using the B3P86 functional with the 6-311+G(d) basis set for Ge and E and aug-cc-pVTZ basis set for Fe and aug-cc-pVTZ-PP basis set for Ru and Os metals. Fig. 2 Shapes of the global energy minimum structures of M@Ge5E5 + with M ¼ Fe, Ru and Os and E ¼ P and As. Geometry optimizations were performed using the B3P86 functional with the 6-311+G(d) basis set for Ge and E and aug-cc-pVTZ basis set for Fe and aug-cc-pVTZ-PP basis set for Ru and Os metals. Fig. 2 Shapes of the global energy minimum structures of M@Ge5E5 + with M ¼ Fe, Ru and Os and E ¼ P and As. Geometry optimizations were performed using the B3P86 functional with the 6-311+G(d) basis set for Ge and E and aug-cc-pVTZ basis set for Fe and aug-cc-pVTZ-PP basis set for Ru and Os metals. RSC Adv., 2020, 10, 19781–19789 \| 19783 This journal is © The Royal Society of Chemistry 2020 View Article Online View Article Online Paper ew Article Online RSC Advances Paper a structural motif is consistently found for both Ru@Ge5P5 + and Os@Ge5P5 + as their ground state, whereas the Ru@Ge10 q cluster does not exist. This result again demonstrates the important role of P atoms in formation of pentagonal prism, in such a way that a multiple doping of P atoms into a germanium host, or replacing of Ge by P atoms, appears to be an eﬃcient approach to generate double ring structures for germanium-based clus- ters. It is interesting to note that the formation of P–P direct connections in each cluster series containing Fe, Ru and Os is in relation to the cluster stability. The most stable structure of Fe@Ge5P5 +, Ru@Ge5P5 + and Os@Ge5P5 + cations contains each only one P–P bond, whereas other isomers having two or more P–P bonds are signicantly less stable (Fig. S1–S3†). The isomers P.Fe.5, P.Fe.6 and P.Fe.7 contain each three P–P direct connections, and they are 10–15 kcal mol1 higher in energy. Similarly, structures containing Ru and Os exhibit three or more P–P direct bonds are calculated to be highly unstable. 2. Computational methods Overall, introduction of Fe, Ru and Os dopants into a Ge5P5 + host establishes a (5/5) hetero-prism double ring structure for M@Ge5P5 + cations, but the Ge and P atoms are mixed in such a way that formation of two or more P–P direct bonds tend to destabilize the resulting clusters. more importantly, a prismatic shape is not observed at all for their lower-lying isomers. Incorporation of a metal of the group Fe, Ru and Os into such Ge5E5 + cations brings in a (5/5) pentagonal double ring prismatic shape for doped M@Ge5E5 + clusters, in which the strings are formed upon mixture of atoms. This appears to be a general tendency for this class of clusters (Fig. 3). The geometric feature of M@Ge5E5 + clusters clearly shows that they prefer a mixed tubular shape rather than form a carbon-free sandwich complex. In fact, the existence of M@Ge5E5 + shows a diﬀerent trend in which both P5 and As5 rings no longer exist. The sandwich structure (Ge5ME5)+ is extremely unstable, even it does not appear as a local minimum on the M@Ge5E5 + potential energy surface. It can thus be concluded that in the global minimum isomer of MGe5E5 +, the metal center is coordinated by both Ge3E2 and Ge2E3 rings without any E5 string. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:3 This article is licensed under a Creative Commons Attribution 3.0 U a single bond character. A similar single bond character is found for Ge–Ge, which exhibits a WBI value of 0.7 in Fe@Ge5P5 +, and 0.5 in both Ru@Ge5P5 + and Os@Ge5P5 +. Connections of Ge with P atoms are characterized by WBI values in the range of 0.4–0.9, also implying a Ge–P single bond. Accordingly, Ge and P atoms form Ge–Ge, Ge–P and P–P single bonds in the Ge5P5 prismatic tube. A similar pattern is observed for the Ge5As5 cages in which Ge–As connections have WBI values of 0.5–0.9 for Ge–As bonds, and 1.0 for As–As and Ge– Ge bonds. It is important to explore the bonding between Ge3E2 and Ge2E3 rings. The connectivities associated with the super- position between both Ge3E2 and Ge2E3 strings is identied as single bond according to WBI results. Therefore, it is not possible to consider the pentagonal Ge3E2 and Ge2E3 rings of M@Ge5E5 + as two independent rings. On the other hand, the M@Ge5E5 + is a tubular cluster rather than a [(Ge3E2) M (Ge2E3)]+ carbon-free sandwich complex. WBI values of 1–2, whereas the WBI of free MGe species is greater than 2. In other words, the strength of a M–Ge bond is reduced when a doped M@Ge5E5 + prism is established. Overall, the WBI analysis indicates that Ge and E atoms either E ¼ P or As, connect together by a single bond whereas a metal atom gives rise to multiple bonds in interacting with the Ge, P and As elements. Although both P2 and As2 dimeric molecules are highly stable, as indicated by large values of their dissociation energies and WBI, the appearance of P–P and As–As connections in a M@Ge5E5 + prismatic cluster tends to destabilize it. Within the most stable isomer of a M@Ge5E5 + cluster, both P–P and As–As connections are identied as single bonds, according to WBI results. Therefore, in order to rationalize the rather negative eﬀect of the P–P and As–As connections on the stability of M@Ge5E5 + cluster, the dissociation energies (DE) of the E–E bonds as evaluated from the homolytic breaking H2E–EH2 / 2EH2, H3Ge–EH2 / Ge3H3 + EH2 and H3Ge–GeH3 / 2GeH3 processes which describe well the dissociation of E–E, Ge–E and Ge–Ge single bonds, are calculated and given in Table 1. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Additionally, the triplet 3A00 (Cs) As.Fe.3 is only 1 kcal mol1 higher than its single state, so that they are competitive for ground state of the Fe@Ge5As5 + cluster. However, this result emphasizes that the existence of hetero-prism containing one As–As connection is a general tendency in M@Ge5As5 + clusters. Within the M@Ge5P5 + clusters, P–P connections have bond length of 2.2 ˚A and WBI values of 1.0 clearly indicating Fig. 3 Geometric shapes of the lowest-energy structure of M@Ge5E5 +. As in the P homologues, the thermodynamic stability of M@Ge5As5 + cations is found again in correlation with the number of direct As–As bonds. In fact, the isomer having one As–As connection is signicantly more stable than those pos- sessing two or more As–As bonds, as shown in Fig. S4–S6 of the ESI le.† In other words, formation of additional As–As bonds tends to destabilize the doped clusters. The above structural identications illustrate the coherent fact that the metal atoms of group 8 involving Fe, Ru and Os induce a great geometrical modication for the Ge5E5 + cations with E being an element in group of 15 (P and As). Both Ge5P5 + P.1 and Ge5As5 + As.1 cations do not exist in a special form, and Fig. 3 Geometric shapes of the lowest-energy structure of M@Ge5E5 +. 19784 \| RSC Adv., 2020, 10, 19781–19789 This journal is © The Royal Society of Chemistry 2020 Paper a single bond character. A similar single bond character is found for Ge–Ge, which exhibits a WBI value of 0.7 in Fe@Ge5P5 +, and 0.5 in both Ru@Ge5P5 + and Os@Ge5P5 +. Connections of Ge with P atoms are characterized by WBI values in the range of 0.4–0.9, also implying a Ge–P single bond. Accordingly, Ge and P atoms form Ge–Ge, Ge–P and P–P single bonds in the Ge5P5 prismatic tube. A similar pattern is observed for the Ge5As5 cages in which Ge–As connections have WBI values of 0.5–0.9 for Ge–As bonds, and 1.0 for As–As and Ge– Ge bonds. It is important to explore the bonding between Ge3E2 and Ge2E3 rings. The connectivities associated with the super- position between both Ge3E2 and Ge2E3 strings is identied as single bond according to WBI results. Therefore, it is not possible to consider the pentagonal Ge3E2 and Ge2E3 rings of M@Ge5E5 + as two independent rings. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. On the other hand, the M@Ge5E5 + is a tubular cluster rather than a [(Ge3E2) M (Ge2E3)]+ carbon-free sandwich complex. For connections containing the metal atom the M–P bonds WBI values of 1–2, whereas the WBI of free MGe species is greater than 2. In other words, the strength of a M–Ge bond is reduced when a doped M@Ge5E5 + prism is established. Overall the WBI analysis indicates that Ge and E atoms either E ¼ P or As, connect together by a single bond whereas a metal atom gives rise to multiple bonds in interacting with the Ge, P and As elements. Although both P2 and As2 dimeric molecules are highly stable, as indicated by large values of their dissociation energies and WBI, the appearance of P–P and As–As connections in a M@Ge5E5 + prismatic cluster tends to destabilize it. Within the most stable isomer of a M@Ge5E5 + cluster, both P–P and As–As connections are identied as single bonds, according to WBI results. Therefore, in order to rationalize the rather negative eﬀect of the P–P and As–As connections on the stability of M@Ge5E5 + cluster, the dissociation energies (DE) of the E–E bonds as evaluated from the homolytic breaking H2E–EH2 / 2EH H Ge–EH / Ge H + EH and H Ge–GeH / 2GeH Table 1 Geometrical parameters (distance in angstrom) and WBI (a. u.) of M@Ge5E5 + prisms Fe@Ge5P5 + Ru@Ge5P5 + Os@Ge5P5 + Fe@Ge5As5 + Ru@Ge5As5 + Fe@Ge5As5 + M–P1 2.3/1.2 2.4/1.6 2.5/1.5 M–As1 2.4/1.1 2.5/1.4 2.6/1.5 M–P2 2.3/1.2 2.4/1.4 2.4/1.6 M–As2 2.4/1.1 2.5/1.4 2.6/1.6 M–P4 2.3/1.3 2.3/1.2 2.3/1.9 M–As4 2.4/1.1 2.4/1.6 2.4/1.9 M–Ge4 2.6/1.0 2.7/1.2 2.7/1.4 M–Ge4 2.6/0.9 2.7/1.2 2.7/1.3 M–Ge1 2.5/1.1 2.6/1.3 2.6/1.5 M–Ge1 2.6/1.0 2.6/1.3 2.6/1.5 M–Ge2 2.5/1.1 2.6/1.4 2.5/1.6 M–Ge2 2.6/0.9 2.6/1.4 2.6/1.6 P2–P3 2.2/1.0 2.2/1.0 2.2/0.1 As2–As3 2.5/0.9 2.4/1.0 2.4/1.0 P1–Ge4 2.5/0.7 2.4/0.9 2.4/0.8 As1–Ge4 2.5/0.9 2.5/0.9 2.5/0.8 P2–Ge4 2.5/0.8 2.5/0.7 2.5/0.7 As2–Ge4 2.5/0.7 2.6/0.8 2.9/0.5 P1–Ge1 2.5/0.7 2.6/0.6 2.6/0.6 As1–Ge1 2.6/0.7 2.7/0.6 2.7/0.6 P2–Ge2 2.5/0.7 2.6/0.5 2.7/0.4 As2–Ge2 2.6/0.8 2.7/0.6 2.7/0.5 P4–Ge4 2.5/0.7 2.6/0.6 2.6/0.5 As4–Ge4 2.6/0.7 2.7/0.6 2.6/0.7 P4–Ge1 2.4/0.9 2.6/0.6 2.6/0.6 As4–Ge1 2.5/0.9 2.6/0.7 2.6/0.6 Ge3–Ge2 2.6/0.7 2.8/0.5 2.9/0.5 Ge3–Ge2 2.7/0.7 2.7/0.8 2.7/0.5 p y This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Table 1 Geometrical parameters (distance in angstrom) and WBI (a. u.) of M@Ge5E5 + prisms ess Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. 3.3 The 18 electron count The stability of the singly metal doped tubes is oen rational- ized by using the classical electron count in which the metal atom receives electrons to gain a fullled d10 conguration. It should however be noted that the Fe, Ru and Os atoms have a general electron conguration of [ns2(n 1)d6], which thus needs 4 additional electrons to ll their (n 1)d10 shell. NBO analysis given above indicates that each Ge5E5 + cage transfers an amount of 3.5 electron to the metal center. In other words, each Ge5E5 + cage eﬀectively provides 4 valence electrons to establish a closed (n 1)d10 subshell for the metal M, and thereby stabilizes the M@Ge5E5 + tubular prism. Following this line of argument, the orbital interaction diagram between the Fe atom and a Ge5P5 + prism is constructed and displayed in Fig. 5. The result constructed for the Ge5P5 + case can certainly be generalized for the other derivatives. Interaction between a metal dopant M with a Ge5E5 + cage is further probed using the NBO atomic charges (Table 2) that illustrate that metal atoms bear a largely negative charge, 3.5 electron. On the other hand, the Ge5E5 + prismatic double ring supplies electrons to the metal dopant, and thereby establishes a negatively charged center Md. Each M@Ge5E5 + prismatic structure can thus be regarded as a [Md (Ge5E5)d+]+ donor– acceptor complex where the metal atom M behaves as an acceptor centre and the Ge5E5 + cage plays as a donor moiety. An analysis of the electron distribution using the electron localization indicator (ELI_D)55 is carried out to further explore the bonding phenomena of M@Ge5E5 + clusters. As shown in Fig. 4, at the bifurcation value of 1.3, a di-synaptic basin, V(E,E), is clearly observed for E–E connections in all clusters consid- ered. Similarly, localization domains are found for either Ge–P or Ge–As connection indicating their covalent bond character. The V(M,Ge) and V(M,E) basins with M ¼ Fe, Ru and Os, and E ¼ P are found at lower bifurcation values suggesting that the connections containing metal atom have an ionic character. This result is consistent with the NBO analysis given above in According to Fig. 5, the 4s-AO of Fe atom involves interaction with the HOMO-9 of Ge5P5 + and produces an S level for Fe@Ge5P5 +. RSC Advances Therefore, formation of Ge–P, Ge–As and Ge–Ge bonds is ex- pected to give raise more thermodynamic stability to M@Ge5E5 + prismatic structure than the P–P and As–As connections. As a result, the most stable isomer of each M@Ge5E5 + cluster contains only one P–P or As–As bond whereas eight Ge–E connections are formed (Table 3). which Ge5E5 + is shown to transfer much electron to the M center. Both NBO and ELI_D analyses illustrate that the elec- trostatic interaction of a negatively charged metal dopant with a Ge5E5 + positively charged tubular moiety contributes to a thermodynamic stabilization of the resulting M@Ge5E5 + hetero-prisms. Therefore, formation of Ge–P, Ge–As and Ge–Ge bonds is ex- pected to give raise more thermodynamic stability to M@Ge5E5 + prismatic structure than the P–P and As–As connections. As a result, the most stable isomer of each M@Ge5E5 + cluster contains only one P–P or As–As bond whereas eight Ge–E connections are formed (Table 3). Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:3 This article is licensed under a Creative Commons Attribution 3.0 U Accordingly, the DEs of the H3Ge–GeH3 bond has a value of 66 kcal mol1 whereas the DE values of H3Ge–PH2 and H3Ge– AsH2 are computed to be 60 kcal mol1. The H2P–PH2 and H2As–AsH2 species have DEs of 50 kcal mol1 which are signicantly smaller than the others. This result points out both P–P and As–As single bonds are consistently weaker than the mixed Ge–P, Ge–As and the pure Ge–Ge counterparts. For connections containing the metal atom, the M–P bonds of M@Ge5P5 + are not only signicantly longer than those of the corresponding M–P diatomic molecules, but their WBI values are also found to be in a range of 1.2–1.9. Similarly, M–As connections of M@Ge5As5 + prism have WBI values varying from 1.1 to 1.9. Hence, metal atoms form, in connecting with P and As atoms, stronger bonds than single bonds. In comparison to free MGe, MP and MAs dimers, formation of M@Ge5E5 + prisms signicantly reduces the strength of the corresponding connection. As shown in Table 2, the M–Ge connections have Table 2 Bond length (d, angstrom) and WBI (a.u.) of MGe, ME, E2 and GeE diatomic molecules Molecule d WBI Molecule d WBI Molecule d WBI Fe–P 2.1 2.6 Ru–P 2.1 3.2 Os–P 2.1 3.5 Fe–As 2.2 2.4 Ru–As 2.2 3.3 Os–As 2.1 4.1 Fe–Ge 2.3 2.3 Ru–Ge 2.2 3.3 Os–Ge 2.1 3.5 Ge–Ge 2.4 2.5 Ge–P 2.2 2.7 Ge–As 2.4 3.1 P–P 1.9 3.6 As–As 2.1 3.5 Table 3 Dissociation energy (DE, kcal mol1) and bond length (r, ˚A) of H2E–XH3 molecules (TPSSh/6-311++G(d,p)) DE (E–X) r (E–X) H2P–PH2 50.3 2.26 H2As–AsH2 47.0 2.47 H3Ge–GeH3 66.2 2.43 H3Ge–PH2 60.7 2.34 H3Ge–AsH2 57.9 2.44 This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 19781–19789 \| 19785 Table 3 Dissociation energy (DE, kcal mol1) and bond length (r, ˚A) of H2E–XH3 molecules (TPSSh/6-311++G(d,p)) Paper View Article Online View Article Online View Article Online 3.3 The 18 electron count The Dxy and Dx2–y2 levels are produced upon inter- action of the HOMO and HOMO-1 with 3d-AOs of Fe. Interac- tion of the LUMO and LUMO+1 of Ge5P5 + prism with 3d-AOs of Fig. 4 ELI_D maps plotted at the bifurcation value of 1.3 for M@Ge5E5 +. Fig. 4 ELI_D maps plotted at the bifurcation value of 1.3 for M@Ge5E5 +. Fig. 4 ELI_D maps plotted at the bifurcation value of 1.3 for M@Ge5E5 +. This journal is © The Royal Society of Chemistry 2020 This journal is © The Royal Society of Chemistry 2020 19786 \| RSC Adv., 2020, 10, 19781–19789 RSC Advances View Article Online Paper Fig. 5 A representative orbital interaction diagram of Fe with Ge5P5 + prism producing MOs containing 18 electrons. Paper RSC Advan Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5 A representative orbital interaction diagram of Fe with Ge5P5 + prism producing MOs containing 18 electrons. A representative orbital interaction diagram of Fe with Ge5P5 + prism producing MOs containing 18 electrons. Fe induces the Dxz and Dyz eigenstates of Fe@Ge5P5 +. The 3dz2 AO of Fe becomes Dz2 of Fe@Ge5P5 + prismatic cluster. The Px, Py and Pz levels of Fe@Ge5P5 + are mainly contributed by the HOMO-3,4,5 of Ge5P5 + cage. Finally, orbital interactions of the Fe dopant with Ge5P5 + prism produce an electron conguration of [S2P6D10] which in fact contains 18 valence electrons. subsequently establishes a 18 electron conguration.38,56 This gives an emphasis that hetero-atoms including P and As not only replace Ge position in a prismatic framework but also provide electrons to full 18 electron conguration. Fe induces the Dxz and Dyz eigenstates of Fe@Ge5P5 +. The 3dz2 AO of Fe becomes Dz2 of Fe@Ge5P5 + prismatic cluster. The Px, Py and Pz levels of Fe@Ge5P5 + are mainly contributed by the HOMO-3,4,5 of Ge5P5 + cage. Finally, orbital interactions of the Fe dopant with Ge5P5 + prism produce an electron conguration of [S2P6D10] which in fact contains 18 valence electrons. This journal is © The Royal Society of Chemistry 2020 4. Concluding remarks A similar orbital interaction is also found for other clusters including RuGe5P5 +, OsGe5P5 + and MGe5As5 + as shown in Fig. S7–S11 of ESI le.† These results show that metal dopants Fe, Ru and Os gain each 4 electrons from the Ge5E5 + cage to fulll its (n 1)d10 level, and thereby induce an enhanced stability for the M@Ge5E5 + prismatic structures, which not only share a general structural motif but also have a same stabilizing mechanism where the metal center gains 4 electrons from Ge5E5 + prismatic cage to establish a 18 electron conguration. It is interesting that the polyanions Co@Ge10 3 and Fe@Ge10 4 pentagonal prismatic clusters, which are iso-valent with Fe@Ge5E5 +, are stabilized by a similar mechanism. They are similar to Ge5E5 + prisms; the Ge10 prismatic host also supplies electrons to fulll 3d10 levels of Co and Fe centers, and In summary, we presented a theoretical investigation on geometry, stability and chemical bonding of the Ge5E5 + and MGe5E5 + cationic clusters with E ¼ P, As; M ¼ Fe, Ru and Os. Structural identications clearly pointed out that the doping of a transition metal atom greatly inuences to geometry of the Ge5E5 + cage. Structurally, the singly doped M@Ge5E5 + clusters are stabilized in pentagonal hetero-prism shape whereas the Ge5E5 + cation is not in any special form. Each hetero-prismatic structure is formed by superposition of the Ge4E, Ge3E2, Ge2E3 and GeE4 pentagons together in prismatic fashion, but the combination of Ge3E2 and Ge2E3 strings peculiarly establishes the global minimum structure for M@Ge5E5 + cations. Inter- estingly, the cluster contains only one E–E connection exhibits the lowest-energy while a structure possesses two or more E–E This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 19781–19789 \| 19787 View Article Online RSC Advances Paper Paper 21 S.-J. Lu, L.-R. Hu, X.-L. Xu, H.-G. Xu, H. Chen and W.-J. Zheng, Phys. Chem. Chem. Phys., 2016, 18, 20321– 20329. bonds is signicantly less stable. Ge–E bonds are in fact stronger than E–E connections. Within the donor–acceptor perspective, with acceptor being the metal dopant, the GeE cage donates around 4 electrons to the M center and then stabilize M@Ge5E5 + clusters. A CMO analysis illustrates that the conventional 18 electron count is eﬀectively recovered in the stabilized M@Ge5E5 + cations. 22 M. Contreras, E. Osorio, F. Ferraro, G. Puga, K. J. References 29 X.-J. Deng, X.-Y. Kong, X.-L. Xu, H.-G. Xu and W.-J. Zheng, ChemPhysChem, 2014, 15, 3987–3993. 1 W. L. Brown, R. R. Freeman, K. Raghavachari and M. Schl¨UTer, Science, 1987, 235, 860. 30 J. Wang and J.-G. Han, J. Phys. Chem. B, 2006, 110, 7820– 7827. 2 E. C. Honea, A. Ogura, C. A. Murray, K. Raghavachari, W. O. Sprenger, M. F. Jarrold and W. L. Brown, Nature, 1993, 366, 42–44. 31 C. Siouani, S. Mahtout, S. Safer and F. Rabilloud, J. Phys. Chem. A, 2017, 121, 3540–3554. 32 X.-J. Hou, G. Gopakumar, P. Lievens and M. T. Nguyen, J. Phys. Chem. A, 2007, 111, 13544–13553. 3 Y. Kamata, Mater. Today, 2008, 11, 30–38. 4 R. Pillarisetty, Nature, 2011, 479, 324–328. 4 R. Pillarisetty, Nature, 2011, 479, 324–328. 33 D. Bandyopadhyay, P. Kaur and P. Sen, J. Phys. Chem. A, 2010, 114, 12986–12991. 5 J. Wang, G. Wang and J. Zhao, Phys. Rev. B: Condens. Matter Mater. Phys., 2001, 64, 205411. 34 Y. Jin, Y. Tian, X. Kuang, C. Lu, J. L. Cabellos, S. Mondal and G. Merino, J. Phys. Chem. C, 2016, 120, 8399–8404. 6 R. B. King, I. Silaghi-Dumitrescu and A. Lupan, Inorg. Chem., 2005, 44, 3579–3588. 7 R. B. King, I. Silaghi-Dumitrescu and M. M. Ut¸ǎ, Inorg. Chem., 2006, 45, 4974–4981. 35 S. Jaiswal and V. Kumar, Comput. Theor. Chem., 2016, 1075, 87–97. 36 S. Mitzinger, L. Broeckaert, W. Massa, F. Weigend and S. Dehnen, Chem. Commun., 2015, 51, 3866–3869. 8 R. B. King and I. Silaghi-Dumitrescu, Inorg. Chem., 2003, 42, 6701–6708. 9 W. Qin, W.-C. Lu, Q.-J. Zang, L.-Z. Zhao, G.-J. Chen, C. Z. Wang and K. M. Ho, J. Chem. Phys., 2010, 132, 214509. 37 R. Ababei, W. Massa, B. Weinert, P. Pollak, X. Xie, R. Cl´erac, F. Weigend and S. Dehnen, Chem.–Eur. J., 2015, 21, 386–394. 38 M. M. Ut¸˘a, D. Cioloboc and R. B. King, J. Phys. Chem. A, 2012, 116, 9197–9204. 10 W. Qin, W.-C. Lu, L.-Z. Zhao, Q.-J. Zang, G.-J. Chen, C. Z. Wang and K. M. Ho, J. Chem. Phys., 2009, 131, 124507. 11 B.-x. Li and P.-l. Cao, Phys. Rev. B: Condens. Matter Mater. Phys., 2000, 62, 15788–15796. 39 E. Urnecˇzius, W. W. Brennessel, C. J. Cramer, J. E. Ellis and P. v. R. Schleyer, Science, 2002, 295, 832–834. 12 A. A. Shvartsburg, B. Liu, Z.-Y. Lu, C.-Z. Wang, M. F. Jarrold and K.-M. Ho, Phys. Rev. The authors declare no competing nancial interest. 25 H. Li, W. Chen, X. Han, L. Li, Q. Sun, Z. Guo and Y. Jia, J. Comput. Chem., 2015, 36, 1919–1927. s Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Acknowledgements 26 B. Zhou, M. S. Denning, D. L. Kays and J. M. Goicoechea, J. Am. Chem. Soc., 2009, 131, 2802–2803. This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 103.01-2019.372 to NMT. 27 J.-Q. Wang, S. Stegmaier and T. F. F¨assler, Angew. Chem., Int. Ed., 2009, 48, 1998–2002. 28 Q. Jing, F.-y. Tian and Y.-x. Wang, J. Chem. Phys., 2008, 128, 124319. Open Access Article. Published on 27 May 2020. Downloaded on 10/24/2024 6:32:11 AM. This article is licensed under a Creative Commons Attribution 3.0 Unported Lice M. Orozco-Ic, J. Barroso, S. Pan, W. Tiznado and G. Merino, Chem.–Asian J., 2018, 13, 1751–1755. The authors declare no competing nancial interest. 4. Concluding remarks Donald, J. G. Harrison, G. Merino and W. Tiznado, Chem.–Eur. J., 2013, 19, 2305–2310. 23 W. Tiznado, N. Perez-Peralta, R. Islas, A. Toro-Labbe, J. M. Ugalde and G. Merino, J. Am. Chem. Soc., 2009, 131, 9426–9431. Conﬂicts of interest 24 A. V´asquez-Espinal, K. Palacio-Rodr´ıguez, E. Ravell, 24 A. V´asquez-Espinal, K. Palacio-Rodr´ıguez, E. Ravell, M. Orozco-Ic, J. Barroso, S. Pan, W. Tiznado and G. Merino, Chem.–Asian J., 2018, 13, 1751–1755. References Lett., 1999, 83, 2167–2170. 40 J. Frunzke, M. Lein and G. Frenking, Organometallics, 2002, 21, 3351–3359. 41 K. Kotrle and R. Herchel, Inorg. Chem., 2019, 58, 14046– 14057. 13 L. Wang and J. Zhao, J. Chem. Phys., 2008, 128, 024302. 14 S. Bulusu, S. Yoo and X. C. Zeng, J. Chem. Phys., 2005, 122, 164305. 42 R. F. Winter and W. E. Geiger, Organometallics, 1999, 18, 1827–1833. 15 S. Yoo and X. C. Zeng, J. Chem. Phys., 2006, 124, 184309. 43 M. Lein, J. Frunzke and G. Frenking, Inorg. Chem., 2003, 42, 2504–2511. 16 V. Kumar and Y. Kawazoe, Phys. Rev. Lett., 2002, 88, 235504. 17 N. D. Phi, N. T. Trung, E. Janssens and V. T. Ngan, Chem. Phys. Lett., 2016, 643, 103–108. 44 T. B. Tai and M. T. Nguyen, J. Chem. Theory Comput., 2011, 7, 1119–1130. 18 X.-J. Deng, X.-Y. Kong, H.-G. Xu, X.-L. Xu, G. Feng and W.-J. Zheng, J. Phys. Chem. C, 2015, 119, 11048–11055. 45 M. Saunders, J. Comput. Chem., 2004, 25, 621–626. 19 J. Wang and J.-G. Han, J. Phys. Chem. A, 2006, 110, 12670– 12677. 46 H. T. Pham, N. T. Cuong, N. M. Tam, V. D. Lam and N. T. Tung, Chem. Phys. Lett., 2016, 643, 77–83. 20 R. Trivedi, K. Dhaka and D. Bandyopadhyay, RSC Adv., 2014, 4, 64825–64834. This journal is © The Royal Society of Chemistry 2020 19788 \| RSC Adv., 2020, 10, 19781–19789 View Article Online View Article Online Paper Paper RSC Advances 47 N. M. Tam, L. V. Duong, H. T. Pham, M. T. Nguyen and M. P. Pham-Ho, Phys. Chem. Chem. Phys., 2019, 21, 8365– 8375. 53 V. T. Ngan, P. Gruene, P. Claes, E. Janssens, A. Fielicke, M. T. Nguyen and P. Lievens, J. Am. Chem. Soc., 2010, 132, 15589–15602. 48 N. M. Tam, V. T. Ngan and M. T. Nguyen, Chem. Phys. Lett., 2014, 595–596, 272–276. 54 M. J. Frisch, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, J. A. Montgomery, et al., Gaussian 09 Revision: D.01, 2009. 49 J. P. Perdew, Phys. Rev. B: Condens. Matter Mater. Phys., 1986, 33, 8822–8824. 55 M. Kohout, F. R. Wagner and Y. Grin, Int. J. Quantum Chem., 2006, 106, 1499–1507. 50 P. J. Hay, J. Chem. Phys., 1977, 66, 4377–4384. 56 M. M. Ut¸˘a, D. Cioloboc and R. B. King, Inorg. Chem., 2012, 51, 3498–3504. 51 K. A. Peterson, D. This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 19781–19789 \| 19789 References Figgen, M. Dolg and H. Stoll, J. Chem. Phys., 2007, 126, 124101. 52 D. Figgen, K. A. Peterson, M. Dolg and H. Stoll, J. Chem. Phys., 2009, 130, 164108. RSC Adv., 2020, 10, 19781–19789 \| 19789 This journal is © The Royal Society of Chemistry 2020
https://openalex.org/W4292478752	https://www.frontiersin.org/articles/10.3389/fgwh.2022.857345/pdf	English	null	Government responses to gender-based violence during COVID-19	Frontiers in global women’s health	2,022	cc-by	9,751	Government responses to gender-based violence during COVID-19 OPEN ACCESS EDITED BY Diana Cruz-Topete, Louisiana State University Health Shreveport, United States REVIEWED BY Martin Hushie, University for Development Studies, Ghana Neil Mitchell, University College London, United Kingdom CORRESPONDENCE Rebecca Gordon Rebecca.Gordon@uws.ac.uk SPECIALTY SECTION This article was submitted to Quality of Life, a section of the journal Frontiers in Global Women’s Health RECEIVED 18 January 2022 ACCEPTED 15 July 2022 PUBLISHED 18 August 2022 CITATION Gordon R, Cheeseman N, Rockowitz S, Stevens LM and Flowe HD (2022) Government responses to gender-based violence during COVID-19. Front. Glob. Womens Health 3:857345. doi: 10.3389/fgwh.2022.857345 COPYRIGHT © 2022 Gordon, Cheeseman, Rockowitz, Stevens and Flowe. 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. OPEN ACCESS EDITED BY Diana Cruz-Topete, Louisiana State University Health Shreveport, United States REVIEWED BY Martin Hushie, University for Development Studies, Ghana Neil Mitchell, University College London, United Kingdom CORRESPONDENCE Rebecca Gordon Rebecca.Gordon@uws.ac.uk SPECIALTY SECTION This article was submitted to Quality of Life, a section of the journal Frontiers in Global Women’s Health RECEIVED 18 January 2022 ACCEPTED 15 July 2022 PUBLISHED 18 August 2022 CITATION Gordon R, Cheeseman N, Rockowitz S, Stevens LM and Flowe HD (2022) Government responses to gender-based violence during COVID-19. Front. Glob. Womens Health 3:857345. doi: 10.3389/fgwh.2022.857345 COPYRIGHT © 2022 Gordon, Cheeseman, Rockowitz, Stevens and Flowe. 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 Rebecca Gordon1*, Nic Cheeseman2, Sarah Rockowitz3, Laura M. Stevens3 and Heather D. Flowe3 1School of Education and Social Sciences, University of the West of Scotland, Glasgow, United Kingdom, 2International Development Department, University of Birmingham, Edgbaston, United Kingdom, 3School of Psychology, University of Birmingham, Edgbaston, United Kingdom Gender-based violence (GBV) signiﬁcantly and substantially threatens women’s health. The COVID-19 pandemic has exacerbated existing risks and patterns of GBV. The impact of COVID-19 on GBV is not inevitable, however, and can be mediated by the policies of governments. TYPE Original Research PUBLISHED 18 August 2022 DOI 10.3389/fgwh.2022.857345 TYPE Original Research PUBLISHED 18 August 2022 DOI 10.3389/fgwh.2022.857345 TYPE Original Research PUBLISHED 18 August 2022 DOI 10.3389/fgwh.2022.857345 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. Government responses to gender-based violence during COVID-19 In this study we developed the Government GBV Response Index to systematically examine how countries (N = 60) performed in response to the pandemic with respect to the government 1) enacting speciﬁc national-level GBV policy; 2) making dedicated COVID-19 speciﬁc funding available; and 3) adapting existing GBV responses to COVID-19 related restrictions and challenges. Most countries (N = 33) delivered fewer than two policy responses. We also performed rapid case study analyses to investigate what might contribute to countries having more comprehensive government policy. We ﬁnd that civil society organizations played a key role in facilitating GBV policy during the pandemic, especially if they are well-funded and well-connected to the government, and if the country has a high-level government ofcial responsible for gender issues. © 2022 Gordon, Cheeseman, Rockowitz, Stevens and Flowe. 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. COVID-19, gender equality, gender-based violence, pandemic, policy, civil society An overview of existing research and understanding on government responses to GBV during COVID-19 Early on in the COVID-19 pandemic, there were calls for governments to make the prevention of violence against women and girls a key part of national emergency response plans, and for shelters and helplines to be included as essential services so they would remain funded and available for use (18). The World Health Organization made similar recommendations for government response plans to include essential services to address GBV, and also noted that health providers, health facilities, community members, and humanitarian response organizations must work to help mitigate GBV impacts arising from the pandemic (19). However, despite these calls, widespread closures of schools and support organizations, coupled with a decrease in government funding, has made protecting and responding to the needs of survivors diﬃcult, and at times impossible, to achieve. For example, domestic violence organizations and safe shelters that have remained open have had an overwhelming amount of GBV cases in some instances, while in others the call numbers have decreased signiﬁcantly because perpetrators are making it diﬃcult for survivors to access protection and other essential services (20). Additionally, decreased funding owing to the economic fallout of the pandemic has caused many organizations to shut down their programmes and facilities (20). Some governments reacted quickly and eﬀectively to the threat of greater violence against women and girls, increasing funding for GBV services, safehouses, and hotlines, while stepping up messaging and communication on these issues (12, 13). However, in some cases governments diverted resources from GBV prevention and response, cut funding to organizations working in the sector and stiﬂed free speech and dissent about these actions (14). In others, government policy was inconsistent and self-defeating, making statements and policies designed to respond positively to GBV while simultaneously reducing the funding for women’s rights organizations (15). One common thread noted by researchers examining gender-based violence during the pandemic was that when government responses were swift and thorough, it oﬀset the worst impacts of the pandemic, weakening the link between lockdowns and increasing GBV (16). This is a critical point for two reasons. First, it serves as an important reminder not to be fatalistic or defeatist about the impact of health crises on GBV - they represent a massive challenge, but governments still have agency about how to respond. Introduction Gender-based violence (GBV) is one of the greatest threats to women’s health and wellbeing today. Although GBV is experienced by both men and women, women experience higher rates of repeat victimization and are much more likely to be seriously hurt or killed (1). Indeed, decades of research have demonstrated that violence against women is prevalent in all contexts; however, during emergencies there can be disruptions to protective structures and services that can lead to increasing levels of abuse and poor responses, exacerbating adverse health outcomes (2, 3). COVID- 19 was particularly concerning in this regard due to social distancing, lockdowns and other restrictions of movement that put women and girls at greater risk of violence inside their own homes (4). Along with school closures, this meant that there were fewer opportunities to occupy safe spaces (5). Furthermore, the emotional and economic stress caused by the crisis is likely to have led to an increase in abuse (6, 7). These interrelated factors led UN Women (8) to describe GBV as a “shadow Frontiers in Global Women’s Health 01 frontiersin.org Gordon et al. 10.3389/fgwh.2022.857345 10.3389/fgwh.2022.857345 pandemic” occurring alongside COVID-19 worldwide, leading to estimates that there would be an additional 31 million cases of GBV globally by 6 months into the pandemic (9). However, whilst this literature has acknowledged the importance of government responses to gender-based violence during health crises, thus far there have been limited comparative studies examining the way in which governments did respond to GBV during COVID-19. Therefore, our research sought to answer the question: What were government responses to GBV during the COVID-19 pandemic? Firstly, this article will explore the pre-existing research and literature on how governments responded to GBV during COVID-19, before presenting a detailed description of our methodological approach to exploring this phenomenon. We next present our ﬁndings and discussion of these ﬁndings before concluding with recommendations for policymakers and activists working in this space. This may make it appear as if increases in GBV during the coronavirus pandemic would be inevitable. But, in reality, the impact of COVID-19 on GBV is not predetermined. To see why, it is important to start by acknowledging that COVID- 19 itself has not caused GBV in any simplistic sense. Instead, lockdown measures implemented to slow transmission rates of COVID-19 during the pandemic have exacerbated existing risks and patterns of behavior (4). Introduction Moreover, it is likely that the impact of COVID-19 can and has been mediated by the policies of governments, the activities of non-governmental organizations (NGOs), civil society organizations (CSOs) and ordinary citizens. As is often the case, civil society plays a major role in aﬀecting change in relation to GBV policies (10). One implication of this is that previous cuts to funding for relevant organizations prior to the pandemic likely inﬂuenced the nature and eﬀectiveness of the response to GBV during COVID-19 (11). An overview of existing research and understanding on government responses to GBV during COVID-19 Relatedly, viewing the gendered pandemic in this way lays bare the culpability of governments, who have the opportunity to oﬀset the greater risk women face during a pandemic but often, due to gender-insensitive systems and policies, actually serve to magnify the risk (17). Second, the positive impact of the measures implemented by gender- sensitive governments provides positive examples of “good basic practices” that policy makers and gender-activists can learn from - and can use to mobilize support for more progressive government responses in future. Rising GBV rates have not simply been organically triggered by pandemic lockdown conditions but have also, in many cases, occurred as a result of a lack of eﬀective government response. One common policy that some countries enacted during lockdown was to ban the sale of alcohol altogether, as in India, the Philippines, and South Africa. Other countries, such as those in Europe and the Americas, left alcoholic beverages available, albeit with restricted sales hours in some locations (21). Due to lockdown restrictions, the limitations on alcohol sales were largely targeted at reducing incidents of domestic violence, as drinking in the home was often the only acceptable place of consumption. However, the ban on alcohol was also targeted at reducing other alcohol-related injuries, such as those Frontiers in Global Women’s Health frontiersin.org 02 10.3389/fgwh.2022.857345 Gordon et al. 10.3389/fgwh.2022.857345 a stratiﬁed sample to ensure representation from diﬀerent continents and from high-, middle- and low-income contexts. This sample of cases therefore reﬂects a broad cross-section of the global experience and provides insights into the extent to which county-level factors such as GDP and women’s representation in parliament relate to the eﬀectiveness of the government’s response to the gendered impact of COVID- 19 responses. from street violence, and to encourage compliance with social distancing measures (22). In South Africa, the alcohol ban was eﬀective both in freeing up hospital beds for COVID patients and in making women feel safer, as their communities were less violent (23). Once the alcohol ban was lifted, emergency visits to hospitals doubled and domestic violence increased, taking up valuable healthcare resources in a country already struggling with providing adequate healthcare for all of its residents (23). p g q Research during this period also focused strongly on the role of civil society campaigns in putting pressure on the government to respond to rising rates of GBV. An overview of existing research and understanding on government responses to GBV during COVID-19 In Kenya, the coalition on violence against Women-Kenya urged the Health Cabinet Secretary to integrate GBV into the daily COVID-19 brieﬁng, and a consortium of feminist organizations petitioned the government for a 30% allocation of COVID-19 funds to the GBV response (24). Ultimately, the pressure that GBV stakeholders and women’s groups placed on the government to pay attention to rising GBV rates led to the late release of guidelines about health care for GBV survivors (25). UN agencies have also promoted global campaigns calling on governments to act on these issues with the message: “Fund, Prevent, Respond, Collect!” (18). In other cases, international NGOs have supported motivated political actors to act on their desires to address rising rates of GBV. For example, Oxfam in Malawi donated four motorcycles to women members of parliament to help them in rolling out campaigns to ﬁght against GBV (26). Rapid case studies Based on our large-n analysis, we also conducted two rapid case studies to better understand the driving factors behind more comprehensive government responses to GBV during COVID- 19. The purpose of these case studies were: to conﬁrm our initial assessment of the government response and better understand the enablers and barriers to an eﬀective government response. Materials and methods Study design In order to eﬀectively critically evaluate government responses to GBV during the pandemic, we sought to utilize a comprehensive and globally used deﬁnition of the term: violence directed against a person because of their gender, which includes physical, sexual, psychological or economic harm or suﬀering (27). However, as noted above we acknowledge that most gender-based violence is inﬂicted on women and girls, by men. Mapping government responses to GBV Our decision to purposively sample 60 countries was based on a number of factors. Due to time and funding constraints, it was not possible to conduct the analysis for all countries impacted by COVID-19, and it is important to keep in mind - in addition to the caveats noted above - that our tracker represents a sample of around one- third of relevant cases. Nonetheless, we have been careful to stratify the sample to ensure representation from diﬀerent continents, from high-, middle- and low-income contexts, and from states with very diﬀerent population sizes and levels of social assistance. However, whilst there is growing evidence on how policy measures that were introduced impacted on GBV, and on the response of civil society mobilizing to campaign for improved governmental approaches to address these impacts, there is a lack of research that has systematically examined government responses on a global basis which our study seeks to address. It is important to note that we were careful to avoid falling into the trap of only collecting data on those countries in which it is easily available, and hence skewing the sample toward higher-income countries. Our sample therefore includes some of the poorest countries in the world, including the DRC, Malawi, Papua New Guinea, as well as countries with considerable conﬂict and instability such as Lebanon, Nigeria, and Myanmar. The full sample is shown in Figure 1 below. While more work needs to be done to conﬁrm that our ﬁndings are repeated in the full set of cases, our sample was chosen to represent a reasonable approximation of the global context. Mapping government responses to GBV Our two rapid case studies were selected based on our analysis of the mapping of government responses. Both countries, the Democratic Republic of Congo (DRC) and New Zealand were chosen because both governments responded to all three measures on our mapping index (explained in more detail To provide a reliable assessment of the global response to GBV since the start of the pandemic, and to highlight examples of good and bad practice, we conducted a rapid mapping of the actions of governments in 60 countries worldwide, with Frontiers in Global Women’s Health frontiersin.org 03 Gordon et al. 10.3389/fgwh.2022.857345 FIGURE 1 Government responses to GBV during COVID-19 overview. FIGURE 1 Government responses to GBV during COVID-19 overview. Data collection below) but have extremely diﬀerent contextual factors that likely mediate government responses, including levels of GBV and government legislative and ﬁnancial capacity. The logic of this approach is to show that even countries with limited resources can pro-actively respond to increases in GBV, and that many countries that could respond eﬀectively fail to do so potentially due to a lack of political will, and to explore the impact of civil society actors and awareness raising. Therefore, these two case studies were considered to provide a useful insight into the factors which may support or hinder eﬀective government responses to GBV during crises. Mapping government responses to GBV Data were collected for 60 countries in July 2021. However, data on government responses to the threat of rising GBV is not always well publicized or held in a central repository. It was therefore important to use a wide range of approaches to identify and check the robustness of data on gender policies and expenditure. Four complementary approaches were therefore utilized to collect the data that underpins the Government GBV Response Index. These included: Frontiers in Global Women’s Health frontiersin.org 04 10.3389/fgwh.2022.857345 Gordon et al. 1. Consultation of existing data sets [e.g., (28)]. 1. Consultation of existing data sets [e.g., (28)]. raising rather than directly to support those responding to the crisis). On this basis, countries could score between 0 and 3, with 0 representing the weakest response to GBV during COVID- 19 and 3 representing the most comprehensive, based on our indicators. It is important to note that the GBV Response Index eﬀectively records oﬃcial changes in policy and allocations of funding – it does not collect data on how eﬀectively these strategies were implemented. We would therefore expect some variation between countries that have similar scores on the Index depending on how planned GBV responses were put into practice2. As we discuss in greater detail below, it is also important to keep in mind that given that there is no global dataset for the existing level of government activity across these three dimensions, it was not possible for us to take into account the diﬀerent “starting position” that countries are likely to have when it comes to preventing GBV. Additionally, due to the challenges of data availability, we did not weight any of the components within our index, due to the likelihood of introducing distortions to the data; however, this would be a valuable avenue for future research. 2. A set of Internet searches with various combinations of keywords, including COVID-19, coronavirus, GBV, domestic violence, policy, response, government funding, and all country names. 2. A set of Internet searches with various combinations of keywords, including COVID-19, coronavirus, GBV, domestic violence, policy, response, government funding, and all country names. In cases where there was ambiguity or references but limited detail about potential government responses to GBV during COVID-19, the following steps were taken: 3. A search of news/press releases from each country’s government website, where available. 4. A search for relevant national media coverage. 1 This related to Protocol/Policy/Legislation that speciﬁcally sought to address/mitigate rising rates of violence, rather than solely protocols to ensure the continued functioning of existing services, which were covered under the third response. When GBV was mainstreamed within COVID-19 legislation, this was considered to be a speciﬁc GBV-related government response to COVID-19 and so this was coded as 1. Rapid case studies The approach to these case studies was to conduct a rapid literature review of available policy and programming documentation, and to conduct ﬁve unstructured interviews across both countries with actors who had been involved in the response either within the government, or civil society/international organizations. These unstructured interviews took place over Zoom and were recorded and transcribed for analysis. Due to the ongoing pandemic, participants are not cited by name as they remain involved in the response. Using these methods, we collected data on three responses that governments could make to try and reduce the impact on GBV. Although these policy responses are not exhaustive, they represent the main menu of options that governments utilized: 1. Were COVID-19 GBV-speciﬁc national protocols/policy/legislation introduced or was GBV mainstreamed within COVID-19 legislation or national responses?1 1. Were COVID-19 GBV-speciﬁc national protocols/policy/legislation introduced or was GBV mainstreamed within COVID-19 legislation or national responses?1 Mapping government responses to GBV It is important to acknowledge that the index is calculated solely on the information and data available through these search terms which were conducted in English. In some cases, these were auto translated by Google, particularly when searches of news/press releases from government websites were being conducted. However, as a consequence of this is that there may be further information that was not found as part of this data collection approach. Additionally, as this was a rapid mapping exercise there may be information that was not easily accessible either through searches or through government websites, and so was not included. To reduce the risk of false negatives the data were shared with experts for veriﬁcation and have proved to be robust. We are therefore conﬁdent of the reliability of the data that forms the backbone of this report. 2 It is also worth acknowledging that in some cases government communication of their responses was more widely available than others. Therefore, there may have been government responses that were not clearly or widely communicated that this database does not capture. Results: Government responses to GBV during COVID-19 2. Was there COVID-speciﬁc GBV funding? There is considerable variation among the 60 countries, demonstrating that comprehensive government responses to the gendered eﬀect of COVID-19 – and other health emergencies – cannot be taken for granted (see Figure 1). 3. Was there a government supported COVID GBV service provision guidance response and adaptation/training/awareness campaign? As evidenced by Table 1, on the positive side, no government scored 0 on the Index, meaning that every government in our sample put in place at least one of the measures set out above. Worryingly, however, only ﬁve countries were found to have enacted all three responses, and most countries were found to have enacted just one response. Overall, a majority We created the Government Response to GBV during COVID-19 Index by awarding one point for each response (or in some cases 0.5 points – for example if some funds were allocated to GBV during COVID-19 but in relation to awareness 2 It is also worth acknowledging that in some cases government communication of their responses was more widely available than others. Therefore, there may have been government responses that were not clearly or widely communicated that this database does not capture. Frontiers in Global Women’s Health frontiersin.org 05 10.3389/fgwh.2022.857345 Gordon et al. TABLE 1 Distribution of countries on the Government responses to GBV index. Government GBV response index Number of countries 0 0 1 27 1.5 6 2 21 2.5 1 3 5 TABLE 2 Government policy responses to GBV during COVID-19. Policy response Frequency of implementation Number of countries that introduced national speciﬁc GBV legislation/protocols which responded to COVID-19 23 (1 was mixed/limited and was coded as 0.5) Number of countries that introduced funding to speciﬁcally address GBV during COVID-19 12 countries (3 had limited/mixed responses to funding) Number of countries responded with updated guidance/increased or adapted service provision/awareness raising or communication campaigns 59 countries (3 had mixed responses which were coded as 0.5) TABLE 1 Distribution of countries on the Government responses to GBV index. TABLE 1 Distribution of countries on the Government responses to GBV index. environment related to gender-speciﬁc responses. There were also some high-income contexts with high gender equality index ranking who appeared to take this strategy (e.g., Norway) that we explore below. Government GBV response index Number of countries Twenty-three countries, some 38% of the sample, implemented pandemic-speciﬁc GBV legislation or national- level protocols. Results: Government responses to GBV during COVID-19 In some ways this is a surprisingly positive ﬁnding. Taking the time to design and pass new legislation and/or national-level protocols amidst a pandemic demonstrates that these governments were both aware of the gendered impact of the pandemic and willing to take steps to prioritize a policy response. For example, in Pakistan, the national government integrated measures to address violence against women and girls under the Socioeconomic Impact Plan and Pakistan’s Preparedness Response Plan for COVID-19. Additionally, Provincial Governments have incorporated gender responsive planning guidelines with a speciﬁc focus on socioeconomic impacts of COVID-19 (28). In Lebanon, the National Commission for Lebanese Women issued a press release on amendments to the Domestic Violence Bill to promote protections for women and children that address increasing domestic violence as a result of the country’s economic crisis and lockdown measures due to COVID-19 (28). In Brazil, new legislation (Law N.14022) was introduced to extend protective measures for women during the pandemic (28). However, it is important to also recognize the shortcomings of government responses during this period. Most notably, we only found evidence of 12 countries that introduced new funding to ﬁnance GBV prevention activities. Countries that deployed this strategy included Australia, Bosnia and Herzegovina, Canada, DRC, Kenya, New Zealand, UK, USA, Uzbekistan and Zimbabwe. These are mostly (although importantly not all) high-income contexts, which suggests that in a number of cases the introduction of new measures was likely hampered by a lack of funds. In the case of the DRC and Zimbabwe this was clearly not the case, and we explore GBV responses in the context of low-income countries in greater depth below. of countries (33 as compared to 27) delivered fewer than two policy responses, meaning that a worryingly high number of governments failed to fully deliver two of three approaches that could have potentially limited the impact of the pandemic on GBV. Of course, which responses governments favored is as important a question as the number of strategies that governments put in place. As noted in Table 2, the most common response was to issue updated guidance and/or raise awareness to addressing higher risks of, and how to respond to rising GBV during COVID-19 (59 countries). This makes intuitive sense – updating guidelines and awareness raising campaigns can often be done by tweaking how existing funding and programming is delivered. Discussion: What explains governmental GBV responses? There are many factors that might inﬂuence governmental responses to GBV at any point, and particularly during crises. Once we developed the Index, we explored whether there were any patterns that could explain the main factors that make it more likely governments respond proactively. Interviews as part of our case studies discussed below suggested that one important factor was the presence of active civil society organizations able to campaign on gender issues because they had direct contact and lines of communication to the relevant government agencies (either pre-existing or established at the outset of the pandemic). This meant they could raise awareness and put pressure on political leaders to take stronger action on GBV. For example, in March 2020, the Agency for Gender Equality Bosnia and Herzegovina (BiH) conducted a survey with Civil Society Organizations (CSOs) running shelters in BiH. Based on CSO feedback the Agency developed the Plan of Intervention measures to support CSOs running shelters, which became an integral part of the Plan of interventions of the Ministry of Human Rights and Refugees in BiH (28). Income Level (1 = low, 2 = lower middle, 3 = upper middle, 4 = high) [Source: (32)]4 When we move to look at pre-existing levels of gender inequality, the relationship is even weaker. As depicted in Figure 3, there was no statistically signiﬁcant relationship between gender inequality and Government Response to GBV during the pandemic (Spearman’s rho = 0.110, p =0.41). Many countries with higher levels of gender inequality performed as well, or the same, as countries with lower levels. Gender inequality is here measured using the UNDP Gender Inequality Index, which takes into account inequality in reproductive health, empowerment and economic status (33). We have already discussed the point that some countries, such as Norway, may have implemented fewer measures in response to COVID- 19 as they already had strong provision, which may help to explain the absence of a relationship in some cases, but even if we exclude these exceptional performers the relationship is marginal. Results: Government responses to GBV during COVID-19 This option therefore represents the path of least resistance – the strategy that is least likely to be opposed on the grounds of cost or time. The majority of the countries that appear to have deployed this strategy were low income and lower middle income contexts (including Bangladesh, Ethiopia, LAO PDR among others); however, there were a number of upper middle income countries that took this approach (including Albania, Armenia, and Jordan), although these tended to also be countries with Gender Inequality Index ranks below 50, which may indicate a more diﬃcult legislative The full evaluation for each response for each country, and a justiﬁcation for the score given, can be found in the data ﬁles for this project. When interpreting these scores, it is important to keep in mind that in some countries that had already established extremely comprehensive GBV laws prior to the pandemic there was no attempt to introduce new COVID- speciﬁc legislation as existing legal frameworks were seen to be suﬃcient. In some of these cases, such as Norway, we consider that this reﬂected a genuine sense that existing legislation was suﬃcient, rather than a derogation of duty – especially as the Norwegian government were proactive in providing donor support to low-income countries to address GBV during the pandemic (29). As a result, there are some cases in which governments that did not develop new legislation or funding did Frontiers in Global Women’s Health frontiersin.org 06 10.3389/fgwh.2022.857345 10.3389/fgwh.2022.857345 10.3389/fgwh.2022.857345 Gordon et al. seek to meaningfully adapt and utilize existing laws and funding mechanisms to eﬀectively address GBV during the pandemic. This was not the case, everywhere, however, which raises the question of what explains variation in government responses to GBV increases? to GBV Index against the relevant factor, as in Figure 23. In each case, a rising diagonal line from bottom left to top right indicates the presence of a strong relationship. Where Figure 2 is concerned, we look at the income level of the government/country concerned, using the World Bank classiﬁcation that divides countries up into low, lower middle, upper middle-, and high-income countries (31). The graph illustrates that there is a slight increase in how countries perform on the Government Responses to GBV Index as they become wealthier, but this relationship is not statistically signiﬁcant (Spearman’s rho = −0.078, p = 0.551). 3 As noted above, it is not possible to take into account initial levels of support for GBV related interventions because there is not a dataset that collects this information for our set of cases. To the best of our knowledge, the closest dataset that exists is Htun and Weldon (10), but this only goes up to 2005, does not include all of our cases, and focuses on CEDAW ratiﬁcation and regional level agreements, rather than levels of domestic funding and legislation. 4 Our decision to use the World Bank classiﬁcations was due to the consideration of their greater stability during a period of economic ﬂuctuation for many countries, which may have inﬂuenced the accuracy of this analysis were we to use other indicators, such as GDP per capita. Results: Government responses to GBV during COVID-19 In other words, there is no evidence from these data that wealth was the key determining factor that shaped gender-sensitive responses to the pandemic. Discussion: What explains governmental GBV responses? We explore this further in the ﬁnal part of the paper, but in order to make sure that this “civil society awareness raising eﬀect” is not simply a by-product of countries in which the conditions are more suitable – and to better understand the diﬀerent forces that shape gender-sensitive policies – we ﬁrst look at three other factors that the literature and past experience suggest may be related to government policy toward GBV: government income, on the basis that leaders with more resources have more room for maneuver, existing levels of gender inequality, on the basis that these are likely to reﬂect the position of women in society and the capacity of anti- GBV groups to eﬀectively mobilize, and the representation of women within the political system, on the basis that countries with more women in prominent positions may be able to galvanize greater support for a gender-sensitive response from within the political elite (30). Levels of violence pre-pandemic may also be related to government policy toward GBV. However, due to the lack of systematic data collection regarding GBV worldwide and diﬀerences in reporting rates between countries, pre-pandemic violence could not be correlated against this Index. Gender Inequality Index Score [Source: UNDP (33) Gender Inequality Index Rank]. Thirdly, we consider whether the representation of women within political roles can lead to more comprehensive and eﬀective policies related to gender issues, including GBV. To do this, we explored whether there is any link between a country’s political parity score and their GBV response during COVID-19. The political parity score is an aggregate measure In each case, we illustrate the presence (or absence) of the relationship by plotting the Government Responses Frontiers in Global Women’s Health frontiersin.org 07 Gordon et al. 10.3389/fgwh.2022.857345 FIGURE 2 Comparing income level to government responses to GBV during COVID-19. FIGURE 3 Comparing gender inequality index to government responses to GBV during COVID-19. of the representation of women in a country’s government, and hence, measures the likelihood that women leaders can Political Parity Index [Source: Council of Foreign Relations Political Parity Score, (34)]. FIGURE 2 Comparing income level to government responses to GBV during COVID-19. FIGURE 3 Comparing gender inequality index to government responses to GBV during COVID-19. Political Parity Index [Source: Council of Foreign Relations Political Parity Score, (34)]. Discussion: What explains governmental GBV responses? of the representation of women in a country’s government, and hence, measures the likelihood that women leaders can shape policy. Again, the relationship was not statistically signiﬁcant (Spearman’s rho = −0.07, p = 0.583). As can be seen in Figure 4, as countries with low levels of political parity have had similar responses to the pandemic as those with high levels. Given that three of the most likely factors we identiﬁed are not statistically signiﬁcantly related to how governments responded to the threat of rising GBV during the pandemic, it is clear that the explanation lies elsewhere. While this study cannot go into depth on every possible driver of government Frontiers in Global Women’s Health frontiersin.org 08 Gordon et al. 10.3389/fgwh.2022.857345 FIGURE 4 Comparing political parity index to government responses to GBV during COVID-19. FIGURE 4 Comparing political parity index to government responses to GBV during COVID-19. FIGURE 4 Comparing political parity index to government responses to GBV during COVID-19. the impact of COVID-19 on GBV. It has also collaborated with UNFPA to develop appropriate response measures based on their study on the impacts of COVID-19 in the country (28). policy, there is considerable evidence that the role of civil society and non-governmental organizations, and their ability to galvanize political governments into action (10), has played an important role in ensuring that governments put gender- sensitive responses to the pandemic on the agenda. Although pre-existing data which maps the robustness of civil society was not consistently available to be able to explore this in relation to our Index, research has demonstrated that civil society has been considered to improve outlines in relation to gender-based violence directly, as a result of service provision (35), as well as playing an important role for monitoring and pressuring governments into action (36). In the Philippines, a particularly diﬃcult context since the election of the populist President Rodrigo Duterte, civil society actors including women’s rights organizations worked with progressive legislators to push for a bill that would criminalize child marriage. The legislation passed the Senate in November 2020 and received the support of the House of Representatives in September 2021 – becoming law – protecting young girls from a worrying increase in child marriages during the pandemic (37). Discussion: What explains governmental GBV responses? Similarly, the Egyptian government was also supported by UN Women, UNFPA and other actors, to update its Standard Operating procedures in order to better address the pandemic (ibid). Our rapid case studies (Boxes 1, 2) explore these factors in more detail. Conclusion Having said this, there were active civil society actors and campaigns in all of the countries in the dataset, and there is a large amount of evidence (including that noted above) of their important role in adapting and responding to GBV during the crisis. Our results suggest that while civil society organizations are important to ensuring more comprehensive government policy responses, these organizations must be well funded and supported in order to make inroads and put ﬁrm pressure on political actors. BOX 1 Aotearoa New Zealand. Aotearoa New Zealand has one of the highest rates of domestic violence in high-income contexts, with one in three women reporting physical abuse and one in two reporting psychological abuse from an intimate partner in their lifetime (38). However, New Zealand also had comprehensive and progressive pre-existing legislation to tackle GBV, in particular domestic violence. For example, The Domestic Violence - Victims Protection Act (2018) was a global landmark for holding workplaces accountable for safeguarding survivors of violence (39). Aotearoa New Zealand has one of the highest rates of domestic violence in high-income contexts, with one in three women reporting physical abuse and one in two reporting psychological abuse from an intimate partner in their lifetime (38). However, New Zealand also had comprehensive and progressive pre-existing legislation to tackle GBV, in particular domestic violence. For example, The Domestic Violence - Victims Protection Act (2018) was a global landmark for holding workplaces accountable for safeguarding survivors of violence (39). The response by the Government in New Zealand to GBV during COVID-19 was comprehensive with the introduction of new national protocols (40). Guidelines for the Family Violence and Sexual Violence workforce for COVID-19 were introduced and continually updated, so that family and sexual violence crisis workers could continue operating safely. The government also provided a comprehensive information site related to COVID-19 and GBV, with updated helplines and organizations for citizens to engage with to support with family and sexual violence prevention (41) Targeted funding was also introduced; on 31st May 2020, the Minister for Women announced a 1 million NZD fund for organizations that support women and girls as part of the Government’s COVID-19 response. It operated in the form of a one-of grant to support these organizations in the short term, but the funding was later doubled to 2 million NZD due to the level of demand. Conclusion A trend that quickly became apparent in the mapping of government responses, as mentioned above, was the importance of active and well-connected civil society organizations. As well as evidence of this emerging for the cases of Bosnia and Herzegovina and Kenya, there were multiple other examples in which civil society organizations – most notably women’s movements and organizations – and international NGOs and UN agencies played a key role in galvanizing and supporting government responses. For example, the Ministry of Women’s Aﬀairs in Cambodia collaborated closely with NGOs and donor agencies to develop and distribute communication materials focused on GBV and COVID-19, collect data and document The results of the policy analysis indicate that civil society and non-governmental organizations had an important role in ensuring governments enacted gender-sensitive responses during the pandemic. Organizations that have strong links and communications with the government were particularly well placed to promote and enable political governments to take action. In particular, the results suggest that countries with previous experience of responding to emergencies (e.g., the Christchurch earthquake, the Ebola crisis) were especially well placed and could take eﬀective action because there was Frontiers in Global Women’s Health frontiersin.org 09 Gordon et al. 10.3389/fgwh.2022.857345 10.3389/fgwh.2022.857345 Our data provide detailed information about government policies enacted to control levels of GBV during the pandemic, which enabled us to compare countries and analyse policy responses to GBV during COVID-19. However, the data included only countries in which data were available regarding government GBV policy during COVID-19. As such, the data may not be representative of countries in which GBV government policy enactments are unknown. Therefore, inferences about GBV policy are limited to those countries in which information was available and accessible. Finally, information about the impact of government policy on GBV levels dur ing the pandemic remain unknown because GBV tends to be underreported, especially during emergencies such as the COVID-19 pandemic. There is therefore a need for capacity and precedence in how to respond and awareness of what would be needed to prevent and respond to GBV during the pandemic. Further, there was evidence that countries that have a dedicated minister (or person within a ministry) for GBV were particularly well-placed to respond. Conclusion Finally, this was incorporated into mainstream funding structures, with an additional 200 million NZD allocated in the budget to respond to increased rates of violence during COVID-19 (28). It is important to understand the facilitating and enabling factors which supported this comparatively comprehensive response. One factor mentioned was the fact that there was a collaborative set-up already in place. The Joint Business Venture Unit (JBVU) was set up by the undersecretary to co-ordinate ten ministries to have a uniﬁed response to family and sexual violence. The creation of one access point to multiple ministries (including health, education and others) meant that NGOs in the sector had a well-established line of communication to respond to the changing needs. During the ﬁrst lockdown, open zoom meetings instigated by NGOs within the sector were joined by the JBVU to share information on what was happening on the ground to best coordinate the government-level response. The JBVU also ofered practical action on policies and responses. Those within the sector agreed that public servants within the JVBU were open and responsive to their suggestions and willing to collaborate, acknowledging the unique situation and recognizing the limits of their expertise in this situation. The JVBU were already working across ministries to get more funding for the sector overall, and this allowed them to pivot during COVID-19 to provide one of grants to organizations. These were ﬂexible and many of the usual deliverables and reporting were scrapped in order to enable organizations to efectively respond to the crisis. This ampliﬁed the high trust environment between the government and organizations working within the sector. However, whilst the JVBU and the sector had a strong working relationship during the pandemic, there were challenges within the wider government, namely barriers to getting onto pandemic committees, which meant that the GBV/SRV lens was missing from the centralized crisis response. Some felt that this could have been addressed if there had been a speciﬁc cluster within the emergency response focused on GBV as often seen in other crises. l (28). The well-coordinated responses from organizations working in this sector had an important role to play in highlighting the adjustments and additional services that were needed. For example, UNFPA worked to provide a ‘one stop center’ to help survivors of violence and worked with the government to put in place a helpline for those who could not physically access services. Similar to New Zealand, there was a pre-existing set-up in place coordinating stakeholders working on GBV, the GBV sub-cluster. During COVID-19, this was adapted to meet online and worked as a way to coordinate responses to GBV during the pandemic. Those we spoke to who worked on these issues felt that the government were open minded and receptive to addressing the challenges related to GBV during COVID-19, and ﬁnding solutions to these issues. There were a number of key stakeholders within the government who played an important role in ensuring this comparatively comprehensive response, including those in the Ministry of Gender, Family and Children, the special adviser to the president on sexual violence of youth, the Minister of Health and the First Lady. There were a number of formal and informal conversations, with roundtables to discuss the response and getting the agenda set. However, it was also noted that the government priority was responding to COVID, and whilst there were increases in funding to GBV services on paper, it is not clear whether these have been realized in practice. Indeed, researchers have emphasized that broader contextual issues, particularly related to funding, are likely to mediate government responses to GBV during COVID-19 (13). literature review to support the article and supported the writing and analysis. HF supported the writing and analysis. All authors contributed to the article and approved the submitted version. governments to make greater investments in national statistical capacity to systematically collect and analyse gender data and use it to monitor and inform policy interventions. Funding The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. This research was funded by a grant award from the Institute for Global Innovation at the University of Birmingham. Ethics 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. The studies involving human participants were reviewed and approved by University of Birmingham. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conclusion espo d o e c s s s a p ed e g us e o e be ee e go e e a d o ga a o s o g e sec o However, whilst the JVBU and the sector had a strong working relationship during the pandemic, there were challenges within the wider government, namely barriers to getting onto pandemic committees, which meant that the GBV/SRV lens was missing from the centralized crisis response. Some felt that this could have been addressed if there had been a speciﬁc cluster within the emergency response focused on GBV as often seen in other crises. NGOs were also pro-active and coordinated in their approach to raising issues with the government. Pre-pandemic, the family and sexual violence sector had begun to work more closely together prior to this period, holding a joint conference in November 2019. This meant that there were good pre-existing relationships and a collaborative mindset which were activated quickly during COVID-19. This coordinated collaboration led to the online meetings between those in the sector and the government mentioned above. NGOs also set up sub-committees (which are still in operation) to focus on diferent issues and consistently share information with the government. Finally, pre-existing experience of adapting and responding to the Christchurch earthquake was considered to have laid a foundation for approaches to crisis response. Christchurch had highlighted that responses to GBV during emergencies were not integrated within the wider response; although this was not ‘solved’ by COVID, there was strategic learning from this response. For example, the government had recognized that communities know what to do best and how to respond, and so the learning from this was shared in multiple ways. In addition, the importance of pre-disaster relationships between the police, civil defense and women’s refuges was highlighted during this event (42). Those in government also reﬂected on the fact that this had already stress-tested some of the issues that arose during COVID-19. However, it was also widely acknowledged that whilst the response was driven and facilitated by an active civil society sector, and openness and willingness to listen from government, that a continued lack of funding and capacity of the family and sexual violence sector was a remaining barrier to a comprehensive response to GBV during the crisis, and beyond. Frontiers in Global Women’s Health 10 frontiersin.org Gordon et al. Conclusion 10.3389/fgwh.2022.857345 BOX 2 Democratic Republic of Congo. BOX 2 Democratic Republic of Congo. The DRC is known to have a high prevalence of GBV; 68% of women report lifetime exposure to physical, sexual or emotional violence (17). The Ebola epidemic (2018-2020) saw an increase in the reported risk and experience of GBV, both due to the fact that gender norms became further entrenched and because a fear of catching Ebola meant that women and girls experiencing abuse did not access services. The challenges were exacerbated by a lack of ﬁnancial resources available to women and girls, and a lack of communication from the government (ibid). There were also reports of increased sexual and gender-based violence nationally during COVID-19, which were particularly severe in North Kivu and Goma, with school closures and ﬁnancial difculties being argued to put girls at increased risk of abuse (43). Violence against women and girls was speciﬁcally included in the national programme to address COVID-19, Programme Multisectoriel D’Urgence D’aténuation des impacts de la Covid-19 en RDC with speciﬁc outputs, indicators, activities and a dedicated budget (28). This plan was introduced to address the efects of the pandemic, in particular economic stability and recovery but also for civil society working to address other areas, including GBV. Additionally, an online network of psychologists and social workers was set up to provide psychosocial support to GBV and COVID-19 survivors and to refer them to appropriate services, which was accompanied by increased awareness campaigns at the national, regional and community level (28). 19. WHO. COVID-19 and violence against women: What the health sector/system can do, WHO. Available online at: https://apps.who.int/iris/handle/10665/331699 (accessed July 25, 2022). 19. WHO. COVID-19 and violence against women: What the health sector/system can do, WHO. Available online at: https://apps.who.int/iris/handle/10665/331699 (accessed July 25, 2022). 20. Kingkade T. Police see rise in domestic violence calls amid coronavirus lockdown 2020. NBCNews. Available online at: https://www.nbcnews.com/news/ us-news/police-see-rise-domestic-violence-calls-amid-coronavirus-lockdown- n1176151 (accessed July 25, 2022). 2. Aolain F. Women, vulnerability, and humanitarian emergencies. Michigan J Gender Law. (2011) 18:1. doi: 10.2139/ssrn.1611818 3. Marsh M, Purdin S, Navani S. Addressing sexual violence in humanitarian emergencies. Glob Public Health. (2006) 1:133– 46. doi: 10.1080/17441690600652787 3. Marsh M, Purdin S, Navani S. Addressing sexual violence in humanitarian emergencies. Glob Public Health. (2006) 1:133– 46. doi: 10.1080/17441690600652787 21. UNOHCHR. Kenya: Monitoring Human Rights Impacts of COVID-19 in Informal Settlements. UNOHCHR (2020). 4. Bradbury-Jones C, Isham L. The pandemic paradox: the consequences of COVID-19 on domestic violence. J Clin Nurs. (2020) 29:2047–9. doi: 10.1111/jocn.15296 22. Oﬃce of National Statistics. Oﬃce UNCsFKC, Prevention DoV, Control NCfIPa, Prevention USCfDCa, Statistics KNB. Violence against Children in Kenya: Findings from a 2010 National Survey. Summary Report on the Prevalence of Sexual, Physical and Emotional Violence, Context of Sexual Violence, and Health and Behavioral Consequences of Violence Experienced in Childhood. Nairobi, Kenya (2012). 5. Rockowitz S, Stevens LM, Rockey JC, Smith LL, Ritchie J, ColloﬀMF, et al. Patterns of sexual violence against adults and children during the COVID- 19 pandemic in Kenya: a prospective cross-sectional study. BMJ Open. (2021) 11:e048636. doi: 10.1136/bmjopen-2021-048636 23. Lufkin. The impact of banning alcohol during Covid-19, BBC Worklife. Available online at: https://www.bbc.com/worklife/article/ 20200917-the-impact-of-banning-alcohol-during-covid-19 (accessed July 25, 2022). 23. Lufkin. The impact of banning alcohol during Covid-19, BBC Worklife. Available online at: https://www.bbc.com/worklife/article/ 20200917-the-impact-of-banning-alcohol-during-covid-19 (accessed July 25, 2022). 6. CARE. Gender-Based Violence (GBV) and COVID-19: The Complexities of Responding to “The Shadow Pandemic.” A Policy Brief. Available online at: https:// www.care.org/wp-content/uploads/2020/07/care_gbv_and_covid-_policy_brief- ﬁnal.pdf (accessed July 25, 2022). 24. Obiria M. Kenya: How Feminists Shaped Kenya’s Response to GBV Amid COVID-19. Daily Nation. Available online at: https://allafrica.com/stories/ 202109140070.html (accessed July 25, 2022). 7. United Nations. Policy Brief: The Impact of COVID-19 on Women. United Nations. Available online at: https://www.unwomen.org/en/digital-library/ publications/2020/04/policy-brief-the-impact-of-covid-19-on-women (accessed July 25, 2022). 25. John N, Roy C, Mwangi M, Raval N, McGovern T. COVID-19 and gender- based violence (GBV): hard-to-reach women and girls, services, and programmes in Kenya. Gender Develop. (2021) 29:55–71. doi: 10.1080/13552074.2021.18 85219 8. UN Women. 19. WHO. COVID-19 and violence against women: What the health sector/system can do, WHO. Available online at: https://apps.who.int/iris/handle/10665/331699 (accessed July 25, 2022). Press Release: As impact of COVID-19 Intensiﬁes, UN Women Calls for Concrete Actions to Respond to the Concurrent Shadow Pandemic. UN Women. Available online at: https://www.unwomen.org/en/news/stories/2020/11/ press-release-16-days-of-activism-against-gender-based-violence (accessed July 25, 2022). 26. Nation Online. Oxfam boosts MPs GBV ﬁght. Available online at: https://www.mwnation.com/oxfam-boosts-mps-gbv-ﬁght/ (accessed July 25, 2022). 26. Nation Online. Oxfam boosts MPs GBV ﬁght. Available online at: https://www.mwnation.com/oxfam-boosts-mps-gbv-ﬁght/ (accessed July 25, 2022). 9. United Nations Populations Fund. Millions More Cases of Violence, Child Marriage, Female Genital Mutilation, Unintended Pregnancy Expected Due to the COVID-19 Pandemic. UNFPA Website (2020). Available online at: https://www. unfpa.org/news/millions-more-cases-violencechild-marriage-female-genital- mutilation-unintended-pregnancies 27. Istanbul Convention. Council of Europe Convention on Preventing and Combating Violence Against Women and Domestic Violence (CETS No. 210). (2011). Available online at: https://www.coe.int/en/web/conventions/full-list?module= treaty-detail&treatynum=210 (accessed July 25, 2022). 28. UNDP. Gender Tracker. Available online at: https://data.undp.org/ gendertracker/ (accessed July 25, 2022). 10. Htun M, Weldon SM. The civic origins of progressive policy change: combating violence against women in global perspective, 1975– 2005. Am Polit Sci Rev. (2012) 106:548–569. doi: 10.1017/S00030554120 00226 29. Norway in Kenya. Norway Supports Response to Sexual and Gender Based Violence Worsened by Covid-19 Pandemic. Available online at: https:// www.norway.no/en/kenya/norway-kenya/news-events/news2/norway-supports- response-to-sexual--gender-based-violence-worsened-by-covid-19-pandemic/ (accessed July 25, 2022). 11. Dlamini NJ. Gender-based violence, twin pandemic to COVID-19. Critical Sociol. (2020) 47:583–590. doi: 10.1177/0896920520975465 11. Dlamini NJ. Gender-based violence, twin pandemic to COVID-19. Critical Sociol. (2020) 47:583–590. doi: 10.1177/0896920520975465 12. Bloﬁeld M, Khalifa A, Madera N, Pieper J. The shadow pandemic: policy eﬀorts on gender-based violence during COVID-19 in the global south. GIGA Focus. (2021) 6:1862–3581. 12. Bloﬁeld M, Khalifa A, Madera N, Pieper J. The shadow pandemic: policy eﬀorts on gender-based violence during COVID-19 in the global south. GIGA Focus. (2021) 6:1862–3581. 30. Cowper-Coles M. Women Political Leaders: The Impact of Gender on Democracy. The Global Institute for Women’s Leadership: Kings College London (2020). 31. Hamadeh N, Van Rompaey C, Metreau E. New World Bank country classiﬁcations by income level: 2021–2022, World Bank Blogs. Available online at: https://blogs.worldbank.org/opendata/new-world-bank-country-classiﬁcations- income-level-2021-2022 (accessed July 25, 2022). 13. Erskine D. Not just hotlines and mobile phones: GBV Service provision during COVID-19. UNICEF Note. Available online at: https://www.unicef.org/media/ 68086/ﬁle/GBV%20Service%20Provision%20During%20COVID-19.pdf (accessed July 25, 2022). 32. World Bank Classiﬁcations. The World by Income and Region. Available online: https://datatopics.worldbank.org/world-development-indicators/the- world-by-income-and-region.html (accessed July 25, 2022). 14. Kelly A, Pattisson P. ‘A Pandemic of abuses’: human rights under attack during Covid, says UN head’ The Guardian. Available online at: https://www.theguardian. Author contributions RG co-designed the research, undertook the data collection, and led the writing and analysis. NC co-designed the research and supported the writing and analysis. SR and LS undertook a Frontiers in Global Women’s Health frontiersin.org 11 10.3389/fgwh.2022.857345 Gordon et al. References 1. Women’s Aid. Domestic abuse is a gendered crime. Women’s Aid. Available online at: https://www.womensaid.org.uk/information-support/ what-is-domestic-abuse/domestic-abuse-is-a-gendered-crime/ (accessed July 25, 2022). 40. Alfers, L. Social Protection Responses to COVID-19: Measures to tackle gender-based violence amid the global pandemic. WIEGO (2020). 41. NZGOVT. United against COVID-19. Available online at: https://covid19. govt.nz/ (accessed July 25, 2022). 42. True J. Gendered violence in natural disasters: Learning from New Orleans, Haiti and Christchurch. Aotearoa New Zealand Soc Work. (2013) 25:78– 89. doi: 10.11157/anzswj-vol25iss2id83 38. Fanslow JL, Robinson EM. Sticks, stones, or words? Counting the prevalence of diﬀerent types of intimate partner violence reported by New Zealand women. J Aggression Maltreatment Trauma. (2011) 20:741–59. doi: 10.1080/10926771.2011.608221 39. Weatherall R, Gavin M, Thorburn N. Safeguarding women at work? Lessons from Aotearoa New Zealand on eﬀectively implementing domestic violence policies. J Ind Relat. (2021) 63:568–90. doi: 10.1177/00221856219 96766 43. CASS. The impacts of the COVID-19 outbreak response on women and girls in the Democratic Republic of the Congo. Social Sciences Analytics Cell: CASS (2020). org/news/senate-philippines-abolishes-child-marriage.html (accessed July 25, 2022). 38. Fanslow JL, Robinson EM. Sticks, stones, or words? Counting the prevalence of diﬀerent types of intimate partner violence reported by New Zealand women. J Aggression Maltreatment Trauma. (2011) 20:741–59. doi: 10.1080/10926771.2011.608221 39. Weatherall R, Gavin M, Thorburn N. Safeguarding women at work? Lessons from Aotearoa New Zealand on eﬀectively implementing domestic violence policies. J Ind Relat. (2021) 63:568–90. doi: 10.1177/00221856219 96766 19. WHO. COVID-19 and violence against women: What the health sector/system can do, WHO. Available online at: https://apps.who.int/iris/handle/10665/331699 (accessed July 25, 2022). com/global-development/2021/feb/22/human-rights-in-the-time-of-covid-a- pandemic-of-abuses-says-un-head (accessed July 25, 2022). 33. UNDP (n.d.) Gender Inequality Index (GII), UNDP Human Development Reports. Available online at: http://hdr.undp.org/en/content/gender-inequality- index-gii (accessed July 25, 2022). 15. Oxfam International. Women rights organisation hit harder by funding cuts and left out of COVID-19 response and recovery eﬀorts. Oxfam International. Available online at: https://www.oxfam.org/en/press-releases/women-rights- organisations-hit-harder-funding-cuts-and-left-out-covid-19-response ( d J l 25 2022) 15. Oxfam International. Women rights organisation hit harder by funding cuts and left out of COVID-19 response and recovery eﬀorts. Oxfam International. Available online at: https://www.oxfam.org/en/press-releases/women-rights- organisations-hit-harder-funding-cuts-and-left-out-covid-19-response (accessed July 25, 2022). 34. Council of Foreign Relations. Political Parity Score, Women’s Power Index. Available online at: https://www.cfr.org/article/womens-power-index (accessed July 25, 2022). 16. Mittal S, Singh T. Gender-based violence during COVID-19 pandemic: a mini-review. Front. Global Women’s Health. (2020). doi: 10.3389/fgwh.2020. 00004 35. García-Moreno C, Zimmerman C, Morris-Gehring A, Heise L, Amin A, Abrahams N, et al. Addressing violence against women: a call to action. Lancet. (2015) 385:1685–1695. doi: 10.1016/S0140-6736(14)6 1830-4 17. Stark L, Meinhart M, Vahedi L, Carter SE, Roesch E, MoncrieﬀIS, et al. The syndemic of COVID-19 and gender-based violence in humanitarian settings: leveraging lessons from Ebola in the democratic republic of Congo. BMJ Global Health. (2020) 5:e004194. doi: 10.1136/bmjgh-2020- 004194 36. Mercer C. NGOs, civil society and democratization: a critical review of the literature. Prog Develop Stud. (2002) 2:5–22. doi: 10.1191/1464993402ps 027ra 37. PGA. Senate of the Philippines passes bill that abolishes child marriage. Parliamentarians for Global Action. Available online at: https://www.pgaction. 37. PGA. Senate of the Philippines passes bill that abolishes child marriage. Parliamentarians for Global Action. Available online at: https://www.pgaction. 18. UN Women. COVID-19 and Ending Violence Against Women and Girls. New York: UN Women (2020). 12 frontiersin.org Frontiers in Global Women’s Health Frontiers in Global Women’s Health 12 Gordon et al. 10.3389/fgwh.2022.857345 10.3389/fgwh.2022.857345 org/news/senate-philippines-abolishes-child-marriage.html (accessed July 25, 2022). org/news/senate-philippines-abolishes-child-marriage.html (accessed July 25, 2022). org/news/senate-philippines-abolishes-child-marriage.html (accessed July 25, 2022). org/news/senate-philippines-abolishes-child-marriage.html (accessed July 25 2022) g 25, 2022). 43. CASS. The impacts of the COVID-19 outbreak response on women and girls in the Democratic Republic of the Congo. Social Sciences Analytics Cell: CASS (2020). 13 Frontiers in Global Women’s Health 13 frontiersin.org

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